EFFECTS OF a4LL-TRANS RETINOIC ACID -4LONE -AND IN COIbIBINATION WITH DEXAMETHASONE ON THE DEVELOPING LIkIB SKELETON

OF THE FETAL MOLISE

Yick Basso

-4 thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Depanment of Pharmacolog University of Toronto

0 Copyright by Nick Basso, 1997

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EFFECTS OF .ALL-TRANS RETINOIC .ACID ALONE .AND IN CONIBINdATION WITR DEXAMETH.4SONE ON THE DEVELOPING LIMB SECELETON

OF THE FETAL !VIOLrSE

blasters of Science. 1997. Nick Basso. Grriduntc Dcpanmcnt of Phrimiacolog. Cni \ . cr s i~ of Toronto

.Ml-trans retinoic acid ( R A ) is a potent teratogen in humans and in sevrral mammals. with

effects on limb and craniofacial development We studied all-tram R A teratogenicity in CD-I

mice and the etrects of desamethasone (des) treatment on the all-trans RA-induced

malformations We present here the first evidence sug_resting that dex can ameliorate the effects

of all-trans RA on long bone foreshortening in the developing limb In response to a single dose

of 50 mgkg all-trans R A on dav 11.5 post coitus (pc). malformations in fetuses included

deflections of the ulna and radius and foreshortening of the distal long bones of the limbs.

blalformations in fetuses treated on day I3.5. 14.5- or 1 5.5 pc were less severe and were limited

to delaved ossification of the tertiary sites of ossification of the humerus and femur Dex. when

ziven from day 10.5 to 12.5 pc. reduced the effects of retinoid treatment on day 1 1.5 pc on limb k

skeletal development but resulted in a pattern of malformations similar to that obsewed afier

exposure to all-trans RA alone on dav 13.5 pc. Treatment with dex from day 13.5 to 15.5 pc

resulted in marked amelioration of long bone foreshortening induced by all-trans KA treatment on

day 1 I . 5 pc.

We hypothesized that the amelioration of retinoid-induced malformations by dex were a

result of des-induced downregulation of the expression of the retinoic acid receptor P (RU((3).

To evaluate the relationship between gene activation and all-trans RA-induced limb skeletal

malformations. we esamined which ceIl tvpes in the developing limb bud responded to all-trans

R A using the R A response element (RARE) hsplacZ transgenic mouse mode!. Exposure to all-

trans Ri\ on day 1 1.5 pc did not activate RARE-initiated transcription. while exposure to all-trans

RA on day 13.5 pc activated transcription in the interdigital zones (IDZ) only However. since

recent evidence from the literature indicates that transcription of the transgene is not stimulated to

the same estent by RARa and M R P and is inhibited by M~l. the outcorne of these

evperiments could not have provided the answer sought.

1 thank first and foremost Dr. Johan Heersche and Dr. Bernard Liebgott for giving me the

opportunity to work on this project. I would like to thank them for their patience. their guidance.

and their belief in me. 1 would also like to acknowledge my peers in the lab for their tnendship. 1

express my gratitude to Ted Tufescu for helpin- me to see what was really there and to both Rita

Lees and Antonio Ciaccia for helping me to celebrate the good days while for~ettinp about the

bad. I would also like to thank M s Inka Tertinesg and Ms. Shirley Reimers for their technical

assistance. Dr. Carl Bellows for his help in scientific (and not so scientific) matters. and the rest of

the lab group for making ec-ery dav an adventure in science.

TAABLE OF CONTENTS

Arknowledgmen ts

Table of Contents

List of Figures

List of Tables

List of Abbreviations

Chapter 1 General introduction

1 . I THE RETiNOlDS

1 . 1 . 1 Vitamin .A

I . ! .2 Retinoic Acid

1 1.3 Nuclear Receptor Proteins

1.1.4 Cytoplasmic Binding Proteins

1.3 EFFECTS OF RETINOIC ACID ON CARTILAGE

1 2 . 1 Retinoic Acid and Chondrogenesis

1 -2 .2 The RCJ 3 . 1 CS Cell Line

1.3 EFFECTS OF RETIVOIC ACID OW THE FET-AL SKELETOK

1 3 .1 Retinoic Xcid Embryopathy

1.3.2 .Animai Models

1.3.; Craniohcial versus Limb Malformations in Humans and Anirnals

1.4 RETINOIC ACID FECEPTOR E.WRESSION D W G L M 3 DEVELOPkiENT

1 . 4 I Limb Development in the Mouse

I A 2 The Retinoic Acid Receptors

1 4 . 3 Retinoic Acid Receptor Alpha

1.4.1 Retinoic Acid Receptor Beta

1 4.5 Retinoic Acid Receptor Gamma

1.5 GLCCOCORTICOIDS

1.5.1 The RoIe o f Glucocorticoids in Growth and Development

1 .5.2 Effects of Glucoconicoids on Cartilage

1.5.3 Effects of GIucocorticoids on the Fetal Skeleton

1.6 INTERACTIONS BETWEEN RETINOIDS .&;'ID GLCCOCORTICOIDS

1.6.1 Combined Effects of Retinoic Acid and Dexamethasone

1.6.1 Effects of Dexamethasone on RXR Expression

1 h . 3 Receptor Interactions

1 7 OBJECTIVES

Chapter 2 The Effects of Treatrnent with Dexamethasone on .Ail-Trrns Retinoic Acid-lnduced Limb Skeletal Malformations in the Fetal Mouse

2.2.1 Dmgs and Vehicles

3.2.2 Animal Treatrnents

2 . 3 Recovery and Inspection of Fetuses

2.2.4 Skeletal Esamination

2 .2 .5 Statistical Analpis

l General Inspection of Fetuses

2.3 .? Skeletal Examination of Fetuses

2.4 DISCUSSION

Chapter 3 Identification of Cell Types in the Developing Limb Bud Responding to Endogenous and Exogenous All-Trans Retinoic Acid L'sing the MREhsplacZ Transgenic Mouse 5 3

3 . 1 INTRODUCTION 64

3 . 2 METHODS 65

3 2 . 1 W h s p l a c Z Transgenic Vice 65

3.2.2 P-Galactosidase Stainino 66

3 2 . 3 Statistical Analysis 67

3 . 3 RESULTS 68

3 . 1 ~~acroscopic Staining Pattern for a-Galactosidase 68

3.3.2 Histological Localkation of X-gal Staining 68

3 .4 DISCL'SSION 70

Chapter 4 Summary and Conclusions 79

References 8-3

LIST OF FIGURES

Chapter 1

1.1 Retinoic Acid Isomers and Metabolites and Their Relative Porencies Lt'ith Respect to Induction of S keletal Abnormalities Within and Berween Species.

1.2 RAR Subtvpe Distribution during Limb Onto_~eny in the blouse.

Chapter 2

Schematic of the Dosing Resirnen for the Al-tram W D e s Combination Studies.

Effect of Dose and Day of Exposure to -41-tram RA on CleH Palate Formation.

Efect of Dose and Time of Exposure to Dex on Clefi Palate Formation

ERect of Treatment with Dex on Clefi Palate Defects hduced bv Exposure to 25 mgkg Ali-trans RA on Day 1 1.5 or 13 5 pc.

Effect of Treatment with Dex on Clefi Palate Defects Induced bv Esposure to 50 m g k g .Ml-tram R A on Day 1 1.5 or 1 3 5 pc.

Effects of Exposure to XII-trans R A on Day 11.5 pc on the Appendicular Skeleton.

Effects of Esposure to .dl-trans RA on Da? 13.5 pc on the Appendicular S keleton.

Effects of Co-treatment with Dex on Da! 11.5 pc .AI-trans RA-mediated Limb Skeletal \4alformations.

Chapter 3

3.1 P-galactosidase Stainine in the RAREhsplacZ Transgenic Mouse.

3.2 Day 1 1 5 pc RAREhsplacZ Trans~enic blouse Hindlimb Sections Stained with X-gal.

3.3 Effect of Esposure to NI-tram RA on Day 11.5 pc on Limb Bud Area and the Number of .Y-gal Positive Cells in the Limb Bud.

3.4 Da). 13.5 pc RAREhsplacZ Transgenic Mouse Hindlimb Sections Stained with X-gal.

3.5 Effect of Exposure to NI-trans R.\ on Day 1 1 .j pc on Interdigital Zone .kea and the Number of X-gal Positive Cells in the Interdigital Zones.

LIST OF TABLES

Chapter 2

2 . Effect of Dose and Day of Exposure to Ail-trans R A on Da? I 1.5 pc-type 57 Appendicular S keletai Malformations.

2.2 Effect of Dose and Day of Exposure to .Ail-trans RA on Da? 13. i pc-type 6 I Appendicuiar S keletal Malformations.

2 .3 Effects of Dex Treatment on Limb Skeletal b~alfomations Induced by 25 62 mg'kg .Ail-tram M.

2.4 Effects of Deu Treatrnent on Limb Skeletal 4Ialformations Induced by 50 59 my'kg Al-trans RA.

LIST OF ABBREVIATIONS

1 k i s retinoic acid

4-hydroxyretinoic acid

4-oxoretinoic acid

5-bromo-4-chloro-3-indoyl P-D-galacropyranoside

9-cis retinoic acid

all-trans retinoic acid

apical ectodermal ridge

P-galactosidase

cellular retinoic acid binding protein

cellular retinol binding protein

cryptic glucocorticoid receptor response element

desamethasone

fetal cellular retinoid binding protein

~lucocorticoid receptor - jlucoconicoid receptor response element b

d~cosaminogIycan - interdigital zone

perosisome proliferator activated receptor

post coitus

progesterone receptor

retinoic acid

retinoic acid embryopathy

retinoic acid receptor

13-cis RA

4-hydroy R A

4-oso RA

S-gal

9-cis RA

aIl-trans R A

AER

P-gaI

C R B P

CRBP

cGRE

des

CRBP(F)

GR

GRE

GAG

IDZ

PP-AR

P C

PR

RA

RAE

R U

retinoic acid receptor response element

retinoid receptor

retinol binding protein

thyroid hormone receptor

t hyroid hormone receptor response elernent

vitamin A deficiency

vitamin D receptor

zone of polarizing activity

RARE

R?CR

RBP

TR

T E

v.AD

C'D R

ZP.4

CHAPTER ONE

G E N E M L INTRODUCTION

1.1 The Retinoids

1. l . I Vitamin .A

Since its discovery. many roles have been ascribed to vitamin A and its naturallv occumng

analogs. which include the alcohol retinol. the aldehyde retinal (retinaldehyde). and the carboxylic

acid retinoic acid (RA). In the adult mammal. deficiency of vitamin A (VAD) results in biindness.

weight loss. testicular and ovarian atrophy. keratinization of squamous epithelia. and marked

changes in bone remodeling processes (Gangulv c'r c d . . 19SO: Navia ol . 1980; Lotan. 1980:

Goodman. 1984). Hypervitaminosis r\ results in scaling of the skin. nausea. headache. hair loss.

bone fractures. and a deterioration of the seneral condition (Look rr d.. 1995). Vitarnin A has a

well-defined role in the visual systern in the f o m of 1 l-cis-retinal. which serves as the

chromophoric substrate in the visual pigment rhodopsin (Chabre rr d. . 1989). Vitamin .A is also a

major regulator of tissue gro~vth and differentiation. Because of this. both fetal V.W and fetal

hvpervitaminosis X result in defects in the cardiac and central nervous systerns. as well as

malformations of the craniofacies and the appendicular skeleton.

In mammals. vitamin A is not produced endogenously and can only be obtained naturallv

from plant-derived p-carotene and long chah retinyl esters derived from animal tissues. Once in

the body. vitamin A is stored mainly in the liver in an inactive fom as retinyl-palmitate and other

retinyl esters

1 .1.2 Retinoic Acid

Retinyl esters can be cleaved in the liver to release retinol. which may conjugate with

retinol binding proteins (RBPs) or remain in a free. unbound fom. Retinol-RBP conju, 'rates are

transponed to tar-et tissues complexed with pre-aibumin (Chen er d.. 198 1). In addition to

exerting its own metabolic efects. free retinol can be converted to RA via oxidation in the liver as

well as in the target tissues. R A can also be formed from the oxidation of apo-carotenals. via the

asymrnetric cleavage of p-carotene. or ingested directl~ (Yapoli rr r d . . 1988). R A has a short

biological haif-life and cannot be reduced to retinol I I I iViw Therefore. RA does not accumdate

in the liver

RA is metabolized via glucuronidation. which is its major metabolic pathway (Stephens-

Jernagin rr r d . . 1983). It is also subject to cpochrome P-150 mediated metabolism. which

generates both oxidized (4-0x0 RA: Roberts er a / . 1992) and hydroxylated metabolites (4-

hydrouy RA. Martini rr d. 1994) A minor pathwav involved in RA merabolism is side chain

oxidation. which generates Few R A species. It should be noted that in organ and tissue culture

systems. where RA is not rapidly metabolized as it would be in the liver. the activity of R A is an

order of magnitude higher than that of retinol. This suggests that it is Rk rather than retinol.

which is the active form of the vitamin ( Thaller el d . 1957).

Apart frorn havin- many overlappins roles with retinol. RA has a very important

morphosenetic role of its orvn dunng embryonic and fetal development RA is produced durin-

development in the chick linib bud in the zone of polarizing activity (ZPA) Released from this

site. RA diffuses across the lirnb bud esrablishing a gradient within the limb which contributes to

pattern development and anteroposterior asis detemination (Thaller et c i / . 1957) Within the

cell. the effects of RA are mediated via two major groups of molecules ( 1 ) the nuclear receptor

proteins and (2) the cytoplasmic binding proteins.

I . 1.3 Nuclear Receptor Proteins

There are two classesss of nuclear receptors for RA, both of which belons to the steroid-

thyroid hormone superfarnily of ligand-activated transcription factors. These two goups are the

retinoic acid receptors ( W s ) . which have equal binding affinities for all-trans RA and its less

active isomer 9 4 s RA and the retinoid N receptors (KXRs), which have a higher binding affinity

for 9-cis RA. Within each group of receptors. three types exist. These are RUu. M D . and

R a ! (Giguère rr al.. 1987: Petkovich et al.. 1987: Zelent r t al.. 1989). and R?(Ru. W B . and

Rm! (Lied r i n/.. 1992: MangeisdorE 1990: Yu er r d . . 199 1 ). These receptors esist in the ce11 as

homodimers or heterodimers. They can heterodimerize with each other as well as with other

receptors within the steroid-thyroid hormone superfamily. includinp the vitamin D receptor

(VDR). the thvroid hormone receptor (TR) and the peroxisome proliferator activated receptor

(PP-AR) (Busse Cr d.. 1992; Kliewer LI!.. 1992). The RARs and the RYRs have diflèrent

spatial and temporal patterns of expression in tissues and likely regdate distinct events during

development and ditferentiation. Since both tWRs and LXRs are ligand-activated transcription

factors. eac h affects gene expression directly by binding to specific retinoic acid recept or response

elements (RARES) in the promoter regions of target genes.

11 .4 Cytoplasmic Binding Proteins

There are also two major categories of cytoplasmic binding proteins. which are different

From the retinol bindins proteins used for transpon. Cytoplasmic retinol binding proteins (CRBP-

I and CRBP-II) bind retinol oniy and are expressed ubiquitously escept in muscle (Chytil d..

1979). Cytoplasmic retinoic acid binding proteins (CRAE3P-1 and CRU3P-II) bind R4 only and

are espressed in al1 tissues except muscle. kidney. small intestine. liver. h g . and spleen (Chyil et

r d . . 1979). A third and rninor type of cytoplasrnic binding protein. CRBP(F). is espressed

exclusively in the fetus and binds both retinol and RA with equal afinity (Omori ri trl.. 198 1) .

These proteins are highly conserved between species and have putative roles in retinoid storage.

transpon. metabolism. and in regulating free retinoid concentration (Boylan rr O / . . 199 1: Dollé er

r d . . 1990. Ruberte er O / . , 199 1 ).

As with Rk in the developing chick limb bud. a gradient of C W P is established at the

same time but in opposite direction to the gradient of R A (Momss-Kay, 1992). This regulates the

levels of free RA and is believed to be important for proper limb patteming. During development

in the mouse. C W P is strictly localized to the brain and the limb buds. and is expressed only

between davs 1 1 and 14 post coitus (pc), which is the penod coincidinr with limb ontogeny This

restricted pattern of expression further supports the importance of CRABP in regulating levels of

RA in the developing limb bud (Momoi rr 01.. 1990). ..Uthough CRABP expression is

upreplated in response to increased levels of Rk it is believed that the teratogenic levels of R A

resulting from esogenous retinoid exposure saturate this mechanism. resultin- in high levels of

free R A and subsequent developmental malformations (Momss-Kay. 1991).

1.2 Effects of Retinoic Acid on Cartilage

1 2 1 Retinoic Acid and Chondrogenesis

In mammals. administration of R A during pregnancy causes abnormalities in limb and

craniofacial skeletal stmctures (Langman ri c d . . 1967: Werler rr d.. 1990: Lynberg cf ol.. 1990:

Kochhar. 1967) which are the resultant manifestation of the initial teratogenic effect of RA on the

chondrogenic process. III iitro. the inhibitory effects of RA on chondrogenesis in both the limb

bud and the facial mesenchyme are well docurnented (Tickle ct d.. 1985; Kochhar. 1973;

Wedden. 19S7). R A has been shown to inhibit cartilage direrentiation in chick limb bud

mesenchymal cells (Paulsen et tzl.. 1988: Moon er al.. 1986) and in chick facial primordia

(Wedden. 1987). and has also been shown to inhibit cartilage matrix production and stimulate

cartilase matris degradation (Horton. 1 987). In cultures of mouse interdigital tissue. which if

isolated from the day 12.5 pc foot plate will develop into cartilage and soft connective tissues,

exposure to a dose of 10 to 50 ngml all-trans R A inhibits this differentiation and promotes cell

death (Lee rt al. 1993).

Cnder some conditions. however. RA also appears to stimulate differentiation of

chondropro~enitors. .Uthoush RX inhibits chondrogenesis in chick frontonasal mass

mesenchvrne. in mandibular cultures treated with the same dose of EU [O 1 to 1 nglrnl (0.3 to 3 3

niL1) all-trans RA] there is a stimulato~ effect on cartilage formation (Langille rr L I / . 1989) In

this svstem. the mechanism for stimulation mav be separate from the mechanism of inhibition since

stimulation results only if R A is present during the first 24 h of culture and thereafier Inhibition

will occur. even if RA is absent korn the culture medium in the first 24 h (Solursh ri tr l . . 1978).

In the developing chick limb bud. both stimulation and inhibition of chondro_~enesis are

also seen. This appears. however, to be dose related as opposed to being time dependent. In this

system. when RA is applied directlv to the pre-axial marsin of the limb bud. enposure to IO-' bl

all-trans RA results in the appearance of polvdactyly (supemumerary digits). A higher dose (i.e.

10" 41) results in svndactyly (loss of digits: Sumrnerbell. 1983) As with the mandibular cells in

culture stimulated by KA in the first 74 h of culture. the effect of a low dose of RA in rhis system

may be due to a stimulation in the recruitment of undifferentiated mesenchyme in the periphery of

the anlasen to the chondro-enic iineare (Summerbell. 1983). In summary. the effects of R A on

chondrogenesis appear to be bot h stimulatory and inhibitoq. depending on tissue type. tissue

origin. dose to which the tissue is exposed. and the lengh of exposure of the tissue to R A

1.3.2 The RCJ 3. I CF Cell Line

In the monopotential chondro_genic celi line RCJ 3 . 1 CS. isolated from the multipotential

mesenchynal cell line RCJ 3.1. culturing for 10 days under standard conditions resuits in the

formation of cartilase nodules (Grigoriadis er al.. 1989). In this cell line. the effects of IW on

cartilage formation are strongly inhibitory. tUthough RA does not affect cellular proliferation in

t his system. its effect s include inhibition of chondroblast differentiation. inhibition of

olycosamino~lvcan synthesis (a major component of the cartilage matris). and stimulation of - - d

cartilase proteoglycan degadation by differentiated chondroblasts and chondrocytes (Lau et al..

1993 ).

The effect of natural and synthetic retinoids was assessssed on the RCJ 3.1 C5 cell line to

evaluate the role of different RAR types in the inhibition of chondrogenesis. The retinoids used

were all-tram RA. which binds RARU. RMP. and FUR-! with similar aflinities (K., - 1 O-"%.

Hashimoto. 199 1 ). 9-cis RA.. which binds preferentially to RYRs (Hevman. 1992). .Am SO. a

synthetic retinobenzoic acid. which binds to RARu and RARp with similar affinities but does not

bind to RN?! (Hashimoto rt irl.. 199 1 ). and its isomer .Am 580. a syntheric RARU ayonist

(Delescluse kJt trl.. 199 1 ). In this svstem. the order of potency of these d m g correlated with the

sequence of RARu afinity but not with the sequence of afinity for RARP. RA&. or the R Y R s

(von Schroeder rr id.. 1994). The sequence of potency of these drugs. with respect to their

effects on cartilage differentiation in this system. parallels the sequence of dysmorphozenic

activitv of these drugs in the chick limb bud. These results support the hypothesis that the

teratogenic effects of retinoids irr i r i i~o are related to their effects on chondrogenesis i ~ r r9irrw

(Kistler et al.. 1981) and that the pleiotropic effects of RA rnay be due to the specific R U

populations expressed within target tissues.

1.3 Effects of Retinoic Acid on the Fetal Skeleton

1 .3 . l Retinoic Acid Embryopat hy

In humans. all-trans R A (Retin A, tretinoin) and 1 3 4 s RA (Accutane. isotretinoin) are

highly effective dmgs used in the treatment of severe acne and other dermatological disorders.

Since the besinning of the use of retinoids for this purpose. in 1983. it has become apparent that

women who become pregnant while on oral RA therapy exhibit significantly increased rates of

spontaneous abonion and premat ure delivery In surviving infants. a c haracteristic set of

congenital defects. collectively tenned Retinoic Acid Embryopathy ( R a ) . are eshibited. These

defects include abnormalities of the cardiac and central nervous systems. the thymus and

parathyroid glands. the long bones of the appendicular skeleton. and the craniofacies (Lammer

L I / . . 1985; Rosa cJl d.. 1983. Braun c.! d . . 1984).

13-cis M. the dmg of choice for dermatolopical disorders. crosses the placenta readily

and accumulates in the liver as the metabolite 4-0x0- 1 3 4 s RA. Following daily esposure of 13-

cis RA during the tirst trimester of pregancy. high levels of all-trans R A can also be found in the

brain (Benifla rr d.. 1995). In a rabbit mode1 for human RAE. it was shown that an embryotosic

recimen - of oral retinpl palmitate ( 10 m & / d a y from day 7 to 12 pc) resulted in a 100-fold

increase of all-tram R A levels rvithin the embryo on dav 12 pc with only a maryinal increase in the

concentration of all-trans RX in the materna1 plasma. Non-pregnant women. whose endolenous

plasma levels are similar to that of the rabbit (5 to 8 nmoVL). also show only a marginal increase

in RA levels afier high vitamin A intake. This indicates that a high vitamin A intake may be

associated with an elevated nsk for teratogenic effects in humans even if matemal blood plasma

levels do not indicate an increase in the concentration of all-trans RA (Tzimas cv t r i . . 1996).

Aithoush cases of RG-like malformations have been reported due to megadose vitamin A

8

supplementation. proper epidemiolo~ical studies have not been perforrned. therefore tùnher

evaluation is required (Rosa rr tri.. 1986).

In humans. the major teratogenic vitamin A analog is 13-cis RA (isotretinoin). In

hamsters and other rodents. the sensitivitv of the embryo to this analog is 16-fold lower than in

humans (Willhite rr al.. 1986) and it is therefore seldorn used in laboraton models of retinoid

teratogenicity The relative potencies of all-trans RA. and it ' s isorners and metabolites are listed

in Figure t . 1 -4lthough topical exposure of 13-cis RA in humans is not associated with an

increased risk of congenital malformations (Jilk rr rd.. 1993). oral esposure dunng the tirst

trimester of pregnancy causes malformations of the brain. craniofacies. thymus. and

cardiovascular system (van Malde-em C r LI/.. 1992: Werler ri trl.. 1990). Cleft palate and limb

defects. which are seen to a high extent in al1 animal models. are seen in only a subset of infants

displaying RAE (Lamrner. 1991; Willhite C r ni.. 1986). In humans. the mûst prevalent

malformations seen are duplication and reduction defects of the cartilage of the extemal ear

(Lammer. 199 1 ).

Epidemiological studies of 402 pregnant rvomen esposed to 13-cis R A showed that.

although the majonty of pregnancies ended in abonion (7'0 spontaneous). 37O.0 were camed to

term. Of the 15 1 infants bom. 4S0;o were normal. 47O.i1 were bom with RAE displaying va-ing

degrees of severity. and SO;b were bom with non-RAE abnormalities (Dai cv rd.. 1992) .-\lthough

in this system. the majonty of women were exposed to continued doses of 1 3 4 s RA dunng the

first trimester of pregnancv. cases involving exposure to only one capsule of 13-cis RA (40 to 80

ri) during the second to fifth week of pregnancy were also reported Studies such as this reinforce Y

the concept that a suitable. clinically viable treatment for retinoid exposure iir rrrero is needed.

1 . 3 . 2 .Animal blodels

In both hamsters and mice. critical periods of susceptibility to RA of vanous organs have

been demonstrated. With respect to limb skeletal malformations in hamsters. it was shown that a

single dose of 120 m~gkikg all-trans RA on day 10.5 pc resulted in marked malformations of both

the fore and hindtimbs (Shenefelt. 1972). These malformations (which included limb

foreshortening. absent/fÜsed digits. and malformed joint articulations) were seen in 9S0b of

suwiving fetuses one day before term. Treatment with the same dose of all-trans RA on dav I 1 5

pc produced none of these effects. however the major effect from exposure to the dru, * seen on

this day was cleft palate. which was obsewed in 889% of surviving fetuses. Effects of all-trans RA

discussed in this study also included marked skull and brain malformations. however. these were

seen only afier esposure to all-trans RA before da? 9.5 pc.

Bv relating these observations to events in the development of the limb. it \vas shown that

the period during rvhich the limb bud displays a sensiiivitv to R A occurs before there are anv

identifiable precursors of the structures that wiil show the effect. On day 10.5 pc in the hamster.

mesenchymal cells are just beginning to condense and have not ver differentiated into

chondrocytes nor formed the canilaginous anlagen (the precursor to bone. which is the structure

arected) (Shenefelt. 1972). blalformations in the limb may then result frorn a RA-mediated

decrease in the pool of proliferatiny mesenchymal cells. as a direct inhibitory effect on

chondrogenesis. or frorn a combination of these two processes (Lee c.1 c d . . 1991).

In mice. sirnilar malformations have been observed. Kochhar ( 1967) noted that a sub-

lethal dose of 100 m g k g all-trans FU given to pregnant mice on day 8. 9. or 10 pc resulted in a

wide variety of malformations including clefl palate. tnincation of the tail and ribs. and spina

bifida occulta. Administration of this dose on day 10.5 to day I I pc resulted in a strikingly

different pattern of malformations. resembling those observed in the limbs of the hamster. with

malformations of the forelimb reaching a maximum incidence on day 1 I pc and malformations of

the hindlimb reaching a maximum incidence on day 1 1.5 pc (Kwasigroch el L I / . 1986).

The observation of such narrow windows of susceptibility in this and other studies is made

possible because of the short biological half-life of all-trans RA ( 1 hour). which. even af er high

doses. bezins to disappear from tissues in less than an hour. This makes the use of all-trans RA.

rather than 13-cis EU (which has a half-life of one day) more suitable for the study of the

teratogenic effects of R A during discrete emb-oloijcal stages of development (Kochhar. 1967)

Sulik rr id. ( 1988) have identified a sequence of susceptibility of the ernbryo to the teratogenic

effects of RA which coincides with the normal developrnental timetable of the ernbryo.

Embryonic developrnent is staggered in a cephalocaudal direction over a 12 hour period. Within

this period. development is also stagjered in the prosimodistal direction. with respect to limb

outgrowth. and also along the anteroposterior axis. The narrow window of susceptibility during

which development is affected by retinoids hnher supports the use of all-trans RA. rather than

1 3 -cis R A or 9-cis RA. in mode1 svstems investigatine the teratogenic effects of retinoids.

With respect to the limb. Kwasigroch el tri. ( 1986) postulated that esposure to high doses

of R A results in a respecification of the positional information received by cells. leadin, cr to a

disruption in avis development. causing both reduction and duplication malformations in the

canilage anlagen By culturing the day 1 1 pc murine limb bud for three days in the presence of

1 O pg/ml ail-trans RA. they found that the size of the resultant anlagen was decreased due to a

disruption by RA of the distal migration of the mesenchyme along the proximodistal asis Thus

the effects of RA on the deveioping limb bud include not only disturbances in the patterning of the

limb. but also an interference with the outgowth of the lirnb.

1 - 3 . 3 Craniofacial versus Limb MaIfornations in Hurnans and himals

With respect to the effects of retinoids on the developinp fetus. digerences esist between

what is obsrrved in humans and what is observed in laboratory animals. The largest discrepancy

is the fact that in humans. limb malformations in infants exposed to R A i ~ i rirem are rare (R ino r f

c d . . 1991) while in animals. limb malformations are prominent. However. craniofacial

abnormalities are seen in humans and laboratory animals to a similar estent. There are two

possible explanations for these observations.

Firstly. in humans. limb development occurs between the fifih and eishth week of

pregnancy. occumng at a time when most women have realized their condition and have

discontinued retinoid use. Primary craniofacial development occurs between weeks two and five

in humans and may coincide with continued retinoid use. as seen in most cases of R4E in

unplanned pregnancv (Dai et id., 1992). The half-life of 13-cis RA is - 18-24 h and is therefore

eliminated quickly. so that later effects fi-om retinoid use are not seen (Rosa er ol. 1956) In

anirnals. the tirnes of retinoid evposure are specifically chosen to study certain malformations. In

the mouse. it is generally accepted that the window of susceptibility of the craniofacies to RA is

between day 6 and 9 pc. while the window of susceptibiiity of the developing limb is between day

10 to 14 pc (Kochhar. 1973). It should be noted. also. that there are no reported cases of RAE

due to exposure to EL4 durin3 the second and third trimesters of pregnancv. which is most likely

due to a discontinuation in use of retinoids by pregnant wornen rather than from a lack of efect.

Secondly. the craniofacial mesenchyme is derived from two sources (cells from the neural

crest and the branchial arches) and each may respond to RA in a different way dependent upon

RAR expression patterns in each ce11 type. This makes the patteming of the craniofacies highly

complex and rnuch harder to study. The limb offers a relatively simplified system to stud)? the

efYects of R4 and RM expression on patterning and canilage development i l i iiw.

1.4 Retinoic Acid Receptor Expression During Limb Development

14.1 Limb Developrnent in the Mouse

The tirst sign of lirnb development. occumng between day 9 and I O pc in the mouse. is the

appearance of a thickening mass of ectoderm along the surface of the embryo called the apical

ectodermal ndge (.AER). This ndze of ectoderm directs the proximodistal gowth of the limb and

is likely to be the first lirnb structure which is affected by esogenous RA. Sulik c.1 td. ( 1988)

demonstrated that a dose of 100 rn~& ali-trans RA given to mice on day 9.5 pc resulted in

duplication and reduction deforrnities in the radius and the digits of the fetuses Csing Nile blue

sulfate. which is a Iysosomotropic vital dye. Sulik rr t t / . fùrther showed that the onsin of these

malformations could be traced to excessive cell death in the AER. Estrapolating from this studv.

Sulik ri r d . argued that the promotion of cell death by Rh mav also be involved in the genesis of

malformations observed in the radius and ulna as a result of exposure on da? 1 1.5 pc. This day

coincides with the emergence of two distinct cartilagenous structures (the presumptive ulna and

radius) from the mass of condensed rnesoderm in the distal pan of the limb. Damage of the AER

bu RA rnay also have effects on ce11 migration and/or cell signaling involved in limb outgrowth.

which could also explain RA-induced malformations of the digits and of the distal Ions bones

(Sulik èr d.. 1988).

Apan fi-om the proximodistal asis. the limb must also establish the anteroposterior asis

during development. This axis allows for proper patteming of the paws and the distal long bones.

Pattern determination is govemed by the ZPA which is a region of the distal antenor

mesenchyme and ectoderm in the limb bud and emerges shortly afier the .AER The ZP.4

produces and emits a si-naling molecule which difises across the limb setting up a gradient

across the limb which establishes the anteroposterior a i s . It has been shown that the topical

application of iL4 on the opposite side of the limb bud can mimic the ZPA and alter gene

expression accordingly (Lohnes rr cri.. 1995). In the developing chick embryo. it is knorvn that

the ZPA produces F U during avis determination (Thaller et c d . . 1987). however. in mammals it

has not been established whether R\ or a related retinoid is the responsible morphogen. or

whether this role belongs to the Hox-4 complex or other homeotic genes (Winter rr t i f . . 1993).

In the patteming of the developins paw. which first resembles a round. tlattened paddle.

another Ievel of complexity e'tists. In the rnouse. day I I pc marks the emerrence of canilaginous

structures within the paw called the digital rays. Between day 1 1 and 13 pc. the areas between

the digital rays. knorvn as the interdisita1 zones (IDZ) underso programmed cell death to fùrther

shape the paw and reveal the emerging digits. If cell death within the IDZ is blocked. which can

be accomplished by removing these regions fiom the paw and maintaining them in culture for 3

days. cartilage will f o m (Lee er c d . . 1994). Conversely. in the presence of RA. ce11 death in the

ID2 mav be promoted. both il, and itr \ ? i w . resulting in either poiydactyly or syndactyly ( Lee

dl LI/ . 1994).

The central ponion of the developing limb bud begins as a mass of undifferentiated

mesenchyme By day I I pc. this mesenchyrne begins to condense in the core. giving risr to the

earliest form of the limb bones. .Mer mesenchymal condensation has occurred. mesenchvme

within the condensate begins to differentiate into chondroprogenitor cells (Solursh. 1983) and

eventually forms cartilage. It should be noted that although mechanisms yovemin- mesenchymal

differentiation into chondrocytes are poorly understood. it is known that difierentiation and limb

outgrouth occur simultaneously and therefore. the proximal mesenchyme becomes chondrocytic

before distal mesenchyme (Solursh. 1983). This process continues fi-om day I 1 to day 13 pc and

results in the appearance of a full cartilaginous skeleton in the limb. Once this occurs, the

cartilagenous anlagen. which provide the foundation for the formation of the adult skeleton. begin

to be rnineralized. This process occurs afier the penod of susceptibility of the limb to has

ended. fùnher illustrating t hat the prirnary and secondae target s for KA-induced teratogenesis are

mesenchyme and cartilage respectively.

1.4.2 The Retinoic Acid Receptors

M a . RARP. and RU+/ are al1 encoded by separate genes Analysis of sequence

homolog reveals that. although there is considerable difference among receptor types within a

given species. there is strong homolog within a receptor type amonp species (Husmann rr dl..

199 1 : Lohnes el d.. 1995). Within each FUR type. isoforms (RARU 1 and u2: RhRP 1. Pz. p3.

and Pd; RAR-;l and :/l) are generated via altemate splicing and differential promotor use

(Husmann el d.. 199 1 ). Of these isoforms. the expression of only the second of each type (a?.

pz. -9) is regulated by RA. Each isoform of a given RAR gene is composed of a common motif

diverging onlv in the 5' untranslated and N-terminal A-regions The interspecies homology within

the A-region. a re~ion to which transcription activation iùnctions have been ascribed. supports the

argument that each isoform has an evolutionary significant (and therefore specific) role. The

prevalent form of the R.-\R both irt i7iri-o and i i t iwo is as a R W R N R heterodirner The

regulated expression of specific heterodimeric combinations may contribute to the pleiotropic

effects of Rh (Luo rr c d . . 1995: Lohnes rr a/.. 1995). Target çenes for the M s include the

M R s themselves. the Hos genes (which also play an important role in limb patteming). and

elements of the RA si~naling pathway (Le.. CFWBP-II).

In the early limb bud al1 three W s . and most of their isoforms. are expressed. The

expression patterns for each M subtype during forelirnb development in the mouse are

illustrated in Figure 1.2. R4Ru and W! are ubiquitously expressed from day 9.5 to 11.5 pc,

and may be. at this time. fünctionally redundant (Dolle rr al.. 1994). Later in the development of

the limb. patterns of expression for both receptors become restricted and mutuallv exclusive. with

RARy localizing to the condensed mesenchyme in the core of the limb bud. and RARa localizing

to the periphery. including the I D 2 Increasing the complevity of this shifi is the observation that

RMQ2. rvhich is highly expressed in the early limb bud. disappears and is almost completely

replaced by RARy 1 during cartilage formation (Kastner rr d.. 1990). W P 2 . the prevalent

RARB isoform in the limb bud. may also be involved in the transduction of the RA sigal since it

is the onlv isoform to display an extremely restricted pattern of expression throuyhout limb

development. In the early stages of development. RARD2 is found only in the proximal

mesenchyme and later complet el- relocalizes to the ID2 (Mendelsohn rr trl.. 1 99 1 ) RXRu and

RWB. the presumptive heterodimeric partners of the RARs are espressed ubiquitously

throughout limb development and becorne restncted only afier dav 16.5 pc (Dolle L'I d. 1994).

Thus the period of limb development which coincides with day 10 to I i pc (the window of

susceptibility of the lirnb to R A ) is a very dvnamic one with respect to R4R isoform expression.

Csing targeted deletion techniques. many tvpes of R;UI knockout mice have been

rrenerated to studv RAR fùnction. In mice homozygous nul1 for one isoform only. there are no *

obvious malfonnations. suggesting that at least one isoform of each receptor type is niinimally

required for proper development Knockout mice homozygous nuIl for one receptor tvpe display

minor defects ?et are still viable and fertile. suggesting that there may be a high degree of

functional redundancy among RAR family members (Li et al.. 1 993: Lohnes rr ul.. 1 993 ; Lohnes

rr al.. 1994: Kastner rr LI/.. 1994). It is only in compound (or double) homozygous nul1 mice

(lacking one or more isoforms from two different receptor types) that limb malformations

recapitulating VAD syndrome are seen (Lohnes et O/.. 1994). This mav suggest that although

some receptors are functionally redundant. others exist which cannot be hncrionallv equivalent.

1 . 4 . Retinoic Acid Receptor .Alpha

NI vertebrate ce11 lines tested to date express endogenous M. This is most likely due to

the rather ubiquitous and unrestricted expression of RARu (Husrnann C r L I / . 1994). Hoxever.

during chondrocyte maturation. RARu is not espressed. Furthemore. it is the downregulation of

this receptor which is thought to be of major importance in the promotion of the maturation of

chondrocvtes Cnder conditions of RX excess. RARU (as well as isoforms of RARP and Ra*!)

are upre_julated. which may in tum delay or inhibit diflerentiation of chondrocytes in the limb

mesenchyme (Jiang C r c d . . 1991: Mendelsohn el c d . . 199 1 ).

.Uthough transgenic mice homozygous null for Wct display only webbed dizits (and no

other obvious malformation). mice homozygous nul1 for both RARu and RARy exhibit a wide

range of malformations including limb cartilage duplications and reductions in distal limb

structures. broad dizits. supemumerary digits and ectopic cartilage in the IDZ (Lohnes P r ci l .

1994). It should also be noted that mice homozvgous nuIl for RW! alone also display fewer

obvious malformations. This suggests. then. that during early stages of development the presence

of one of these foms of the receptor is critical for proper limb development. To further iilustrate

this point. Lohnes rr d. ( 1994) demonstrated that the addition of a single copy of the RrUiul

eene in RM2uRA.R-y double null mice was sufficient to block the permissive conditions leading to CI

encess c hondrogenesis.

In a double nul1 W u R A R P I mouse. ectopic cartilage formation in rnanv structures

similar to those seen in the RARcc'R;UI./ mutant were observed. (Lohnes et c d . . 1995). Since it is

known that dunng limb development. W P and RR:/ are expressed in mutuallv restricted

patterns. the increase in chondrogenesis in these mice may also be linked to the absence of RARU.

Taken together with the preiious study. this suppms a role of RARU in the inhibition of

chondrogenesis.

1.4.1 Retinoic Acid Receptor Beta

.Uthough W u ? . W P 2 . and RAR9 are al1 inducible by RA. it is onlv RrUIP? whosr

levels remain elevated 9 to 12 11 afier an initial exposure of 100 mdkq all-trans RA (Soprano ri

d.. 1994). Ligand bound RARP7 is able to induce its own expression by binding to R A R E P Z

(which is also used in the transcription of fWRP1) Funhermore. Soprano ci d. ( 1994) have

shown that this elevation occurs only during the gestational period of teratogenicity of RA. To

further examine the relationship between a sustained elevation of RARP? levels and a teratogenic

outcome. the efects of 100 m g k g all-trans RA was compared to those seen afier esposure to I O

rny'kg all-trans RA. 100 m& i 3 4 s M. and 3 u 100 m-k g 13-cis RA (3 hours apart) For

both 100 mgkg ail-trans FU and 3 .r 100 m g k g 13-cis R A (both highly teratogenic doses).

RARD2 mRiVA kvas elevated 14-fold in the limb and remained elevated for over 6 hours. The

non-teratogenic doses of 10 m g k g all-trans RA and 100 mgkg l k i s RA increased M R P 2

mRVA levels in the limb IO-fold. bu t failed to sustain that elevation afier 3 hours. Not only does

this imply a role for RARD2 in IW-mediated teratogenesis. it also illustrates that in addition to the

initial rise in R4RP2 m%VA transcription. the duration of that rise is also an important

determinant in teratogenic outcome. The increase of W P I in response to RA may also be the

major determinant in separating mildly teratogenic doses of RA from those that are highly

teratogenic. Since RARs heterodimerize with RXRs. it has also been postulated that the sharp

rise in N P 2 following exposure to RA may decrease the amount of fiee R?(R available to

heterodimenze with other receptors. This possible indirect effect of exosenous RA may also

contribute to its role in fetal dysmorphogenesis (Soprano et d. 1991).

By generating a F U R D homoqgous nul1 mouse line. it has been s h o w thar the absence of

RARP does not affect fenilitv or viability of mice. Aiso. like mice null for R a u and RW:.

mice nuIl for RARB do not display any striking abnormalities. Luo rt t r l (19%) have (ùnher

shown that RARP ~ u l l rnice treated with terato-enic doses of RA have the same malformations as

observed in wild type mice exposed to the same dose of RA. From this. Luo et rd have

hypothesized that RARP mav not b r an important mediator ofthe RA signal. However. it is more

likely that none of the three RAR receptor types by themselves plav a dominant role in

development. but rather work in concert to achieve their effects. I t has been s h o w using in situ

hybridization that RARP CO-localizes with RARu 1 (Rubene ri LI/.. 199 1 ). Although RARu 1

levels are not significantlv increased in the R4RP knockout rnouse as compared to the wild type

(Luo ci trl.. 1995). RARu 1 may still be able to compensate for the absent R A R P This ïiew is

supported by the observation that RARU l / W B double null mice display severe malformations

and -enerally die 30 minutes afier birth (Ruberte ri tr l . . 199 1 ).

1 . 4 . Retinoic Acid Receptor Gamma

RARy is found in the rnouse embryo as early as day 8 pc and continues to be e'cpressed

from that point onward. In the early stages of development. there is unifom expression of RAI?/

in the mesenchvme of the limb buds. the pharyngeal arches. and the frontonasal mass. At later

stages (beginning on day 13.5 pc). RARy becomes restricred to cartilage and squamous

keratinizing epithelia (Look ri d.. 1995). This localization is highly suggestive of a role for

in the support of chondrogenesis and differentiation. In mice homoqgous nul1 for R*{.

there are no marked malformations observed. and when challenged with teratogenic doses of RA

on days 8 to 9 pc. no further malformations are noted (Look ri r J . 1995) This resisrance to

some M-mediated abnormalities funher implies a role for W & y in RA-induced teratogenesis

In situ and RV.Ase protection analysis demonstrate that RARP and RW! 1 are expressed

in a reciprocal fashion. Husmann rr L I / . ( 199 1 ) showed in at least 3 cell lines ( C L 1. NIH 3T3.

and HeLa cells) that RWil blocked the stimulatory effect of endo-enous ligand and R4R

(probably RARu) on transcription of a reporter construct containins RAREDI. Likewise. RARy 1

blocked the stimulatory effect of transiently transfected RmP2 and RARy2 on transcription of

the same reporter in these cell lines. The amount of inhibition was dose-dependent and kvas based

on the ratios of RUiPl or RAR-!? to RAR-fl. It is ihought. since both RARD2 and RARyI bind

to RARE32 (Kastner rr dl.. 1990). that this cornpetition for binding may play an important role in

chondrogenesis and in the determination of the reciprocal expression patterns of these receptors.

R4.RPI and M,2. which both stimulate transcription frorn R W P 2 are espressed in

overlapping regions It should also be noted that during cartilage formation. there is a

downregulation of M i 2 and an upregulation of RW{I (Kastner a t r i . 1990) It would appear

then. that although RARU and R4RP are strongly associated with inhibitov effects on

chondrogenesis. RARyl seems to play a role in the promotion andior maintenance of the

c hondrogenic process.

L.5 Glucocorticoids

1 .5.1 The Role of Glucocorticoids in Growth and Development

Glucocorticoids are synthesized in and secreted fi-orn the adrenal cortes and play

important roles in manv metabolic processes Glucocorticoids are involved in the physiological

response to stress. the perinatal activation of gluconeogenesis. and in ot her developmental

processes including the qrowt h and differentiation of the adrenal medulla. the lune. the liver. and

in osteoblasts (Baxter et t r l . . 1979; Anderson et d.. 1986: Bellows et d. . 1989). Glucocorticoids

are also invotved in the induction of severat enn/mes with roles in differentiation and maturation

of many organ systems during embryonic and fetal development (Zimmermann et d.. 1993 ).

The effects of glucocorticoids are rnediated primarilv via intracellular glucocorticoid

receptors (GR) which belong to the steroid-thyroid hormone superfamilv. Ligand actij-ated GR

can cross the nuclear membrane to bind to glucoconicoid response elements (GREs) in the

reyulatory reyions of target genes. thus having a direct effect on gene transcription. Like RARs

and RYRs. GRs are found in many ce11 types and in manv tissues. with levels of GR varying

considerably amon3 many tissue types (Ballard L J ~ c d . . 1 974).

In the rnouse. mRNA for the GR has been detected as early as day 9.5 pc. suggestinr an

important role for glucocorticoids dunng embryonic development (Cole et cil.. 1993). Prenatally.

the glucocorticoid cortisol promotes maturation of many organ systems. For esaniple. cortisol

induces the synthesis of the protein surfactant (which is required for proper lung function).

promotes the closure of the ductus artenosus in the hean. plays a role in the hnctional maturation

of islet cells in the pancreas. and promotes hnctional rnaturity of the liver (Ballard. 1979). The

GR is also found in cells of the cartilage lineage but the actions of glucocorticoids there are

unclear (Blondelon rr t r l . . 1980).

1 3 . 2 Effects of Glucocorticoids on Cartilage

hi \ 7 w . glucoco~icoids have been shown to inhibit cartilage growth and bone formation

(Silbermann. 1983 ). III i9~iro. however. the synthetic ~lucoconicoid de': stimulates of the

formation of cartilage nodules in cultures of fetal rat calvaria cells (Bellows CI c d . . 1989) and in

chondrogenic ce11 lines (Cirigoriadis ri LI / . . 1989). Whether the stimulation of chondrogenesis 111

wi-o has anv relevance for chondrogenesis iir iV i i1o has been addressed using cultures of

decapitated fetal mice isolated on day 1 1 3. 12.5. and 13.5 pc (Zimmermann rr c d . . 1993 ). In this

studv. it was shown t hat the addition of 1 O-' Y[ dex to cultures of day 1 1.5 pc fetuses resulted in a

maximum induction of cartilase production. Stiniulation of cartilage formation was also seen in

cultures of the limb buds This same dose had a smaller etfect on cultures isolated from day 12.5

pc fetuses and had no efect in cultures isolated fiom day 13.5 pc fetuses. Zimmermann et c d .

hypothesized that the stage-dependent induction of cartilage in this system is directly related to

the stage of limb development br i * i i p o from which the cultures were isolated. Cultures isolated

frorn dav 1 i 5 pc reflect the stage I ~ I i* i iVo during which the differentiation of mesenchynal cells

into chondrocytes begins. which occurs afier the mesenchyme in the core of the iimb bud

condenses Bv da? 13 5 pc. chondrogenesis in the limb bud has reached a maximum and ma?; not

be Iùrther stimulated by the addition of dex (Zimmerman PI d.. t 993).

In the RCJ 3.1 C5 cell line. the monopotential chondrogenic cell line discussed previously.

de't increases cartilage nodule formation (Gngonadis ri al.. 1989). This phenomenon is dose-

dependent. with an EDsii equivalent to - 10 nM and a maximum stimulatory dose of 10" M. The

other effects of dex in this culture system match those previously seen in cultures of other

chondrogenic cells obtained fiom vanous sources. These include a stimulation in the formation of

chondroitin sulfate (Calcagno. 1970). a stimulation of the precocious accumulation of calcium and

matrk vesicles. and a stimulation of GAG and proteo_olycan synthesis. DNA synthesis. and

cellular proliferation (Lewinson ct d.. 1981: Kato et a/.. 1985. Tekano et d.. 1985). In RCJ 3 1

C i cells. dex has also been shorvn to counteract the inhibitory effects of RA on canilage

differentiation such that the dose-response curve for the effects of R A on cartilage formation was

shifted to the right by an order of magnitude in the presence of I O niM dex (Lau rr id.. 1993)

1 . F 3 Effects of Glucocorticoids on the Fetal Skeleton

Like retinoids. physiological amounts of glucocorticoids are required for normal

embryological and fetal development . However. alt hough excess retinoid exposure dun n a

gestation results in severe teratogenic consequences. there have been no documented cases of *

teratogenic effects due to elevated matemal exposure to glucoconicoids in humans In fact. the

pharmacological use of dex for asthma (0.0 14 to 0.043 m g k g body weight: C. P. S. 4lanual. 1993 )

during pregnancy is an accepted practice. In rodents. however. ihere have heen man- studies

documenting stunted general. skeletal. and visceral growth (Reinisch rr a/ . . 1978: Loeb. 1976) in

fetuses as a result of maternai esposure to glucocorticoids during pregnancy Doses tested.

however. u-ere up to 5 times higher than those used in humans (0.2 m g k g des in rats as

compared to 0.04 mgkg deu in humans) Specific effects. includin- clefi palate and w a y ribs.

resulting atter esposure to hi& doses of glucocorticoids ( O 2 to 50 m g k g des) have also been

reported (Greene c'f c d . . 1975).

To assessss specific periods of susceptibility to glucocorticoid-induced teratogenesis in

rats. LaBorde cl c d . ( 1992) administered 0.2 or 0 .1 mg/kg/day dex to dams on either days 9 to 14

or 14 to 19 pc (mid and late gestational periods. respectively) and noted abnorrnalities in day 20

pc fetuses. I t was found that. in addition to a 50% incidence of clefi palate as a result of 0.4

mgkjday des given dunng the mid-gestational period. dex also severely decreased the mass of

al1 organs e'rcept the brain. Administration of dex durinq this penod also decreased ossified bone

lensqh. Measurements of cartilage length. however. were not made. Des e'rposure during the late

gestational period did not affect the formation of the palate or the long bones. but did result in the

appearance of wavy nbs. rUthough matemal to'ricity. in the form of del-induced anoresia. was

also seen. pair-feeding expenments showed that the decrease in weight and lack of nutrition in

dams \vas not responsible for any of the obsewed effects in fetuses.

1.6 Interactions Between Retinoids and Glucocorticoids

1 6 Cornbined Effects of Retinoic Acid and Dexamethasone

Both synergism and antagonism between retinoids and glucoconicoids have been reponed

from work done in many experimental systems. RA and dex act synergistically to inhibit growth

of myeloma cells I I I vitro by hrther decreasing EU-induced reduction of the expression of the IL-

6 receptor. which in turn reduces the normal IL-6 response (Chen rr LI/. 1996) It has also been

demonstrated that RA and des can synergistically induce S 14 sene transcription in 3 T3-F-I-l l .A

adipocytes in culture (Lepar rr 4. 1997) Like dex. RA can also increase the translocation of the

glucocorticoid receptor from the cytosol to the nucleus in liver cells (Adouin-Chevallier ri d.

1995) Whether this is sufficient to induce the proteolytic degradation of the receptor in the

nucleus. however. is unknotvn.

Des and R A have antagonistic effects on the expression of the epidermal growth factor

receptor in the fetal rat lung (Oberg rr O / . . 199 1 ) as well as the expression of CRBP in the lung of

both the fetal and adult rat (Rush et al.. 1991). In human anicular canilage. RA and dex display

differential effects on the expression of the monocyte chemoattractant protein- 1 (MCP- 1 ).

Nthough RA can stimulate expression of MPC-1 and dex inhibits it. cornbined. there is a net

reduction of MCP- 1 (Villiger et al.. 1991).

In the rat clonal preosteoblast ce11 line L M 201. differential effects have also been

observed. In this system FU stimulates the expression of alkaline phosphatase mR11A. while dex

inhibits the Rh-mediated increase (big rr L I / . . 1989). In cultured human skin fibroblasts. des was

show to decrease hvaluronate synthesis and. combined with RA Funher reduce the RA-rnediated

reduction of glycosaminoglycan synthesis (Smith. 1990). Likeu-ise. dex and R A have been s h o w

to work syner~isticallp to decrease type I and type III procollagen mR,iL'A svnthesis in this system

(Oikarinen et c r i . . 1 989).

Interactions between retinoids and glucocorticoids are aiso obsened clinically. Topical

application of RA can prevent corticosteroid-induced skin atrophy without having an- effect on

its anti-inflammatorv properties (Lesnik. 1989). In another studv. the efect of topical application

of RA was shown to be blocked bv - svstemic - corticosteroid therapy in kidney transplant recipients

( De Lachamiere t.r c d . . 1 990) .

1.6.2 Effects of Dexarnethasone on RAR Expression

Recently it was shown. in adult rat liver. that dex decreases cellular RARP mWA. in tum

causing a reduction in nuclear receptor protein (Pallet cl c d . 1996). Whether deu also decreases

RARu and/or in this system is unknown. Since there have been no reports identieing

RAR genes containing full glucoconicoid response elements in their resulatory domains. the

decrease in F U R D was ascribed by the authors not to be a GR-mediated change in RAR gene

transcription but rather a dex-mediated reduction of fiee RA levels available to sustain induction

of the W s . This seems possible since des is known to modulate the peripheral metabolisrn of

RA. For instance. it has been shoun that dex increases the cytochrome P-150 mediated 1-

hydroxylation of RA (Martini rr al.. 1994). which is thought to be a protective mechanism as it

divens RA fi-om the cytochrome P-450 mediated 4-oxidation pathway. which yields a putatively

teratogenic metabolite of RA (Roberts et (71.. 1992). CVhether this mechanism also reduces the

amount of enterohepatically circulating RA-~lucuronide is not known. This tvpe of deu-mediated

decrease in RARP mRNA is also seen in postnatal rat lunr tissue In this -stem. dex has no

effect on R M u or R a ! mRNA levels (Grumrner rr LI/.. 1995).

Dex also increases the expression of liver specific RXRu in 3 rat hepatoma ce11 lines.

which could possiblv augment the effect of 9-cis RA (Wan et d.. 1994). KXRs. rvhich enhance

the binding of KARS to their cognate RARES. may be crucial in the determination of the effects of

RA and of other hormones It has been postulated that the dex-mediated increase in RXRs ma?

shift the equilibrium between RXR-RAR heterodimers and kW-RSR homodimers to favor the

formation of the latter (Wan r t al.. 1994. Pallet et ul.. 1996). Bv increasing RYR-RSR

homodimers. the pool of heterodimers wouid be decreased. which in tum would decrease FUR

binding to RiXEs. This may then diminish the expression of genes housing these RARES.

includins the genes encoding the KARS. .Mthough homolo~ous reculation - of RARs and RXRs bu

RA is knorvn to occur. whether these receptors can be heterolo~ousl~ regulated by strroid and

thyroid hormones. such as des. remains to be proven conclusiveiy

1 6 3 Receptor Interactions

Recentlv. the identification of a c-ptic ~lucocorticoid response element (cGRE). which

conveys glucoconicoid responsiveness only in the presence of a ligand bound TR (or related

receptor). has prompted the search for the existence of cross-talk mechanisms between rnembers

of the steroid-thyroid hormone receptor superfamily in overlapping target cells (Leers rr al..

1994). In this study. it was shown that a reporter gene regulated by a thyroid hormone response

element (TRE) can also confer a glucocorticoid response in addition to. and dependent upon, a

thyroid hormone response. The glucocorticoid action is mediated via the cGRE. which remained

non-functional in the presence of glucoconicoids unless activated in conjunction wirh a TRE.

Using a fragment of the human RAREa. it was funher shown that the 30-fold induction of the

transcription of the reporter gene. mediated via the addition of RA. was further stimulated 3-fold

by the addition of dex. It should be noted. however. that no cGREs have yer been found in the

qulatory regions of KA-responsive genes. .Uthough much of this work has been done usin9 the

TR and the GR as mode1 receptors. the authors have also success~lly replicatea their work

substituting the RAR for the TR and/or the progesterone receptor (PR) for the GR Funhemore.

although the TR and the GR alone could not activate the reporter consttuct containing the human

W u fragment. in the presence of ligand-bound G R the specificitv for the M u \vas

overmled and activation of transcription bv ligand-bound TR was shown.

In another study lookin- at the receptor/response element interaction of sreroid-thvroid

hormone receptors. Bedo t . r ( ~ / . ( 1989) showed that either thvroid hormone or RA were required

to mediate the induction of the rat growth hormone gene bv g1ucocorticoids. This mav explain

how the expression of growth hormone is potentiated by glucoconicoids. even though no fùll

GRE has been identifieci within the growth hormone jene.

Aithough the mechanisms of response element recognition are still poorly understood. it is

important to note that most tissues respond to thyroid hormone. ~lucocorticoids,

mineralocorticoids. progestins. and retinoids simultaneously dunng growth and differentiation (Qi

z . 9 ) . Since simultaneous activation of thesr receptors is most likely a normal occurrence

i t ~ 1 7 ~ it is only logical to hypothesize that CO-regdation of the responses of tissues to these

agents. at the level of the receptor. is also likely to occur.

1.7 Objectives

It was previously determined in the monopotential chondrogenic cell line RCJ 3 . 1 C i that

the glucoconicoid deitamethasone could counteract the inhibitory effects of all-vans RA on

cartilage differentiation such thar the dose-response curve for the effects of all-tram RA on

cartilage formation was shified to the right by an order of magnitude in the presence of 1 O nM dex

(Lau et tri.. 1993). it has also been determined that the inhibitory effects of all-tram RA on

cartilage deïelopment I I I i V i m have an NI iqii70 counterpan in that the development of the

appendicular canilaginous skeleton is also inhibited (Kistler rr L I / . . 1984). Our objective \vas to

hrther eïaluate the effects of all-trans RA on the development of the limb and to deterniine

whether des could counteract these all-trans RA-mediated abnormalities in ktaI mice.

We aiso used the RAREhsplacZ transgenic mouse to determine in which cells of the developing

limb bud concentrations of endogenous RA were suficiently hish to detect .A P-gaiactosidase (13-

gai) response. and to see if this response could be upregulated by esosenous ail-trans RA. We - also used this mouse to invesrigate the relationship between gene activation and RA-induced

malformations in the limb as a result of esposure to all-trans R A on either davs 1 I 5 or 13.5 pc.

Figure 1. I Retinoic Acid Isomers and Metabolites and Their Relative Potencies Wit h Respect to Induction of Skeletal Abnormalities Within and Between Species.

mouse hamster human

'nuinbers are espressed in m g k g and represent a 1009,0 teratogenic dose

' Jiang C r c d . . 1 994 h Shenefelt, 1972 ' Dai ri c d . . 1992

Figure 1.2 R a Subtype Distribution during Limb Ontogeny in the Mouse.

RARy

- - - - -

day 1 1 . 5 ~ ~ day 12.5 pc day 13.5 pc

Patterns of expression for RARU. M P . and RARy in the murine forelimb during development are shown. Black areas represent areas espressin2 relatively high levels of the receptor and white areas represent areas with no receptor expression. Light and dark grey areas represent low and mid-levels of receptor expression. respectively. RdZu distribution is representative of bot h RARul and M a l isoforms. RARP distribution is representative of R A R P Z only. which is the major RARP isoform in the developing limb bud. RARy distribution is representative of RAR:/I on dav 1 1.5 pc but this isoform is alrnost completely replaced by RARy2 by day 13.5 pc.

CHAPTER TWO

THE EFFECTS OF TREATMENT WITH DEXAMETHASONE ON .4LL-TRAXS RETlNOIC ACID-INDUCED LIMB SKELET.AL

MALFORiVIATIONS IN THE FET.AL MOUSE

2.1 Introduction

Previous experiments (Lau et c d . . 1993) usinp the monopotential chondrogenic ce11 iine

RCJ 3 . I C5. have shown that the inhibition of cartilage formation by all-trans R A could be

antagonized by dex 111 iin-o. In this chapter. we describe experiments investigating whether this iir

i V i m phenornenon has an i r i i ~ i o counterpart. in that the developmental defects induced bv ail-

trans R A in the canilaginous bone precursors are similarly antagonized by d e s We have tested

the hypothesis that glucsconicoid treatment can prevent or ameliorate the teratoyenic effects of

retinoids.

2.2 blethods

3.2.1 Dmys and t'ehicles

.Ail-trans RA was purchased from Sigma Chemical C o (St. Louis 510) and suspended in a

corn oil vehicle in three stock solutions ( 13.5. 6 .25 . and 1.5 mdml). - These suspensions were

stored in light-impermeable tubes and kept at -70°C when not in use. One solution of deu ( O 05

mgml final concentration) was prepared and diluted in saline as required. This solution \vas kept

at -FUC when not in use.

2 . 2 . 2 Animal Treatments

Timed pregnant female CD-I mice (Harlan Sprague Dawlev. Indianapolis IN) were

housed in separate cages and were given Purina Rodent Chow and tap water ta/ lihiritni duriny

the course of the esperiment. The mice were given two d a y afler transport to acclimatize to a

regular I 2 hour light-dark cycle before use.

To screen for the day of highest susceptibility of the fetus to all-trans RA. a single dose of

either 50. 3. or I O m g k g was administered in a volume of 0.2 mi b'; gavaje on one of days I 1.5.

12.5. 13.5. 14.5. or 15.5 pc (noon of the dav following a successful matins is considered day 0 5

pc). Similarly. to assessss the efects of des on the developing fetus. a single dose of either 0.7.

0.1. 0.05. 0.025. or 0.0 1 m~-r/kg'day was administered intramuscularly in a volume of 0.1 ml

during one of the following dosing penods: days 10.5 to 12.5. days 13 5 to 15. S. or days 16.5 to

1 8.5 pc. On the davs of dnig administration. between 9 and 1 1 a.m.. pregnant rnice were

weighed. given a single dose of either all-trans EU or dex (as outlined above). and retumed to

their cages.

To test the ability of des to ameliorate the teratogenic effects of all-tram RA. a single dose

of 25 or 50 mgkg all-trans RI\ was administered to pregnant mice on one of either day 1 1 5 or

13.5 pc in conjunction with a dose of O. 1 rn@g/day dex on either days 10.5 ro 12.5 or days 13.5

to 15.5 pc. Pre-nant mice receiving either all-trans RA or des alone on one of the above specified

days were used as positive and negative controls. respectively On the days of dru-

administration. between 9 and 1 1 a.rn.. pregnant mice were weighed. given a sin-le dose of either

all-trans RA or dex or both (as outlined above). and returned to their cages The trearment

regimens are outlined schematically in Figure 2.1

3.2.3 Recovery and Inspection of Fetuses

On day 18.5 pc. between 1 and 3 p.m.. pregnant mice were weighed and sacniiced by

cenical dislocation. Ut en were O pened and records of implantation sites and fetal resorpt ions

were made. Fetuses were removed. dissected t e e of their membranes. weighed. and placed in

p hosphare buffered saline. Subsequently. fetuses were examined for esternal malformations. t heir

age established by the niethod of Gruneberg ( 1943). and fixed for five days in 95O, ethanol. .&fier

fixation. feruses were skinned. eviscerated under a dissecting microscope. and exarnined for cleli

palate .A palate was regarded as being clef? if a lack of fusion of an? pan of the palatal shelves

alon3 the midlinr was displayed.

Following evisceration. fetuses were placed in 10090 acetone for two days to remove

remaining deposits of adipose tissue and subsequently double stained with alizarin red S for bone

and alcian blue for canila-e as outlined by McLeod ( 1980). Brieflv. eviscerated fetuses were

placed for 3 days in a solution composed of 0.1% alizarin red S in 959'0 ethanol. 0.3% alcian blue

in 70°0 ethanol. acetic acid. and 70Y0 ethanol ( 1 : 1 : 1 : 17). Stained fetuses were then partially

cleared in lof; KOH until the skeleton became visible undemeath the sofi tissues (approsirnately 2

days). Fetuses were then placed in increasing concentrations of glycerol (20°/0. 50°h. S0°,6) in lo i ,

KOH until fùlly cleared. They were stored in 1 OOOh glvceroi.

1.2.4 Skeletal Examination

Fetuses were examined under a dissecting microscope to assessss malformations of the

appendicular skeleton. Long bones from treated litters were judged a-ainst controis on four

parameters: ( 1 ) curvature. (7) overall lengh. ( 3 ) degree of ossification. and (4) gross

morpholog of the region of overlap between the cartila-inous plu- and the bone diaphysis

(herein called the 'gow-th plate' for simplicitv). A I malfomations were assessssed from the

ventral aspect to better view al1 Ion- bones and joint articulations within a aiven limb

simultaneously In only one instance ( l ad of ossification of the lesser trochanter of the femur)

were malfomations assessssed from the dorsal aspect.

Long bone curvature was resarded as being any protrusion from or bowing of the bone

such that the center of the bone was situated either fùnher pre-auially (radius. tibia) or tùrther

post-axially (ulna) than the ends in the viewinr plane. Total limb length was assessssed by

cornparing treated limbs with limbs from control liners. Bone length was deemed abnormal if it

was less than two thirds the lengh of the analogus bone from a control animal. The degree of

ossification of the long bones was deemed abnormal if less than half the length of the bone (Le.

the middle iOO.a) was stained wlth alizann red. In the teniary sites of ossification in both the

humerus and the femur (points of muscle attachrnent including the deltoid tuberosity and the

Iesser trochanter. respectively) any ponion stained with alcian blue kvas reyarded as an

abnormality of that site. Abnormal growth plates were judged as those that had uneven

metaphyseal and/or diaphyseal surfaces (such that the growth plate appeared cone-shaped as

opposed to being flattened) and stained with both alizarin red and alcian blue (which resulted in a

dark purple to black coloration).

3 - 2 5 Statistical .balysis

Statisrical analvsis was performed on a per litter basis on the assumption that it is the

mother and not the fetus that is the experirnental unit (Gaylor. 1975). Cornparisons of

quantitative data were made bv rneans of a two-tailed. unpaired S tudent 's t-test using p<O 05 as

the minimum level of significance.

2.3 Results

2 . 1 General Inspection of Fetuses

Neither litters treated with aIl-trans RA nor litters treated with des. at an- dose or dav of

administration used. displayed an increase in fetal monalitv. resorption. or average weight as

compared to controls.

Developrnent of clefi palate as a result of exposure to all-trans R A u j ~~~~~~~o was tirne-

dependent. with the mêuimum effect seen as a result of administration of all-trans ft-\ on day 1 3 S

pc (Figure 2 . 2 ) . .A single dose of either 10. 25. or 50 mgkg all-trans RA administered to dams on

day 13.5 pc resulred in a 509.0 incidence of clefi palate within litters. The days of susceptibility of

the palate to euogenous all-trans R A extended over a four da' period. with an incidence of cle%

palate formation of 39% on day 1 1.5 pc and 36% on day 14.5 pc for the 50 m g k g dose. This

dose of ail-trans RA only elicited cleft palate in 10°h of Fetuses within a litter when siven on da?

15.5 pc.

With respect to des. a dose of 0.2 rngkgday +en on days 10.5 to 12.5. 13.5 to 155. or

16.5 to 1 8 . pc elicited clefis in 30%. 46%. and 1 8 O . 0 of fetuses within a litter respectively (Fisure

2.3 ) .A dose of O. 1 mg/kg/dav dex given durin2 anv of the 3 time periods within this study \\.as

less terato-enic. with between 17% and 39i1 o f fetuses within a litter displaying cleH palates.

Doses lower than O 1 rngkdday either did not penurb or onlv slightly perturbed the formation of

the palate on days 10.5 to 12.5 and days 16.5 to 18.5 pc but appeared to have an effect similar to

t hat of the O 1 rny'k-Jday dose when adrninistered on days 1 3.5 to 1 5.5 pc.

Based on the results described above, we chose to evaluate the effect of treatment with

O 1 rng/kg/day dex @en on day 10 5 to 12.5 or 13.5 to 15.5 pc on cleft palate defects induced by

all-trans RA treatment (75 and 50 mgkg) on days 1 1 3 and 13.5 p c The results are shown in

Figures 2.1 and 2.5. Treatrnent with dex alone (0.1 mg/kg/day) on days 10.5 to 17.5 or days 13.5

to 15.5 pc elicited clefi palates in 15% and 2196 of fetuses respectively (Figure 2.4). Treatment

with 25 mgkg all-trans RA on day 1 1.5 pc and dex on days 10.5 to 12.2 pc ('CO-treatment')

displayed a mean incidence of clefi palate formation of 6'0. whiie treatment with dex on days 13.5

to 15.5 pc ('post-treatment') resulted in a rnean incidence of clefi palate formation of 20O.0.

Neither of these values were significantly different frorn the values for fetuses esposed to 25

mgkg ail-trans RA on day 1 1 5 pc alone ( l7O.0). Litters treated with 25 m g k g all-trans RA on

day 13. S pc and de'; on days 10.5 to 12.5 pc ('pre-treatment') showed a marked decrease in clefi

palate formation (20L) as did dex Frorn dav 13.5 to 15.5 pc ('CO-treatment.). which reduced defi

palate formation to 15O6 (compared to 5 19% in litters treated with all-trans R A alone).

As s h o w in Figure 1.5. litters treated with 0.1 rn@g/'dav dex from day 10.5 to 12.5 or

day 13.5 to 15.5 pc exhibited clef? palates in l j O , O and 2 I a , o of fetuses respectively Co-treatment

with des oflitters receivin- 50 rny'kg all-trans K A on day 1 1.5 pc resulted in a mean incidence of

clefl palate formation of SjO,o. which \vas not significantly different from the value of S J O , ~ for

fetuses esposed to all-trans RA alone. Post-treatrnent with deu also had no eKect on ail-tram

RA-induced cleft palate formation. In animals receiving 50 m d k g - all-trans R A on da? 13 5 pc

however. pre-treatment with des resulted in a sigificant decrease in clef palates (7' O). compared

to 71O.0 in litters treated with all-trans M alone. However. CO-treatment with dex had no efect

on cleti palate formation.

2.3.2 Skeletal Esamination of Fetuses

General inspection of the axial skeleton revealed no differences in bone shape or bone

length between control fetuses and fetuses treated ivith all-trans RA or dex at any dose or day of

administration. In the appendicular skeleton. however. all-trans R4 treatment induced a variery

of limb malformations. The types of malformations generated depended stronglv on the time of

ail-trans R A exposure. We have classified the abnormalities resulting from earlv exposure to a[]-

trans R A (days 11.5 and 11.5 pc) as 'day 11.5 pc-tvpe' maiformations and those resulting from

late exposure to al!-trans R A (days 13.5. 14.5. and 15 .5 pc) as -dav 13.5 pc-type' malformations.

Malformations seen in the day 18.5 pc fetus as a result ofexposure to all-trans R A on da'

1 1 5 pc are illustrated in Figure 1.6. In the forelimb. abnormalities resulting from rxposure to 3

and 50 m g k g all-trans RA included pre-axial deflections of the radius (Figure 2.6 3 and 2.6 5 ). In

the hindlimbs. detlections of the tibia were obsemed as a result of esposure to 50 m g k g ail-tram

RA only (Figure 2.6 6). as was foreshortening of al1 appendicular long bones (Figure 2 6.5 and

2.6.6) .

A quantitative evaluation of the incidence of day 1 1 5 pc-type appendicular skeletal

malformations in mice treated with all-trans RA on day 1 1.5 to 1 5 . 5 pc is shown in Table 2.1.

With respect to esposure to ail-trans RA on da! 1 1.5 pc. a dose of 10 mgkg failed to elicit an?

malformations. At a dose of 25 mgkg all-trans FQ the hindlimb was affected to a lesser evtent

than the Forelimb ( Y0 0 and 22O b incidences of malformation of the ulna and the radius versus S0 O

and 7'0 incidences of malformation of the fibula and tibia). M e r exposure to 5 0 m g k g ail-trans

M. this \vas reversed (49O and 2 o . 0 incidences of malfonnations of the uina and radius versus

5j0a and 3 9 O . 0 incidences of malformations of the fibula and tibia). Horçever. with respect to

administration of both 25 and 50 m g k g all-trans Rr\. the proportion of ulnar and fibular

malformations was always greater than radiai and tibia1 malformations. The incidence of these

malformations was considerably less when all-trans RA was administered on dav 12.5 pc and no

day 1 1 5 pc-type malformations were seen when all-trans RA was administered on day 13.5 pc or

later.

Malformations seen in the day 1 8.5 pc fetus as a result of esposure to 75 mgkg all-trans

RA on day 13.5 pc are illustrated in Figure 2.7. These included widened growth plates within the

appendicular long bones and areas of delayed ossification in the humerus and femur (Figure 37.3

and 2.7.4). Malformations resulting from exposure to 50 m g k g all-trans M on day 13.5 pc were

similar. Day 13.5 pc-type malformations were not seen as a result of exposure to all-trans RA on

day 11.5 pc and only to a small extent on day 12.5 pc (a 940 maximum incidence of

malformation). A quantitative evaluation of the incidence of dav 13.5 pc-tvpe appendicular

skeletal malformations in mice treated with all-trans RA on day 1 1.5 to 15.5 pc is shown in Table

2.2. IO mg'kg all-trans R A given on davs 13.5. 145. and 15.5 pc resulted in delaved ossification

of sites on the humerus and the femur (16Oi I ~ ? . / O . and 1 I O , O respectively) and widening of the

crowth plates I -89'0). Malformations resulting from exposure to 25 m d k + - - 2 all-trans R A on an- of

these 3 days were on average increased 3-fold. There was no fünher increase in incidence of

malformation as a result of exposure to 50 mgkg. Aithough both delayed ossification and growth

plate abnormalities were seen to a similar extent afler eiiposure to 50 mgkg all-trans R4 on day

13.5 pc (-50Sh and 43?/0 respectively). there was a noticeable difference in dav 1 5 . 5 pc treated

animals in the incidences of these two types of defects (-489.0 and I?',O respectivelv).

We next studied the effects of treatment with O. 1 rng/kg/dav des on all-rrans KA-induced

malformations. In preliminary studies. it was found that a dose of 0.7 rngkddav dex given from

either day 10.5 to 12.5 or 13.5 to 15.5 pc did not elicit malformations in the appendicular skeleton

but did elicited clefi palates in - 4 i 0 / b of fetuses. .At O I mgkglday dex. the incidence of cleft

palate formation was -7?,&. however. this dose had no effect on limb skeletal morphology. We

therefore decided to use 0.1 m@g/dap dex to determine whether dex could ameliorate the effects

of all-trans IW on limb skeletal malformations. We decided to administer dex either from day

10.5 to 12.5 or from 13.5 to 15.5 pc since these periods overlapped with the highest days of

susceptibilitv of the developing limb to all-tram RA exposure (dav 1 1 5 and i 3 . 5 pc).

Dex treatment from dav 1 O. 5 to 1 3.5 pc ( -CO-treatment' ) clearly affected malformations

induced by 25 and 50 m g k g all-trans RA on day 11.5 as illustrared in Figure 2.8. Co-treatment

with dex and 25 rn&g all-trans R A and dex resulted in malformations sirnilar to da' 13 3 pc-type

malformations. These included widened growth plates and delayed ossification of sites on the

humerus and the femur (Figure 2.8.1 and 28.2) . Den reduced bot h day 1 1 . 5 and 13.5 pc-tvpe

malformations resulting from 50 mgkg all-trans RA administration (compare Figure 2 . 8 3 to

1.6.5 and Figure 2.8.1 to 2.6.6). In the forelimb. dex treatment ameliorated the eiTects of all-trans

IU on bone length but not on radial or ulnar deflections. In the hindlimb. des ameliorated all-

trans RA-induced effects on femoral length but not on tibiai or fibular foreshonening The effects

of treatment with des from day 13.5 to 15.5 pc (.post-treatment') on ail-trans RA-induced

malformations did not differ from those illustrated in Figure 2.5.

A quantitati~~e analvsis of the effects of treatment with des on malformations resulting

from esposure to 72 mgkg all-trans R A on days 11.5 and 13.3 pc is shown in Table 2 . 3 . I and

2.3 2 respectively In this experiment. 25 mgks all-trans R A alone resulted ir. a 28' 0 incidence of

ulnar detlection but failed to elicit malformations in the radius (as was observed in the tirst set of

experiments) Co-treatment with dex and 25 mgkg all-trans ELA on day I I 5 pc decreased ulnar

detlections (a da? 1 1.5 pc-type malforniation) by 609. and increased dav 13 5 pc-tvpe

malformations by 5 1°o (Table 2 - 3 1 ). This shifl €rom day 1 1.5 to day 13 .5 pc-tvpe malformations

was also seen as a resuit of post-treatment with des. With post-treatment. however. there was a

90% reduction in day I 1.5 pc-type malformations but only a 249.0 incidence of day 13.5 pc-type

malformations.

Treatment of pregnant animals with dex from dav 1 O. 5 to 12.5 pc ('pre-treatment') prior

to administration of 25 mdkp all-tram RA on day 13.5 pc had no effect on all-trans RA-induced

ossification defects but decreased gowth plate abnormalities by 7406 (Table 2 . 3 . 2 ) . There was

also an 8-fold increase in da- 1 1.5 pc-type malformations. Treatment with des from days 13.5 to

15.5 pc and 25 mgkg ail-trans R A ('CO-treatment') similarly increased day 11.5 pc-tvpe

malformations and reduced al1 day 13.5 pc-type defects.

Co-treatment of animals with de't and 50 m g k g all-trans Rr\ on dav 11.5 pc slightly

reduced radial. tibial. humeral. and femoral day 1 1.5 pc-type malformations but had no eflect on

either ulnar or fibular day 1 1.5 pc-type malformations (Table 7 .4 1 ). This combination. horvever.

increased day 13.5 pc-type malfxrnations 3 to 5-fold. .A shifi in the pattern of malformations

from the day I 1 .S pc-type to the day 13.5 pc-type was also seen as a result of post-treatment of

animals with dex and all-trans RA. This treatrnent resulted in a decrease in both da' 13 5 and day

1 1.5 pc-type malformations as compared to the total number of malformations resultinq from

either CO-treatment with de'c and all-trans RA or treatment with all-trans R A alone. It is

interesting to note that following a dose of 50 mgkg all-trans RA on day 1 1.5 pc the number of

bone deflections and forshortenings was similar in each of the bones esamined. Co-treatment

with dex decreased bone deflection and foreshortening of the radius and the tibia to a similar

e'ctent. but had little effect on the morpholog of the ulna and fibula. Post-treatrnent u-ith des.

however. decreased all-trans RA-induced bone foreshortening to a greater estent t han bone

deflection, particularly in the ulna and the radius.

Pre-treatment with des and 50 mgkg all-trans RA on day 13 5 pc reduced day 13.5 pc-

tvpe malformations by -7 1 ° , ~ without any effect on day 1 1.5 pc-type malformations (Table 2 .42) .

Co-treatment with des and all-trans R A similarly had no efect on day 1 I .i pc-type malformations

but reduced the frequency of day 13.5 pc-type malformations by -59SG. Day 11.5 pc-type

malfonnations are not shown in this table since there were none obsemed.

2. J Discussion

In this senes of experiments. we focused on the teratogenic effects of all-trans RA on

palate formation and formation of the appendicular skeleton and how these could be ameliorated

by treatment with the corticosteroid dexarnethasone. The first parameter we esamined was clefi

palate. Administration of ail-trans RA resulted in clefts of the secondary palate. which was

strongly dependent on the day of administration. We did not observe clefting of the prima-

palate as a result of ail-trans R A administration. The origin of clefis of the seconda? palate

depend upon the phase of development during which the teratogen is administered. Firstly.

growth of the palatal shelves. which occurs primarily dunng day 11.5 pc in the mouse. ma! be - inhibited. resulting in shelves that cannot make contact in the rnidline (Abbott et d. 1991).

Secondly. shelves of length sufikient to establish contact may not fuse as a result of a defect in

the îùsion process This process. which involves initial contact of the palatal shelves. apoptosis of

the peridemi. and fusion of the esposed epithelia to form one solid structure. beeins on day 13 5

pc in the rnouse. Complete fusion of the underlying mesenchvme begins afier this and is

completed bv - dav - 16.5 pc (Abbott et trl. 199 1 ). With all-trans RA. al1 doses +en during the days

of susceptibility of the palate resulted in incidences of rhese defects ranginp between 2 i 0 , , o and

5040 and were t herefore deemed teratogenic.

Treatment with dex from days 10.5 to 12.5 pc or from davs 1 3.5 to 1 5 5 pc increased the

incidence of cleft palate. likely resulting from a lack of palatal fusion. Clefl palates also resulted

Bom des administration from days 16.5 to 18.5 pc. possibly as a result of a rupture of the palate

afier the initial iùsion due to a dex-dependent defect in the merging of the underlying mesenchyme

(Greene et ttl.. 1976). As with clefis ansing afier exposure to ail-trans R4. al1 clefis seen in this

set of esperiments were seconda- clefis. In this experiment. a dose of 0.7 mgkgda- de': fi-orn

days 13.5 to 15.5 pc was found to induce cleft palate in -45?& of fetuses within a litter. but had no

effects on the appendicular skeleton. The lower doses tested (0.1 and O O5 mgkgday) resulted in

approximately 25% incidence of clefi palate formation and also had no effccts in the appendicular

skeleton. In controls. the incidence of clef3 palate was - 5 O We have chosen a dose of O I

r n g k ~ d a v dex in the all-trans RNdev antagonism experiments based on these results

Neither CO-treatment or post-treatment with O 1 rn~gkglday dex had anv efect on ail-tram

RA-mediated cleft palate formation as a result of exposure on da- 1 1 5 pc. .A possible

explanation for the absence of antagonisrn and/or synersy between these two agents may be that

all-trans RA inhibits grow-th of the shelves (and thus prevents fusion). which cannot be corrected

by dex. If dex acts on the fusion process and not on outgrowth. which is compatible with the data

presented above. no effect would be expected once shelf growth is inhibited.

The eKect of 25 m g k g ail-trans RA exposure on da): 13.5 pc on clefi palate formation

was ameliorated by both pre-treatment and CO-treatment with des. However. the effect of 50

mgkg all-trans RA was counteracted by pre-trearment only This interaction may be linked to

RARP expression. which in mesenchymal cells is upreylated by ail-trans RA. and possibly

downregulated by deu (Lohnes ci c d . 1995: Pallet rt cd.. 1995). Rowe ri d ( 199 1 ) found that

espression of RARP is highly restricted to the antenor portion of the maxillary mesenchyme in

chick facial priniordia. Following administration of all-trans RA to the det-eloping chick embryo.

RARP expression rises dramatically in both the anterior and posterior portions of the ma'tillary

mesenchyme and may be responsible for the abnomal morphology (resulting in both clef3 palate

and cleft beak) obsen-ed (Rowe et ~ 7 1 . . 1 99 1 ).

The restricted pattern of RARP expression and the change in expression observed in

response to all-trans EU in the chick mesenchyme (Rowe rt al.. 1991) reflect the pattern of

l W R P espression in the limb bud It seems likely that the processes which control the

development of the facial pnmordia in the chick and palatal shelf o u t g o ~ h in the mouse ma^

also be the same as those controlling limb bud outgrowth in these species (Richman rr c d . 1997).

If so. this would explain why both structures respond to all-trans ELA dunnq the same windows of

susceptibility and with similar reduction malformations.

We next examined the effects of all-trans RA cxposure on limb deveiopment We first

focused on the teratogenic effects of all-trans RA in the appendicular skeleton and evaluated dose

dependency. time of treatment. and types of defects developed. .-\Il-trans R A exposure on da'

1 1.5 pc at 25 m g k g resulted in a series of malformations that involved defects in bone cuwature

At this dose. there was little effect on overall length of the bones e'rarnined. At 50 mg'kg

however. both long bone foreshortening (defects in bone lengh) and bone deflections (defects in

limb patterninr) were seen. Limb outgrowth is directed by the .4ER which expresses RrU1P7

esclusively on day 11.5 pc (Lohnes rr t r l . 1993). Sulik rr td (1988) showed that all-trans R A

administration. on either day 10.5 or 1 1.5 pc in the mouse results in cell death within the AER.

They hypothesized that the cartilage defects observed in the limb following all-trans RA

administration on either of these daps arose from the disruption ofcell migration in the bud due to

improper signalin- fiom the damaçed M R . This supports the hvpothesis that the mechanism bu

which esogenous all-tram RA exerts its effects on bone outgrowth could be mediated by ligand-

activated RARP action on the AER.

Defects resulting from day 13 5. 11 5. and 15.5 pc administration of all-trans RA were

similar. The most notable was a widening of the growth plates within al1 long bones examined

which likely occurred as a direct effect of all-trans RA on chondrogenesis. The delaved

ossification in the teniary sites of ossification (the deltoid tuberosity of the humerus and the lesser

trochanter of the femur) induced by all-trans RA, however. is more likely related to an inhibition

by all-tram FW effects on muscle formation. Inhibition of myoblast fusion by all-trans RA is

known to occur (Momoi rr r d . . 1991) and since these regions are sites of muscle attachment.

defects in muscle formation may result in ossification defects. In addition. in other srudies usine

the W h s p l a c Z transgenic mouse. X-gal staining was observed within muscle groups in the

limb during days 14.5 and 15.5 pc (von Schroeder rr LI/.. submitted for publication). This

suggests that myoblasts respond to endogenous retinoids during development and may therefore

respond to exogenous all-trans R4. resulting in the malformations obsewed.

It should also be noted that the da'-dependent effects of all-trans RA-induced limb

malformations seen in this set of experiments confirm observations made bv Kochhar ( 1967) and

Kwasigroc h rr d. ( 1986) Kochhar ( 1 967) demonstrated dose-dependent effects wit h all-trans

R A administration (200. 100. and 50 mgkg) on dav 9 pc such that the administration of the

highest dose resulted in 1005'0 tètal resorption. administration of the middle dose resulted in axial

skeletal malformations and clef palate. and the administration of the Iowest dose resulted in clefi

palate only In addition. Kwasigoch C r (71 . (1986) demonstrated that the effects of all-trans RA

strongly depended on the dav of administration such that the marked decreases observed in Ions

bone area as a result of all-trans RA exposure on day 1 1 .5 pc were not obsenred after treatment

with the same dose on day 12.5 pc In the present study. these previous obsen-aiions have been

estended. With respect to the dose-dependent effects of all-trans RA. we have shown that

administration of a dose of 50 mgkg all-trans RA results in long bone foreshonening and

deflection. administration of a dose of 25 mgkg all-trans RA results in long bone deflection only.

and administration of a dose of 10 mgkg all-trans RA results in neither malformation. We have

also shown that the effects of all-tram RA on limb development are stronply day-dependent. with

two morphologically distinct sets of mal format ions resulting from all-tram RA exposure on day

1 1.5 or 13 3 pc. and an almost cornplete absence of malformations obsewed afler treatment with

all-trans RA on day 12.5 pc.

We next investigated whether these defects could be reduced by treatment with dey. With

respect to the 25 rn?& dose. day 1 1 3 pc-type malformations. which consisted onlv of bone

deflections and not foreshortening. were decreased with concorninant increase in dap 13.5 pc-type

malformations by CO-treatment with dex as well. Post-treatment with des resulted in a similar

senes of malformations. Co-treatment of animals with dex and 50 m g k g all-trans R A on da-

1 1 .S pc showed that all-trans RA-induced bone foreshortening and detlection was ameliorated.

but that day 13.5 pc malformations were induced as well. Post-treatment of anirnals treated with

all-trans R A on day 1 1 i pc with dex resulted in marked amelioration of long bone foreshonening

(to a greater estent than that observed for CO-treatment with des and all-trans RA) Also.

induction of day 13.5 pc-type malformations was seen in a smaller proportion of fetuses following

post-treatment as compared to CO-treatment These observations show for the first time that the

effects of all-trans RA on long bone foreshonening can be ameliorated by both CO-treatment and

post-treatment with dex.

Zimmermann rr trl. ( 1993) showed stimulation of cartilage formation by des in organoid

cultures of limb bud cells isolated from da? IO5 to 12.5 pc fetal mice and a sub-maximal response

tiom cultures isolated fiom da- 13.5 pc ktal mice. In our esperiments. da? 13.5 to 15 5 pc

administration of des also resulted in amelioration of bone foreshortening but did not ameliorate

all-trans FU-induced effects on bone detlection to the same estent. The ability of dex to

antasonize the effects of all-trans RA on limb outgowth to a greater degree than on iimb

detlection may be related to the stimulatory effects of des on chondrogenesis. Pre-treatment with

dex had no effect on amount or seventy of malformations induced by exposure to all-trans RA on

day 13.5 pc. Co-treatrnent with dex. however. resuited in marked amelioration of the day 13.5

pc-type all-tram RA-mediated malformations.

With respect to the vanous malformations induced bv all-trans RA in the limb. it would

appear that limb foreshonening is the most severe. as this malformation would result in niice \vith

severely affected limbs. Day 13.5 pc-type malformations. which are likely due to delayed

ossification. may ossifi post-natally In light of the effects of des on ail-trans RA-induced

malformations. it is important to note that although dex treatment following all-trans RA evposure

on day 1 1.5 pc results in an increse in day 13 .S pc-type malfomations. the striking decrease in the

incidence of long bone foreshortening would suggest that the net effects of dex treatment in this

study are beneficial.

With respect to the mechanism of the des-mediated amelioration of all-tram RA-induced

abnormalities. or of the des-mediated shift from day 1 1.5 to 13.5 pc-tvpe malformations. several

esplanations are possible. Firstly. den could be antagonizing the rffects of all-trans RA in this

svsteni bv stimulating chondrogenesis. With respect to day 13.5 pc-type malformations we would

then espect that CO-treatment would result in an increase. and not a decrease. in malformations.

Since this is not the case. it seems unlikely rhat all-trans R A acts through increasing canilase

Formation. If day 13 5 pc-type bone malformations a i se due to prima? defects in muscle

formation (Momoi ~ ' r LI/.. 1990). it is possible that the means by which des antasonizes these

malforrnations is via the stimulation of muscle formation. Another mechanism through which dex

can attenuate all-trans RA action is through induction of cytochrome PJ5O-mediated nietabolism

of ail-trans RA to the inactive 4-hydroxy RA metabolite (Martini P r d . 199.1) This would

decrease the amount of all-trans R4 in the embryo. However. this could only explain the effects

of dex CO-treatrnent.

In our experirnents. the arnelioration of all-trans RA-induced long bone foreshortening bv

CO-treatrnent with dex on day 1 1.5 pc could be based on the opposire effects of these dmgs on the

expression of W P . If d e l were to downregulate RARP. for example. this would result in

amelioration of day 1 1.5 pc-type rnalfonnations. which are likely mediated bv RUPZ action in

the AER and an increase in day 13.5 pc-type rnalfonnations due to unopposed Rr\R:/ efects on

chondrogenesis. This mechanism has been observed in the liver Pallet r? L I / . ( 1995) showed that

the administration of 2 m g k g des twice dailv for three days to 6 week old male rats resulted in

downregulation of R4RP mRNA and decreased RARP protein in the liver. Furthemore. this

study showed that CO-administration of dex and ail-trans RA resulted in a net reduction in both of

these parameters. It is possible that a sirnilar decrese in RARP protein could be seen fiom

treatment with dex followin~ exposure to ail-trans RA.

.Aithough dex has been shown to decrease Mi3 mmA levels in liver and lung tissue both

I I I w o and i j r virro (Pailet d . 1995: Grummer et c d . . 1995). the molecular events surroundine ci

this phenomenon are unknown To date no glucocorticoid response element has been h u n d

within promotor regions of any of the RAR genes. Because of this. it is thought that the decrease

in R U mRKA by de': is due not to an effect of the GR on RAR genes but rather to efects of deu

on R A levels in tarset tissues. Aithough the effects of de': on the metabolism of all-trans RA have

been discussed. other ways in which de'c exerts its effects on RAR mRNA expression likely esist.

For esample. Wan (il. ( 1994) showed that 5 .r 10" M des increased RICRU and RX&; mRNA

2- to 3-fold in a rat hepatoma ce11 line. Since W s and RXRs exist within the ceIl in a balance

between heterodimers and homodimers. this increase in RXR may shifi the balance between these

receptor foms to favor W R X R homodimer formation. This would resulr in a shiftiny of the

pattern of response of target genes. RAREPZ has a high affinity for W R W heterodimeric

receptors but cannot be activated by RYRIRXR homodimers (Pallet r? LI/. 1995). The induction

of RXR/ttXR homodimers by dex can then be directly linked to a decrease in the transcription of

N P . It should be noted that it is this subtype for which a role in the mediation of al[-trans R A

lirnb teratogenesis has been implied in this study.

Figure 2.1 Schematic of the Dosing Regimen for the NI-tram RVDex Combination Studies.

I I I

The dose of des used was O. 1 mg/kg/dav i.m. The doses of ail-trans R A used were either 25 m~&s or 50 mgkg administered bu gavage

- DEI(

I 1 1 1

I I l 1 1 . 1 I -RA

I I

DEX DAYS 10.5 TO 12.5 pc

t

DEM DAYS 13 5 TO 15.5 pc

R A DAY 1 1 . 5 ~ ~

DAY 1 3 . 5 ~ ~

RA - R A R A DAY

11.5 pc D AY 13.5 pc

RA - R A RA RA DAY

11.5 pc D Al'

13 5 pc

Fioure 3.3 Effect of Dose and Dav of Ex~osure to dl-trans R A on CIeft I

Palate Formation.

day of administration

Pregnant CD- 1 mice were administered a single dose of 10. 25. or 50 mgkg all-tram R A on day 1 1 5 . 12.5. 13.5. 14.5. or 15.5 pc Controls received corn oil vehicle only. On da? I S i pc. dams were sacnficed, fetuses remoc-ed. fised. and examined for clefl palate.

*sisnitkant difference from control ( P<O.O 1 ) * *sigrtifkant difference from previous lower dose and from control (P<O O 1 ) Results are espressed as the mean percent of fetuses within a litter displaving clefi palate i sem of n = 3 litters. except for the 25 m g k g dose on days 1 1.5 and 13.5 pc where n = 4 litters and controls. where n = 6.

F e 2 . Effect of Dose and Time of Exposure to De'r on Clefi Palate Formation.

control 10.5-12.5 13.5-1 5.5 16.5-18.5

days of administration

Pregnant CD- 1 mice were administered a single dose of eirher 0.0 1. 0.025. 0.05. O 1 . or O 2 mg/kg/day de'r i .m from either days 10.5 to 12.5. 13.5 to 15.5. or 165 to 18.5 p c Controls received vehicle only On day 18.5 pc. dams were sacrificed. fetuses removed. fised. and exarnined for clel't palate

"significant dittèrence tiom control (P<O.O5) **significant difference from previous lower dose and from control (P<0.05) Results are expressed as the mean percent of fetuses within a litter displayin- clef? palate z Sem of n = 3 litters.

Figure 2.4 Effect o f Treatment with Dex on Clef1 Palate Defects Induced bv Exposure to 1 5 mgkg .Ail-trans RA on Day 1 1.5 or 13 5 pc.

60 r

control R A 1 ISdpc R A 13.5 dpc

17 no des

CS 10.5- 12.5 dpc

Odes 13.5- 15.5 dpc

Pregnant pc or da- day 13.5

- 1 mice were administered a single dose of 25 m g k g all-trans RA on either day 1 1.5 5 pc and received also 0.1 m@aJday des either frorn da? 10.5 pc to 12.5 pc or from 15.5 pc. Controls received corn oil vehicle o n l y Des controls received 0.1

mg/kg/day dex on either days 10.5 to 1 2 5 pc or days 13 .S to 1 5 .5 pc. Ali-trans RA controls received 3 m d k g - all-trans RA on either day 1 1.5 pc or 13 .S pc. On day 18.5 pc. dams were sacrificed. fetuses removed. tixed. and esamined for clefi palate.

"significant difference between - des and - des (PcO.0 I ) Results are expressed as the mean percent of fetuses within a litter displaving clet? palate z sem of n = 3 litters.

Fiyre 2.5 Effect of Treatment with Dez; on Cleft Palate Defects Induced by Exposure to 50 rn~gkncg .MI-trans RA on Day 11.5 or 13-5 pc.

RA 13.5 dpc

no dcs

Hdcs 10.5- 12.5 dpc

O ~ C S 1-3.5- 15.5 dpc

Pregant CD- 1 mice were administered a single dose of 50 rn& all-trans RA on either day I 1 .i pc or day 13.5 pc and received also O. 1 mgkglday dex either from day 10.5 pc to 12.5 pc or from da); 13.5 to 15.5 pc. Controls received corn oii vehicle onlv Deu controls received 0.1 mg/kg/day dex on either days 10.5 to 12.5 pc or days 13.5 to 15.5 pc. Al-trans RA controls received 50 m d k r all-trans R4 on either day 1 1.5 pc or 13.5 pc. On dav 18.5 pc. dams were sacrificed. fetuses removed. fixed. and enamined for cleft palate.

*sianifkant difference between presence and absence of des (P<O.O 1 ) Results are expressed as the mean percent of fetuses wit hin a litter displayin- clefi palate i Sem of n = 3 litters

2 6 1 Control Forelimb 2 6 2 Control Hindlimb

2 ô 4 Hindlimb 25 mqkg

2 6 5 Forelimb 50 mgikg 2 6 6 Hindlimb. 50 mg/kg

Pregiidnt CD-I niice nere administerrd a single dose of all-tram RA an da\ I 1 5 pc On da!, IS 5 pc. mise IL cre sacri t iccd and irtuses ~r ere remm ed F e u s e s Ir ere tïued. skinn;d. e\ isserated. and double iiained N i rh alizai-in rrd for bone and alcian blue for cartilage Subsrquently. the apperidicular skeleton Ir 3s t\âniined hr rnaliormations us1 n3 a d i ss rc t in~ microscope

H. humerus. U. ulna. R. radius. F. femur. T. tibia. Fb. Fibula. arrowhead. pre-axial deflection

Table 2.1 Effect of Dose and Day of Exposure to AiI-trans IW on Day 1 1.5 pc-type Appendicular S keletal Malformations.

1 dose 1 control 1 day of exposure day 11.5 pc ulna radius fibu la tibia dav 12.5 DC

Pregnant CD4 mice were administered a single dose of either 10. 75. or 50 mgk- all-tram RA on either dav 1 1.5. 12.5. 13.5. 14.5. or 15.5 pc. Controls received vehicle only On da? 1 8 . 5 pc. dams were sacrificed, fetuses were fised. stained. and examined for skeletal defects.

O 27.38 = 8.41 * 19.31 = 5 97 O 22.32 = 3.62* 32.53 = 5.64 O 5.2 i = 2.64* 53.2 1 = 9.49* O 7.01 = Ml* 39.82 = 4.93*

ulna radius fibula tibia d 13.5. 14.5. 15.5 pc al1 those listed above

*significant difference frorn previous lower dose (W0.05) Results espressed as the mean percent of fetuses within a litter with defect t Sem of n = 3 litters except for the 25 mgAg dose on day 1 1.5 and 13.5 pc. where n = 4.

O O O O O O O O 5 02 = 2.52* O O 4 21 = 2.33*

O O O

Figure 2 7 Etkcts of Esposurt: to Il-[rails R A on Dav 13 pc on the -4ppendicular Skeleton

2 7 1 Control Forelimb 2 7 2 Control Hindlimb

2 7 4 Hindlimb 25 mg!kg

Prcgiiant CD-I misr nrre administered a sinslr dose of all-rrans RA on da' I 5 ps On da! I S 5 pc. mise n r rc sacri ticcd and fet~ises \r ere remm ed Feiuses \r rre h r d . skinncd. ri isserated. and double stained i r i t h alizariii rrd for bonr and alcian blue for sanilage Subseqiientl!. the apprndtcular skeleton n a s ruaitiined fur nia1 formations using a dissecti ri- niicroscope Jlalforiiiations elici ted froni a dose of 50 nig Ly all-trais R-4 did not diftèr signiticantl? i n appcaransr froni thosr rliciird from a dose of 2 5 nig kg and are nut s t l o ~ ~ l

DT. deltoid tuberosity: LT. lesser trochanter: GP. growth plate: arrowhead. lack of ossification

Table 2.2 Effect of Dose and Day of Exposure to Ail-trans RA on Da- 13.5 pc-type Appendicular S keletal Malformations.

dose day of exposure

control 25 m g k ~ 50 my 'kg

day 11.5 pc ail those listed below

1 femur 3" sites 1 O 32.68 I 6.06* 50.0 1 = 4.39 1

O O O

- - --

humerus 3" siCs femur 3" sites day 13.5 pc humerus 3" sites

--

1 dav 11.5 o c - 1 - 1

-- -

O 2.31 = Lis* 7.54 = 1.67* O 3.21 = O.S9* 9.23 = 2.23*

O 40.54 = 7.37* 55.76 = 8.39

Pregnant CD- 1 mice were administered a single dose of either 10. 25. or 50 mgkg ail-trans RA on either day 1 1.5. 17.5. 13.5. 14.5. or 15.5 pc. Controls received vehicle only On dav 1 S. 5 pc. dams were sacrificed, ktuses were fised. stained. and esamined for skeletal defects.

humerus 3" sites femur 3" sites day 15.5 pc humerus 3" sites femur 3" sites

*si ynificanr ditference from previous lower dose (WO.05 ) Results espressed as the mean percent of fetuses within a litter with defect x sem of n = 3 litters escept for the 25 m g k g dose on day 1 1 .5 and 13.5 pc. where n = 4.

- -

O 39.53 = 2.45* 47 23 = 3.63 O 30.3. 1 = 4.81* 5023 = 6 i 5

O 40.4 1 = 5.94* 43.25 = 3.14 O 26.38 = 4.38* 48.12 = 7 48

Table 2.3.1 Effects of Dex Treatment on Limb Skeletal Malformations Induced by 25 m g k g .dl- trans R A on Day 1 1.5 pc.

dex treatment malformation uhar deflection

Table 2.3 2 Etfects of Dex Treatment on Limb Skeletal Malfornations Induced by 15 m g k g -41- trans RA on Day 1 3 .5 pc.

none day 10.5-12.5 pc dav 13 5-15.5 pc

28.43 14 .76 11.67 r 2.91* 3 33 z 1.67* L

humerus 3" sites femur 3" sites

dex treatrnent I maiformaion

O 47.51 i 4.73* 22.92 =4.64* O 51.66 I 6.5 1 * 34.67 I 6.64*

none day 10.5-12.5 pc dav 13 5-15.5 pc

Pregnant CD- 1 mice were administered a single dose of 25 m g k g all-trans R A on either day I 1.5 pc ( 2 . 3 1 ) or day 13.5 pc ( 2 3 2 ) and received also O. 1 mg/kg/dav dex either frorn dav 1 O 5 pc to II 5 pc or from da- 13.5 to 15.5 pc. Controls received 25 rngkg ail-trans R A on either day I 1 5 pc or 1 ;5 pc.

ulnar deflection humerus 3" sites femur 3" sites

* significant ditference between presence and absence of de'i (PcO.05) Resulrs espressed as the mean percent of fetuses within a litter rvith defect z sem of n = 3 litters

2.37 1 1.45 47.33 r 4.12* 40.47 r 5 C7* 42.12 = 2.04 48.54 r 5.36 3 33 1 1 72* 36.56 s 0 97 39 44 r 5.08 O*

Table 1 .41 Effects of Dex Treatment on Limb Skeletal Malformations Induced by 50 rng'kg AI-trans R A on Day 1 1.5 pc.

des t reat ment l none day 10.5-12.5 pc dav 13.5-155 pc

mal format ion ulna deflection

foreshonening radius deflection

toreshonenins fibuia deflection

foreshortening tibia deflection

foreshort ening humerus 3" sites femur 3') sites

Table 2.1.2 Effects of Dex Treatment on Limb Skeletal Malformations Induced by 50 mgkg M-trans F U on Day 13.5 pc.

des treatment malformation

Pregnant CD-1 mice were administered a single dose of 50 mgkg all-trans RA on either day I 1.5 pc (2.41 ) or day 13.5 pc (3 .42) and recieved also 0.1 mgkgda! des either from da? 1 O 5 pc to 12.5 pc or tiom day 13.5 to 15.5 pc. Controls recieved 50 mgkg all-tram R A on either day 1 I 5 pc or 13.5 pc.

humerus 3" sites femur 3" sites

* significant difference between presence and absence of dex (P<O.O5) Results expressed as the mean percent of tètuses within a litter with defect r sern of n = 3 litters.

none

50.2 1 r 6.53 45.51 I 10.21

day 10.5-12.5 pc day 135-15.5 pc

8.67 r 0.87* 14.67 i 6 .32"

1s 1 1 z 966" 33.67 r 5 .54

CHAPTER THREE

iDENTIFIC.4TION OF CELL TYPES IN THE DEVEtOPlNG LIMB BC'D RESPONDING TO ENDOGENOUS AND EXOGENOCS .-ILL-TRAYS RETINOIC .ACID

CSING THE R4REhsplacZ TRINSGENIC MOL'SE

3.1 Introduction

Because the results presented in chapter 2 susgested that RARP activated in the . G R

might result in the ali-trans KA-induced f~reshonenin~ obsened with 50 mgkg al!-trans R A

civen on dav 1 1.5 pc and because of the possibilit?; that dex may counteract this eCc t of all-tram - RA by downre@ating -0. we investigated IWR activation in the limb bud of day 1 1 5 pc

ernbqos using a transgenic mouse aodel carrying the RAREPZ coupled to the heat-inducible

promotor hsp68 and the lac Z reporter gene (Rossant et al.. 1991 ). We first deterrnined in which

cells of the developing limb bud concentrations of endogenous RA were sufficiently high to detect

a P-~alactosidase (P-sal) response. and then detemined if this response could be upre~ulated by

ervogenous ail-trans R A .

It has been shown previously that the RAREf32 half-site (the firsr half of the RAREP2

palindrome) can initiate transcription of al1 three W s and their vanous isoforms (Sucov et al..

1990). However. subsequent studies revealed that some of the RAR isofoms cannot stimulate

transcription from the full RXREP2 (Husmann et al.. 199 1 ) In other reports it uas shown that in

constructs containing the same response element. eene activation was stronyly stimulated by

RARP and RARu but only marginally stimulated by M! (Najpal et al.. 1992). This apparent

discrepancy and the validity of the ILUlEhsplacZ transgenic mouse mode1 for use in t h e detection

of all-trans RA-responsive ce11 types and tissues are also addressed in this chapter.

3.2 Methods

3 3 1 RAREhsplacZ Transgenic bfice

The mice used in this study contained multiple copies of a RAREhsplacZ transgene. The

construction of the vector containing the transgene. as well as the in vitro activitp of the

construct. has been preblously described (Rossant et al 1991 ). The consrmct contains three

copies of a 3-1 base pair oligonucleotide encoding the murine RAREPZ insened upstream of the

promoter for the mouse heat-inducible hsp68 gene and the lac 2 reporter sene derived tiom E

Coli. The activation of this construct can occur only in the presence of both the R A ligand and

one of the RARs since only the ligand-bound RAR can bind to the RARE Once bound to the

RAREP2. induction of the transcription of the lac Z gene. via the hsp promoter. can begin. The

lac Z gene encodes P - y i . an enzyme whose presence is readily identifiable using X-gal as

substrate ( Figure 3 .1 ).

Males homoqgous for the RrUlEhsplacZ transgene were bred with normal virgin C D 4

females and resulting litters. al1 of which contained the transgene. were used for these

esperirnents. h imals were fed t7d lthitrirn and kept under regular 12 hour light-dark cvcles.

Cnder these conditions. matiny usually occurs at midnight. therefore animals were coupled

ovemisht. Noon on the day following a successfùl mating was designated as day 0.5 pc. On da?

1 1.5 or i 3 5 pc. pregant females were given. by gavage. a single dose of 25 m g k g body weight

all-tram RA (Sigma Chemical Co.. St. Louis MO) in a volume of 0.2 ml corn oil or the same

volume of the corn oil vehicle alone Pregnant females were sacrificed 6 hours afier dosing by

cervical dislocation. This lengh of time was chosen to allow for transcription from the W B 2

and subsequent translation of the lac Z mRNA (Soprano et al. 1993).

3 2 . 2 P-Gal Staining

Fetuses were rinsed in phosphate buffered saline (PBS) and subsequently fixed in 2'0

formaldehvde. 0.2?/0 glutaraldehyde. 0.01?/0 Nonidet P10. and 0.0 l a o Na deosycholate in PBS at

4-C for one hour. M e r fixation. al1 specimens were incubated with the histochemical substrate 5 -

brorno-4-chloro-3-indoyl P-D-galactopyranoside (X-gai: Sigma Chernical Co.. St. Louis MO).

The X-gal (0.5 mgml final concentration) was dissolved in dimethyl suifoxide (40 mg N-gal/ml)

and diluted in PBS containin- 5 mM K:Fe(CN)r. 5 miVl K.Je(CN),. and 2 ml1 bl~C12.6H20.

Hindlimbs were dissected frorn the fised fetuses before staining with S-gal to ensure proper

penetration of the dye. The hindlimbs were incubated in this solution for 1-1- 16 hours at a

constant temperature of 30°C. When the RAREhsplacZ transgene is activated bv ligand-bound

FUR P-gal is produced and is available to cleave the N-sa1 substrate. The cleavage results in a

blue coloration of the cytoplasm by one of the cleavage products. The presence of P--al activitv

can be interpreted as a response to all-trans RA.

Following incubation. hindlimbs were post-fixed in -PO paraformaldehvde in PBS.

dehydrated in successive concentrations of ethanol (30°,0. 70O.0. 90O.0. 100° O ) , cleared in

chloroform and embedded in paraffin wax. Those limbs not embedded were stored at 4'C in 70° O

ethanol until fùnher processing and use. Parafin-embedded limbs were sectioned ( 7 pm) and

stained with hematoylin and eosin (H6-E). For some specimens. adjacent sections were also

mounted but not stained to determine if the H&E was obstructing the X-gal dve. Cells positive

for X-gal were counted using a Zeiss microscope and a ZID.AS image processing system

(magnification 200N). Areas corresponding to several anatomical landmarks were measured and

the number of positively stained cells was expressed per unit area.

3 2 . 3 Statistical halysis

Cornpansons of quantitative data were made by means of a two-tailed. unpaired Student's

1-test usin- p<O.Oj as the minimum Ievel of significance.

3.3 Results

3.3.1 klacroscopic S taining Pattern for P-Galactosidase

On gross inspection of control transgenic fetuses at 11.5 days pc. light stainin- was

observed in the eye and nasolacrimal groove. the base of the head. the back. and the base of the

developing limb buds. P-Ga1 response as observed in transsenic fetuses evposed to 25 m-k- all-

trans RA in utero on day 11.5 pc showed similar pattems of staining with little difference in

intensity. In day 13.5 pc fetuses. darker stzining was obsenred in the eye and nasolacrimal

groove. the brain and head. neck. back. and in al1 of the interdigital webs of the fore and hindlirnbs - ( Figure 3 . 1 ) . Transgenic fetuses exposed to 25 mgkg al!-trans R A in utero on dav 1 3 F pc

displayed similar pattems of staining in al1 body regions escept in the interdisita1 webs and the

head. where staining was much more intense and encompassed a larger area.

3 . 3 . 2 Histological Localization of X-gal Staining

In sections from hindlimbs of control transgenic day 1 1.5 pc fetuses. X-gal staining was

restricted to the apical ectodermal ridge (AER) and proximal mesenchyme (Figure 3 . 2 ) . There

was no X-gal stain in the limb bud mesenchyme. In sections from hindlimbs of ernbryos treated

with 25 m& all-trans RX on dav 1 1.5 pc. no differences were seen in either pattern of staining

or in number of cells positive for ?(-sa1 (Fisure 3 .3 .1 and 3 . 3 2).

In dav 13.5 pc feruses. S-gai staining was restncted to the IDZ only (Figure 3 .4 ) There

was no staining observed in the limb bud mesenchyme. the digital rays. the cartilagenous long

bones and as well as in regions between the bones (the area between the presumptive tibia and

tibula). In transgenic fetuses e~posed to all-trans RA on day 13.5 pc staining within the ID2 was

more intense than staining observed in controls. Aso. as with control transgenic fetuses. no X-

gal staining was obsen:ed within the mesenchyme. the digital rays. or the cartila~enous long

bones. There was no difference in size of the IDZ between controIs and ail-tram R A treated

fetuses (data not shown). and there was a 3 to 5-fold increase in the number of X-gal positive

cells in the ID2 of treated fetuses as compared to control (Figure Figure 3 5.1 and 3 5 . 2 ) .

3.4 Discussion

In an attempt to investisate the relationship between gene activation and all-trans RA-

induced malformations in the limb as a result of esposure on day 1 1.5 or 13 5 pc. we used the

RMhsp lacZ transgenic mouse model. Transgenic mice treated with 25 mgkg all-trans RA cn

day I l 5 pc displayed a pattern of expression for 0-gal that was not different in either area or

intensity of stain from non-treated control transgenic animals In the limb bud mesenchyme. no P-

cal response was observed. Staining was observed only in the proximal mesenchyme and . G R . -

where KARP? is expressed However. staining in these regions did not increase in response to

treatment with all-trans RA. Since exposure to 25 mgkg on da? 1 I 5 pc was s h o w to hav no

efiect on limb foreshortening. it is possible that this dose may not elicit changes in retinoid

response in these reqions. In retrospect. usinq a dose of 50 mgkg all-trans R A would have been

more appropriate.

In control transgenic mice. expression of the transgene on day 13 5 pc was observed only

in the IDZ. again recapitulating the expression patterns for RARP2. Administration of 25 mgkg

all-trans R A on dav 13.5 pc resulted in upregulation of the B-gal gene as seen by an increase in X-

cal positive cells per unit area in the ID2 onlv. This supports a role for RARP7 in digit formation. CI

However. since M a is also expressed in the [DZ. this receptor form may also be involved in

digit formation and this e'rperimental model cannot discnminate between these possibilities.

Previously. it was believed that al1 three RARs and their isoforms were able to stimulate

transcription from the RAREPZ equally. suzgesting that this response elemenr. linked to a

reponer construct. could be used to assess the activation of al1 RARs by endogenous and

exosnous RA. in initial studies (Sucov et al.. 1990). reporter constmcts were made

incorporating either one of two possible M P Z half sites in front of the herpes simples virus-

thymidine kinase promotor. Using this construct. al1 R R isofoms were shorvn to stimulate

transcription of the reporter gene. Work done subsequent to this (Husmann et al.. 199 1 ) usin!

the full R N P 2 linked to the thymidine kinase promotor and the chloramphenicol

acety ltransferase reporter gene demonstrated that RrU1! 1 inhibited transcription from the

complete response element and also antagonized the stimulatory effects of some of the other

RARs (RARU. RW2. RAROI) on transcription from the M P î . This suggested that the half

sites may behave differentlv from the full RAREPî. However. other studies shorved that although

RUPI and W ! l bind with high affinity to the RAREPî. this RARE. which is strongly

activated by M P Z . is only marginaily activated bv RRyl (Hoffmann P r d.. 1990: Nagpal

LI / . . 1991). Since R a ! 1 can onlv partially activate transcription from the R.UZEf32. this su, (r'lest s

that RARyI may anta~onize the effect of W P î on the RARE by binding to and blocking the

binding site.

With respect to the observed patterns of e~pression of RARP2 and RA+,& which appear

to be mutually exclusive. as elucidated by i ~ r .siru hybridization and RXase protection analysis

(Husmann ri L I / . . 199 1 ) it is possible that in cells expressing W! 1. the espression of RARPI. is

inhibited via the above rnentioned mechanism (since the RARP2 gene contains a R W P 2 in its

promotor region) For the same reason. cells expressing R4Ry 1 may not express anv R l R P 2

responsive gene (including the RAREhsplacZ transgene). The obse~at ion that RAR-! 1 and

RARD2 seem to have directly opposing roles in chondrogenesis is of interest in this respect. It

should also be noted that RARyI is the only RAR that supports chondrogenesis. and in that way

can be seen to be in opposition to the other members of the IUR family.

Nthough there is no upregulation of P-gal response in M P I expressing cells in this

case. RARD2 may still play a roie in the mediation of the teratogenic response to ail-trans R A

during early limb development. This is supponed by observations made in the RARdRAFb!

double knockout mouse that indicate that in these anirnals. the distal Ions bones of the lirnbs do

forrn normally but undergo dvsgenesis durin later periods of development (Lohnes et al.. 1995).

Data obtained with the RARP knockout mouse, however. indicates that R- is not required ro

mediate the teratogenic response of all-trans M. In the absence of RUIP. compensation by

M u 1 (which is expressed in overlapping, but not identicai. regions) ma? occur (Luo et al..

1995).

Since RrUlu is also expressed in the IDZ (and kee of the Rwl-mediated antagonism

seen in the lirnb bud mesenchyme). this receptor form rnay also be involved in digit formation

Xlthough M a l and W u 2 have been shown to stimulate transcription from both the

R4REP2 half sites (Sucov et al.. 1990). whether they c m stirnuiate transcription from the Ml

RAREP? to the same degree as M P 2 is questionable. However. the ability of RARu to

stimulate transcription from the full RAREPI in this system is also questionable since in the

peripheral lirnb mesenchyme. where RARu is comparatively hiyhly espressed. a neyligible P-=al

response was seen. This cannot be explained by RAR-11-mediated inhibition of RARu transgene

transcription since this region does not contain W! 1 at this time

It is thought that all-trans RA. through RARu and RARP. mediates some of its effects on

mesenchymal cells via induction of apoptosis. There is considerable evidence supporting a role

for apoptosis in the formation of the diijts and that this process is upregulated in the presence of

all-tram R A (Sulik et al.. 1988). lUthough the mean ID2 area in response to ail-trans RA

treatment was not significantly different from cont rols (Figure 3.51 ). the obsemed increased

number of X-gal positive cells per unit area rnay be indicative of the number of cells within the

IDZ that enter into the apoptotic pathway. This phenornenon may require longer than a 6 hour

period to occur and would therefore not have been seen in this set of experiments.

The observations by Rossant rr tzl. ( 199 1 ) that the expression of f3-gal appears to overlap

with R4RP expression in the RAREhsplacZ transsenic mouse with subtle differences between the

two can be explained by the fact that the transgene will be strongly stirnulated by RARP but

minimally by RARU. Due to their mutually exclusive expression patterns Irr i i iw . RWil cannot

antagonize RARPZ in this system. Thus the RUEhsplacZ transgenic mouse does not appear to

be suitable for the localization of the ceIl types in the developing limb bud responding to

exogenous all-trans R A since only tissues expressing RARP will reliablv transcribe f3-gal and ais0

because of the possible. and varying. expression of p-gal in response to R.*u and RARy2. It is

because of this that the effects of dex on transgene expression in this model were not assessed.

3 1 . 1 Day 13.5 pc untreated transgenic mouse 3.1.2 Day 13.5 pc untreated normal (CD1 ) histochemically stained with X-gai. mouse histochemically stained wit h X-gal.

Da)- 13 5 pc fenises wrre srained \cith N-gal for 14 to 16 hours at 30°C When the RAREhsplacZ Jene is activated by ligand-bound RAR. P-galactosidase is produced and is available to cleave the N- b

yal substrate The cleavagr results in a blue solouration by one of the cleavage products of S-gal Normal CD- I da). 13 5 pc lack endogenous 13-ealactosidase. as indicated by the lack of X-gal staining

3 2 1 Day 1 1 5 pc control transgenic mouse 3 2 2 Day 1 1 5 pc treated (25 mqkg all-trans RA) transgenic mouse

Da' 1 1 5 pc trnrisgenic riiistt 11 ere ei thrr trcrated u ith 25 ing kg all-traiis R.A or vehisle aluns O Iwurs follon i rig dnig adrnini stratiuti. dairis N ere sacri tïcttd and t'ttnistts riii~iowd Fstusss ere incubatd for 14- 16 h w r s N i t l i S-gal ar 3 0 " ~ Folio\\ in- incubation. hind liinbs n rre isolated. postfiued. eiiibrddçd in parar'tiii \ \ a \ and sectioricd at 7 niiii .A. apical ectodsrriial ridge. hl. liiiib bud 11iest3llr: ll\1l'lt3

Figure 3 3. l Effect of Exposure to Al-trans R A on Day 1 1 . 5 pc Limb Bud .kea

control

Figure 3.3 2 Effect of Exposure to .Ail-trans RA on Da- 1 1 . i pc on the Number of ?<-sa1 Positive Cells in the Limb

control

Control and retinoid treated ( 2 5 mgkg all-trans RA on day I I .5 pc) fetal mice containing the RAREhplcicZ transgene were incubated for 14 to 16 hours at 30°C in the presence of X-gal. Following incubation. hind limbs were isolated, fixed, embedded in parafin wax and sectioned at 7 pm. Sections were mounted on glass slides. counterstained with H&E and coverslipped. Using a Zeiss microscope and a ZIDAS image processing system (2OOX magnification) iimb bud area was measured and the number of X-gal positive cells per unit area was counted.

Figure 3 3 . 1 Results are expressed as area ( 1 O-' mm2) = sem of n = 15 sections. Figure 3 3 . 2 Results are espressed as X-gal positive cells per IO-' mm' = Sem of n = I F sections

3 4 1 Day 13 5 pc untreated transgenic mouse 3 4 2 Day 13 5 pc treated (25 mqkg all-trans RA) transgenic mouse

Da' 13 5 pc traiisgnis mise w r r eitlier treaied \r i t l i 25 1112. kg all-iraris RA ar \ehislc alone 6 Iiours ~o l lu i~ i r ig dnig adrriinistration. daim w r e sacriticed and ktuses rerrioi ed Fetuses w r e inc~ibated h r 1-1- 16 hoiirs wi l i S-gal ai 30°C Folloir in r - insubatioii. hiiid Iinibs w r e ~solated. posttiscd. rtiiibeddcd i n parat'tiii iras aiid sectioned dt 7 i t i i i i IDZ. iiiterdi~ital zoiiss. DR. d i g a i ra'x 51. Iimb b ~ i d rriese~ictn rtie

Figure 3 .5 .1 Effcct of Exposure to Ai1 -trans R A on Day 13.5

- pc on interdigital Zone .Ares

4 control

I7 25 n ~ l k g

idz 2 idz 3 idz 4 interdigtal zone

Figwre 3 . j 7 Effect of Exposure to Al-trans RA on Day 13.5 pc on the Number of X-gal Positive Cells in the Interdigital

Zones 30 1 *

idz 3 idz 3 idz 4 interdigital zone

Control and retinoid treated ( 2 5 mgkg all-trans RA on day 13.5 pc) fetal mice containiny the R.1LREhpi~rcZ transgene were incubated for 14 to 16 hours at 30°C in the presence of 'i-gal. Following incubation. hind limbs were isolated, fised. embedded in parafin wax. and sectioned at 7 pm. Sections were mounted on ~ l a s s slides. counterstained with H&E and coverslipped. Csing a Zeiss microscope and the ZIDAS image processing systern (200X magnification) interdigital area was measured and the number of ,Y-gal positive cells per unit area was counted.

Figure 35 .1 Results are expressed as area ( 1 o4 mm') = Sem of n = 1 5 sections. Figure 3 5 . 2 Results are expressed as X-gal positive cells per 1 O~ mm-' = Sem of n = 15 sections.

* significant difference between control and treated sections. (P<O. O 1 )

CHAPTER FOUR

SUMMARY AND CONCLUSIONS

It was previouslv detenriined in the rnonopotential chondrogenic ceIl line RCJ 3.1 CS that

the glucocorticoid dexamethasone could counteract the inhibitoq effects of all-trans RA on

cartilage differentiation such that the dose-response cume for the effects of all-tram RA on

cartilage formation was shified to the rirht by an order of magnitude in the presence of 10 niiI des

(Lau rr d.. 1993). It has also been determined that the inhibitory effects of all-trans RA on

cartilage development I I I i* i rr -o have an itr iviivo counterpart in that the development of the

appendicular cartilaginous bone rudiments is also inhibited (Kistler el d.. 1984). Our objective

was to hrther evaluate the effects of all-trans RA on the development of the limb and to

deterrnine ~vhether des could counteract these alI-trans RA-mediated abnormalities in fetal mice.

We first determined the day of highest susceptibiiity of the fetus to the efects of all-trans

RA on cartilage and the lowest dose of all-trans RA on that da? which yielded a teratoyenic

effect The highest 'non-teratogenic' dose of dex during three periods within linib development

was also determined. Neut. the effects of d e l in pretreatment. co-treatment and post treatment

with all-trans RA were determined and the ability of dex to act as a curative and/or preventative

agent was evaluated.

In response to a single dose of 50 mgkg all-trans RA on day 11.5 or 12.1 pc.

malformations in fëtuses included clefi palate. pre- and post-axial detlections. and foresliortening

of the distal long bones of the limbs. These limb malformations were not seen as a result of

esposure to all-trans IU on day 13.5. 11.5 or 15.5 pc. Malformations prominent in fetuses

treated on day 13.5. 14.5. or 1 S. 5 pc were clefl palate and delayed ossification of the tertiary sites

of ossification of both the humerus and femur and within the growth plates. We therefore

classified the abnormalities resulting from early exposure to all-trans F U (days 1 I . 5 and 1 1.5 pc)

as -day 1 1.5 pc-type' malformations and those resulting from late esposure to all-trans RA (days

13.5. 14.5. and I 5.5 pc) as 'day 13.5 pc-type' malformations.

We have s h o w for the first time that trearment with dex fiom da- 10.5 to 12.5 pc or day

13.5 to 15.5 pc ameliorated all-trans RA-induced day I 1 .j pc-type malformations. Specifically.

post-treatment with de'r significantly decreased all-tram RA-mediated long bone foreshonenina.

This antagonism between dex and all-trans RA rnay be related to dex-induced stimulation of

chondrogenesis following inhibition of chondrogenesis by all-trans RA. With respect to

administration of all-trans R A on day 13.5 pc. treatment with des from da- 13.5 to 15.5 pc

significantly reduced day 13.5 pc-type mslformations. Exposure to all-trans R A on day 13.5 pc

resulted in defects in growth plate morphology. which may be due to direct effects of all-tram R A

on chondrogenesis However. other day 13.5 pc-type malformations (lack of ossification of sites

on the humerus and femur) are more likely due to all-trans RA-rnediated inhibition of rnyogenesis

rather than chondrogenesis. It would be interesting to examine the effects of both day I I .i pc-

type and day 13.5 pc-type malformations on the ~iability of pups afier binh. This would address

whether dav 1 1.5 pc-type malformations are more severe than day 13 5 pc-type malformations. as

hypothesized in t his thesis.

Because the results presented in chapter 2 suggested that R4RP activated in the AER

might result in the all-trans RA-induced foreshortening obsemed with 50 mgkg all-trans RA

ziven on day 1 1.5 pc and because of the possibility that des rnay counteract this effect of ail-trans - R A by downregulating MD. we investisated FWRactivation in the limb bud of day 1 1.5 pc

embryos using a transgenic mouse mode1 canying the RAREP7 coupled to the heat-inducible

promotor hsp68 and the lac Z reporter gene (Rossant et al.. 199 1 ). We first determined in which

cells of the developin- limb bud concentrations of endogenous KA were sufficiently high to detect

a P-galactosidase (P-gal) response. and then determined if this response could be upregulated by

elvogenous all-trans M.

In ageement with previous observations (Rossant rr d.. 199 1 ). this analvsis revealed that

X-gal staining was closelv associated with regions known to express R a P . Interestingly.

however, X-y1 staining was not found in regions knorm to express both RARu and R.UQ This

can be explained since in the transgenic mouse. the transrene will be strongly srimulated bv RARP

and also by RUu and RlU-iI. although to a lesser e'ttent (Husmann rr tri. 199 1 ) Funhermore.

M i l . which binds to the R.uIE:Pî with high afinitv but only weakly activates it. may also

antagonize the effects on P-gai expression by other KARS by blocking the RAREPI (Hoffmann ci

rd.. 1990: Xagpal Cr r d . . 1992). We concluded that this makes the W h s p l a c Z trans~enic

rnouse inappropriate for the studv of the response of al1 the RARs to endogenous and esogenous

RA and/or dex since onlv tissues expressing RARP exclusively will reliably transciibe P-gal. This

transgenic mouse cannot be used to assess the effects of all-trans RA as mediated by RARP alone

either (such as in the day 1 1 .S limb bud. where the effects of all-trans RA on limb patterning and

outgrowth have been linked to R4RP2 expression) (Soprano ri L I / . . 1994) due to the possible. and

varying. expression of p--al in response to RXRu and R A R Q

In conclusion. since W ! 1 appears to have a role contrary to the rest of the RAR family

rvith respect to chondrogenesis. and possibly in other systems. it may be diffic~ilt to isolate a

response element activated by al1 8 RAR isoforms to a similar extent in al1 circurnstances I t is

suggested then. that EUR isoforms be studied individually to hlly linderstand the effects of

endogenous and exogenous RA and dex on each receptor.

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