www.elsevier.com/locate/ydbio

Developmental Biology 265 (2004) 105–112

FLASH, a component of the FAS–CAPSASE8 apoptotic pathway,

is directly regulated by Hoxb4 in the notochord

Richard Morgan,* Alison Nalliah, and Ali S. Morsi El-Kadi

Department of Anatomy and Developmental Biology, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK

Received for publication 7 March 2003, revised 14 August 2003, accepted 16 September 2003

Abstract

The Hox genes are a family of homeodomain-containing transcription factors that confer positional identity during development.

Although their regulation and function have been extensively studied, very little is known of their downstream target genes. Here we show

that Hoxb4 directly induces the expression of FLASH in the notochord of embryos after neurulation. FLASH is a component of the FAS–

CAPSASE8 apoptotic pathway, and blocking its activity, or that of Hoxb4, prevents apoptosis in the notochord.

D 2003 Elsevier Inc. All rights reserved.

Keywords: Hox; Hoxb4; Notochord; FLASH; Apoptosis; Xenopus

Introduction

The Hox genes are a family of highly conserved,

homeodomain-containing transcription factors that confer

cellular identity both in early embryonic development and

in the adult. In vertebrates, zygotic Hox expression

begins early in gastrulation with the anterior expression

limit of each gene forming a nested series along the

anterior to posterior (anteroposterior) axis. The duplica-

tion, depletion or misexpression of individual Hox genes

frequently causes a homeotic transformation, whereby one

part of the body is replaced by another (reviewed by

Burke, 2000; Carroll, 1995; Gehring, 1988; Krumlauf,

1994).

Identifying and characterising Hox target genes is fun-

damental to understanding Hox gene function, but to date,

very few specific targets have been identified. For this

reason we have searched for genes that are directly regulated

by the Hox gene Hoxb4. This is the vertebrate homologue of

the Drosophila Deformed (Dfd) gene, which is expressed in,

and required for, the correct specification of many cephalic

segments. Deformed mutants lack maxillary and mandibular

structures, the head having been transformed to thoracic-like

0012-1606/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ydbio.2003.09.030

* Corresponding author. Fax: +44-208-725-3326.

E-mail address: rmorgan@sghms.ac.uk (R. Morgan).

structures dorsally and deleted ventrally (Merrill et al.,

1987). In addition, Dfd acts as pro-apoptotic gene during

early developmental stages, maintaining the boundary be-

tween maxillary and mandibular head lobes by inducing the

bordering cells to enter the apoptotic pathway (Lohmann et

al., 2002).

Studies in the mouse and in the frog (Xenopus) have

revealed that the expression pattern of Hoxb4 is essentially

quite similar to that of Dfd. Thus Hoxb4 transcripts are

detected in the hindbrain and spinal cord, with a sharp

boundary in the hindbrain between rhombomeres 6 and 7

(Godsave et al., 1994; Graham et al., 1988). The homo-

zygous null mutation of Hoxb4 in the mouse results in a

homeotic transformation of the second cervical vertebrae

from axis to atlas, and defective morphogenesis of the

sternum (Ramirez-Solis et al., 1993). Ectopic expression of

Hoxb4 in early Xenopus embryos results in the deletion of

structures anterior to where Hoxb4 is usually expressed

(i.e., the forebrain, midbrain and hindbrain anterior to

rhombomere 7; Hooiveld et al., 1999). Hoxb4 is also

expressed in the adult, and is required to maintain early

hematopoietic progenitor and stem cell (HPSC) popula-

tions. In contrast to the function of Dfd in early develop-

ment, Hoxb4 acts as a proliferative factor, and its

continued expression in adult HPSC populations is re-

quired to allow continued cell division and prevent differ-

entiation (Antonchuk et al., 2002; Kyba et al., 2002;

reviewed by Morgan, 2002). Hoxb4 also promotes the

Fig. 1. Differential display identifies FLASH as a possible down stream

target of Hoxb4. Hoxb4–GR (which confers dexamethasone dependence

on Hoxb4 activity) was injected into fertilised eggs and activated at

either stage 7 (blastula) or stage 10 (gastrula). Total RNA was extracted

at the tailbud stage and randomly amplified. Identical but independent

amplifications were performed to check for reproducibility. (+), Hoxb4

activated by dexamethasone at the stage indicated; (� ), no dexame-

thasone added.

R. Morgan et al. / Developmental Biology 265 (2004) 105–112106

proliferation of keratinocytes in the developing epidermis

(Komuves et al., 2002).

Attempts to identify Dfd targets have principally taken

the form of screens for mutations that genetically interact

with it (Florence and McGinnis, 1998; Gellon et al.,

1997; Harding et al., 1995). Whilst potentially allowing

many genes to be identified, this approach has the

disadvantage of also identifying genes that are not Dfd

targets at all, but instead interact with Dfd at some other

level, for example in parallel pathways or directly, as Dfd

binding partners. To date then, relatively few bona fide

Dfd targets have been identified, but these include Dfd

itself (Kuziora and McGinnis, 1988), and three other

genes that have important roles in development, Distal-

less (a transcription factor, O’Hara et al., 1993), Serrate

(an extracellular ligand, Wiellette and McGinnis, 1999),

and Reaper, a pro-apoptotic gene that has been shown to

mediate the regulation of programmed cell death by Dfd

(Lohmann et al., 2002).

Definitive experimental evidence that a gene is a bona

fide target of Hoxb4–Dfd is not easily obtained in

Drosophila or in most vertebrate models of development,

because it is difficult to manipulate Hoxb4 expression in

vivo under tight temporal control. In vitro studies using

cell culture have proved useful in this respect though,

and have provided several important findings, most

notably that two components of the AP-1 transcriptional

activation complex, Jun-B and Fra-1, are directly up-

regulated by Hoxb4, and that this is sufficient to account

for the proliferative effects of Hoxb4 over-expression

(Krosl and Sauvageau, 2000). Curiously though, Hoxb4

over-expression also blocks the expression of another

transcription factor involved in cellular proliferation, c-

myc (Pan and Simpson, 1999, 2001).

Like Dfd, Hoxb4 can also positively autoregulate

through HOXB4 binding sites in its own promoter. This

autoregulatory loop is probably highly conserved through-

out the vertebrate lineage, as it has been demonstrated in

the early development of both mice and Xenopus (Gould

et al., 1997; Hooiveld et al., 1999). Generally though, it

is studies in the frog (Xenopus laevis) that have provided

most of the examples of genes that are directly regulated

by Hoxb4 during development. This is largely because

both the up-regulation and down-regulation of Hoxb4 can

be achieved using a hormone inducible form of the

transcription factor (Hooiveld et al., 1999), and antisense

oligonucleotides (Morgan et al., 2003; Theokli et al.,

2003), respectively. Genes regulated by Hoxb4 in this

context include Rap1 (a small GTPase similar to Ras;

Morsi El-Kadi et al., 2002), and Flamingo, thought to be

a component of the Wnt signalling pathway (Morgan et

al., 2003). The expression of both of these genes is

repressed by Hoxb4, confining Rap1 and Flamingo ex-

pression to the anterior part of the nervous system. A

third Hoxb4 target is the transcription factor Irx5, which

is itself involved in early neural patterning. Unlike Rap1

and Flamingo, Irx5 is up-regulated by Hoxb4 (Theokli et

al., 2003).

Here we report that FLASH, a component of the

death-inducing signalling complex in receptor-mediated

apoptosis (Imai et al., 1999), is also a direct target of

Hoxb4. FLASH is expressed exclusively in the notochord,

where its expression is coincident with Hoxb4, and

ablating Hoxb4 RNA in this tissue results in a rapid

and specific loss of FLASH expression. Furthermore, we

show that both FLASH and Hoxb4 are required for the

apoptosis of notochord cells.

Results

FLASH and Hoxb4 have overlapping expression patterns in

the notochord

To search for down stream targets of Hoxb4, we used a

differential display technique to compare gene expression

in embryos which had developed from eggs injected with

Hoxb4 RNA to that in untreated controls. Our attention was

drawn to one transcript in particular because it was signif-

icantly more abundant in Hoxb4-injected embryos (Fig. 1).

We therefore cloned and sequenced the corresponding

cDNA from the untreated, control embryos. Conceptual

translation of the partial open reading frame encoded in this

clone gives a peptide, which is 51% identical to the murine

FLASH protein (accession numbers AY422084, AY422085;

Imai et al., 1999).

To confirm that FLASH expression is indeed up-regulat-

ed by Hoxb4, we injected fertilised eggs with Hoxb4 mRNA

and then examined the expression of FLASH later in

development, at the tailbud stage (Fig. 2). Using a similar

approach, we also examined the affect that Hoxb1, Hoxb5

and Hoxb9 over-expression have on FLASH (Fig. 2). Both

Hoxb4 and Hoxb5 result in a striking up-regulation of

FLASH, whilst both Hoxb1 (a more anteriorly expressed

Hox gene) and Hoxb9 (the most caudally expressed Hoxb

Fig. 2. RT-QPCR analysis of RNA extracted from noninjected control

(‘NIC’) or Hox-expressing embryos. Fertilised eggs were injected with

either Hoxb1, Hoxb4, Hoxb5 or Hoxb9 mRNA (as shown above each lane).

Total RNA was extracted at the neurula stage and examined for the

expression of FLASH, Hoxb4, Hoxb5, Rap1 or ef1a by RT-QPCR. Note

that the Hoxb4 PCR primers do not recognise the injected Hoxb4 RNA and

are therefore specific for the endogenous Hoxb4 transcript. Ef1a is included

as a loading control, and the ratio of target–ef1a is shown numerically for

each gene.

R. Morgan et al. / Developmental Biology 265 (2004) 105–112 107

gene) have no apparent affect on its expression. As a

control, we also studied the expression of three genes that

have previously been shown to be regulated by Hoxb4.

These are Hoxb4 itself, and Hoxb5, which are both up-

regulated by Hoxb4 (Hooiveld et al., 1999), and Rap1,

which is repressed by Hoxb4 (Morsi El-Kadi et al., 2002).

All of these genes respond in the manner suggested by these

previous reports (Fig. 2).

To determine whether the up regulation of FLASH by

Hoxb4 is reflected in their expression pattern in the embryo,

we used whole mount in situ analysis to compare their

expression. FLASH is first detected relatively late in devel-

opment, at the tailbud stage, and is restricted to the noto-

chord (Fig. 3). Hoxb4 is also expressed in the notochord of

tailbud stage embryos, in a region that is coincident with

that expressing FLASH (Figs. 3A, B). Its expression is more

wide spread than this though, being expressed also in the

neural tube and the adjacent mesoderm cells (Fig. 3; God-

save et al., 1994).

Hoxb4 is the only Deformed homologue expressed in the

notochord

Previously reported findings have indicated that the

functions of many vertebrate Hox genes may also be

mediated by closely related Hox genes, which are thought

to have arisen by two independent duplications of an

ancestral Hox cluster (reviewed by Carroll, 1995; Gehring,

1988; Krumlauf, 1994). In the case of the Deformed homo-

logue Hoxb4, three of these duplicates (or ‘paralogs’) are

found in most vertebrates, Hoxa4, Hoxc4 and Hoxd4. As

there is potential functional overlap (‘redundancy’) between

these paralogs, we decided to examine whether any of them

are expressed in the notochord of tailbud stage embryos. We

cloned the likely Xenopus homologue of Hoxd4 (accession

number AY422086; Figs. 4A, B), using redundant PCR

primers based on the highly conserved sequence of the

homeodomain. RT-QPCR analysis of RNA extracted from

isolated tailbud stage notochords (see below) revealed that

Hoxd4 is not expressed in this tissue, although it is

expressed elsewhere in the embryo at this stage (Fig. 4C).

Our cloning strategy did not find likely homologues of

Hoxa4 and Hoxc4. Furthermore, possible Hoxa4 and Hoxc4

candidates are not represented in over 70,000 publicly

available expression sequence tag clones derived from

Xenopus embryonic stages, indicating that these genes are

not expressed, at least not until after the tailbud stage. In

addition, we note that, although the mouse homologues of

Hoxa4 and Hoxc4 are expressed during early development,

they are not present in the notochord (Geada et al., 1992;

Wolgemuth et al., 1987).

The up-regulation of FLASH by Hoxb4 is direct and

independent of protein synthesis

The preceding results indicate that Hoxb4 up-regulates

FLASH expression, but they do not provide any indication

as to whether this is direct (i.e., independent of further

translation), or indirect. To address this, we used a fusion

between Hoxb4 and the human glucocorticoid receptor

(Hoxb4–GR; Hooiveld et al., 1999). The glucocorticoid

receptor binds the heat shock protein HSP90, preventing it

from entering the nucleus. This steric hindrance of nuclear

entry is relieved by ligand binding, in this case the gluco-

corticoid analogue dexamethasone (DEX), which by itself

has no discernible effects on Xenopus development (Gam-

mill and Sive, 1997). Hence the Hoxb4–GR construct

confers DEX dependence on the activity of Hoxb4 (Hooi-

veld et al., 1999).

We injected fertilised eggs with Hoxb4–GR RNA and

allowed them to develop to the tailbud stage. The embryos

were then treated with DEX in the presence or absence of

cycloheximide (CHX), which blocks protein synthesis. We

examined the expression of FLASH and Hoxb4 by RT-PCR

of RNA subsequently extracted from these embryos (Fig. 5).

Hoxb4 positively autoregulates its own expression by a

direct mechanism (Hooiveld et al., 1999), thus activating

the Hoxb4–GR construct should up-regulate Hoxb4 expres-

sion, even in the presence of cycloheximide, as indeed it

does (Fig. 5).

The activation of Hoxb4–GR by DEX results in a strong

up-regulation of FLASH expression. There is also a very

strong up-regulation of FLASH when DEX and CHX are

added together. This implies that the up-regulation of

Fig. 3. Whole mount in situ hybridisation analysis of FLASH (A, C) and

Hoxb4 (B, D) expression. (A, B) Lateral view of tailbud (stage 28)

embryos, dorsal top and posterior to right. The arrowheads mark the limits

of FLASH and Hoxb4 expression in the notochord. (C, D) Dorsal to ventral

sections through A and B, respectively, midway along the axis. The dorsal

side is uppermost. (E) Schematic view of the sections in C and D to identify

the major anatomical features. en, endoderm; nc, notochord; nt, neural tube.

R. Morgan et al. / Developmental Biology 265 (2004) 105–112108

FLASH by Hoxb4 involves a direct mechanism, at least in

part.

Blocking the expression of the endogenous Hoxb4 gene

results in a significant decrease in FLASH transcription

Previously we described the use of an antisense strategy

to block Hoxb4 translation in early Xenopus embryos

(Morsi El-Kadi et al., 2002). This makes use of a ‘mor-

pholino’ oligo (MO), which is chemically modified to

prevent its degradation by endogenous nucleases. The

MO was injected into fertilised eggs and is subsequently

distributed throughout the embryo where it can block

translation of Hoxb4 RNA by binding specifically to the

ATG start codon and surrounding nucleotides (Morsi El-

Kadi et al., 2002).

We chose to use a similar approach to see whether

blocking Hoxb4 translation in the notochord also blocks

FLASH transcription. Unfortunately though, this approach

is not practical for many reasons, including the highly

variable segregation of the MO into later notochord cells,

and the difficulty in interpreting any results in the back-

ground of a Hoxb4-negative embryo. For this reason we

chose to dissect the notochord from live tailbud stage

embryos and culture them in the presence of the MO

(Fig. 6). Subsequently, total RNA was extracted from the

isolated notochords and gene expression was analysed by

RT-QPCR (Fig. 6). Notochords cultured with the anti-

Hoxb4 MO showed a significant reduction in Hoxb4

expression, as previously described in whole embryos

(Morsi El-Kadi et al., 2002). This is probably a conse-

quence of the autoregulatory properties of this gene

(Hooiveld et al., 1999). Likewise Hoxb5, a direct regula-

tory target of Hoxb4 (Hooiveld et al., 1999), is also down-

regulated (Fig. 6), whilst the expression of Hoxb9 is

unaffected. Rap1 has previously been shown to be down

regulated by Hoxb4 in whole embryos (Morsi El-Kadi et

al., 2002), and its expression is activated in isolated

notochords by the antisense Hoxb4 MO (Fig. 6). Hence

the ectopic application of the Hoxb4 MO on notochords

has the same affect on gene transcription as it does when

introduced into whole embryos by microinjection.

As expected from its expression pattern, FLASH tran-

scripts are detected by RT-PCR in isolated, untreated

notochords (Fig. 6). The application of the antisense

Hoxb4 MO results in a significant reduction in the

amount of FLASH mRNA present in notochords, whilst

no affect is observed when the control antisense MO is

used.

Both Hoxb4 and FLASH are required for apoptosis of

notochord cells

The notochord is a transient structure, and significant

portions of it do not form any identifiable structure in the

adult. In addition, we have shown here that it specifically

expresses FLASH, a component of the FAS-mediated cell

death pathway. For this reason we examined whether

notochords from tailbud stage embryos undergo apoptosis,

using whole mount TUNEL staining. This procedure entails

labelling the short DNA fragments that result from the

fragmentation of chromosomes, a highly characteristic,

and terminal, event in apoptosis.

Whole mount TUNEL staining of isolated, untreated

notochords reveals a significant degree of apoptosis in this

structure (Fig. 6B). To determine whether this was depen-

dant on FLASH, we treated notochords with an antisense

oligo that blocks its translation. This results in a very

marked reduction in apoptosis. Likewise, notochords treated

with the anti-Hoxb4 morpholino oligo also show reduced

apoptosis, whilst the control morpholino oligo has no

significant effect (Fig. 6B).

Discussion

The function of Hox genes in the establishment of the

anteroposterior axis, both along the body and subsequent-

ly in the limbs, are among some of the most extensively

studied problems in developmental biology. Later devel-

opmental functions of Hox genes are somewhat less well

characterised though, due in part to the difficulty in

studying late Hox defects in isolation. At the simplest

level, the function of Hox genes is to activate or repress

Fig. 4. Hoxd4 is not expressed in the tailbud stage notochord. (A, B) Sequence comparisons of Xenopus HOXD4 and HOXB4 proteins compared to those from

other species, as shown. The Genebank accession numbers are shown next to each gene name. (A) The N-terminal part of the homeodomain. (B) The N-

terminal part of the HOX protein starting from amino acid number 20. (C) RT-QPCR analysis of RNA extracted from both isolated notochords and whole

embryos at the tailbud stage. Ef1a is included as a loading control, and the ratio of target to ef1a signal is shown for each gene. Dr, Danio rerio (Zebrafish); Hs,

Homo sapiens (human); Mm, Mus musculus (mouse); Xl, Xenopus laevis (frog).

R. Morgan et al. / Developmental Biology 265 (2004) 105–112 109

a large group of target genes that together specify the

identity and fate of those cells that express them. Here

we have shown that the late expression of Hoxb4 in the

notochord directly activates the transcription of the

FLASH gene, and that the expression of Hoxb4 and

FLASH are coincident within the notochord.

FLASH contains a death-effector-domain-recruiting do-

main (DRD) through which it can bind to the death-

effector-domain (DED) of capsase 8, a component of the

tumour necrosis factor (TNF) mediated apoptosis pathway

(Imai et al., 1999). TNF fails to induce apoptosis in the

absence of FLASH, indicating that it is an essential

component of this pathway. FLASH is also required for

the anti-apoptotic effects of TNF that occur in some cells,

where it participates in the activation of the NF-kB

transcription factor (Choi et al., 2001). The data pre-

sented here though suggests that FLASH acts as a pro-

apoptopic gene in the notochord, as blocking its transla-

tion significantly reduces the number of apoptopic cells

(Fig. 6). This is particularly interesting, especially as the

onset of FLASH transcription occurs only at this relative-

ly late developmental stage. In developmental events that

occur before the tailbud stage, the notochord has a key

role in establishing the dorsal ventral patterning of the

overlying neural tube and somites (reviewed by Altmann

and Brivanlou, 2001; Cossu et al., 1996). By the tailbud

stage though, this pattern has been established and is no

longer dependant on interactions with the notochord.

Relatively little is known about the fate of the notochord

subsequently, except that significant portions of it prob-

ably degenerate, with the remainder going to form the

tissue of the intervertebral discs (the nuclei pulposi;

reviewed by Gilbert, 2000). The tailbud stage notochord

is thus in a transitory state between an active signalling

centre and what is essentially a redundant, regressive

structure. FLASH expression may then be part of a new

programme of gene transcription that realises this change;

it will be interesting to see whether other Hoxb4 target

Fig. 5. RT-QPCR analysis of RNA extracted from control (‘untreated’) or

Hoxb4–GR expressing embryos. The embryos were treated with

dexamethasone (DEX) and cycloheximide (CHX), either alone or in

combination, as shown. Ef1a is included as a loading control, and the ratio

of target to ef1a signal is shown for each gene.

R. Morgan et al. / Developmental Biology 265 (2004) 105–112110

genes are specifically regulated in the notochord at the

same time.

In this context it is note worthy that the pro-apoptotic

gene reaper is directly up-regulated by Deformed (Dfd),

the Drosophila homologue of Hoxb4 (Lohmann et al.,

2002). Dfd maintains the boundary between maxillary

and mandibular head lobes by inducing the bordering cells

to enter the apoptotic pathway. Hence there seems to be a

clear functional parallel between the role of Dfd and

Hoxb4, in that both can activate apoptosis in contexts

where redundant embryonic structures need to be removed.

We should perhaps be cautious in taking this parallel too

far however, as the notochord bears no embryonic rela-

tionship to the cells that separate the head lobes in

Drosophila, and in addition, Dfd activates an apoptopic

pathway, via modulation of the reaper gene, that is distinct

from that activated by Hoxb4.

Whilst there is a clear similarity between the function

of Dfd and Hoxb4 in the context of the late develop-

ment of the notochord, it is notable that this is at

variance with other reported functions of Hoxb4 in

vertebrates, where its expression increases cell prolifer-

ation (Antonchuk et al., 2002; Komuves et al., 2002;

Kyba et al., 2002). How can this apparent paradox be

explained? It is worth reiterating that although Hoxb4 is

expressed in both the posterior neural tube and meso-

derm, as well as the notochord, FLASH expression is

confined to the latter. This would indicate that addition-

al, notochord specific genes have to be expressed to

activate FLASH expression, and thus Hoxb4 activity is

specifically modified in this particular context. There are

a few examples of genes that are only expressed in the

notochord at the tailbud stage, a particularly poignant

one being the Brachyury transcription factor (Hayata et

al., 1999; Smith et al., 1991). Further studies of the

regulatory interaction between genes in notochord de-

velopment will undoubtedly aid our understanding of

this fascinating problem.

Experimental procedures

Differential display analyses

Fertilised Xenopus eggs were injected with 500 pg of

Hoxb4–GR RNA. Dexamethasone was added at either

stage 7 or 11, and the embryos were then allowed to develop

until stage 28. At this point total RNA was extracted and 1

Ag was used to make cDNA by reverse transcription using a

poly-deoxythymidine primer (T15). Two percent of this

reaction was then randomly amplified by PCR using a

single primer (5VCAG ATT GGT GCT GGA TAT GC 3V),with 2 rounds of amplification at 94jC, 45jC and 72jC for

30, 30 and 60 s, respectively, and then 30 rounds of

amplification at 94jC, 60jC and 72jC for 30, 30 and 60

s, respectively. The PCR products were resolved by elec-

trophoresis on 2% agarose for 4 h at 200 V (4jC) and

visualised by ethidium bromide staining. Differentially dis-

played bands of interest were cut out the gel and the PCR

products were extracted by Qiaquick PCR Purification Kit

(Qiagen) and eluted in 50 Al water. The purified PCR

products were PCR-re-amplified and gel-purified if neces-

sary, cloned into pGEM-T easy vector (Promega), and

sequenced.

RNA extraction and RT-QPCR

Total RNAwas extracted from whole embryos or isolated

notochords using an RNeasy mini kit (Qiagen). One micro-

gram of RNA was used in subsequent reverse transcription

reactions. This was mixed with a poly-T15 oligo to 5 Ag/ml

and heated to 75jC for 5 min. After cooling on ice, the

following additional reagents were added; dNTPs to 0.4

mM, RNase OUT (Promega) to 1.6 U/Al, Moloney Murine

Leukemia Virus Reverse Transcriptase (M-MLRvT) Rna-

seH-point mutant (Promega) to 8 U/Al and the appropriate

buffer (supplied by the manufacturer) to �1 concentration.

The mixture was incubated for 1 h at 37jC, heated to 70jCfor 2 min and cooled on ice.

QPCR reactions were all performed in a total volume

of 25 Al. For each we used 1 Al of the M-MLRvT

reaction (as described above), 0.2 nmol of each primer

and 20 Al of pre-mixed QPCR components (Sigma). All

reactions were cycled at 94jC, 60jC and 72jC for 30,

30 and 60 s, respectively, for 30 cycles. The primers

used for FLASH amplification were as follows: forward-

FLASHU: 5VGAA CCC GTG CAA GCT GTT AT 3Vandreverse-FLASHD: 5VCTC CCC TCA GAT TCA TGC TC

3V, and for Hoxd4 amplification: forward-HOXD4U: 5VACC CCC AGC CAT AGT TTA CC 3V and reverse-

HOXD4D 5VGTG GAT CTG CCT TTG GTG TT 3V. Thesequences of the other primer pairs can be found on the

Fig. 6. Blocking Hoxb4 translation results in the reduced expression of

FLASH, and blocks programmed cell death in the notochord. (A)

Isolated notochords were cultured either in 0.1 MMR buffer either

alone (1), with an antisense Hoxb4 morpholino (2), an antisense FLASH

morpholino (3), or a control (scrambled sequence) morpholino (4). Total

RNA was extracted from the notochords after 3 h and analysed for the

expression of FLASH, Rap1, Hoxb4, and Hoxb5 by RT-QPCR. The

ratio of each gene is expressed as a proportion of the loading control

signal (ef1a). (B) Whole mount TUNEL analysis for programmed cell

death in the notochords treated as described in part A (1–4). Scale bar:

0.25 mm.

R. Morgan et al. / Developmental Biology 265 (2004) 105–112 111

internet at http://www.sghms.ac.uk/depts/anatomy/pages/

richhmpg.htm/.

QPCR was performed using the SYBR green labelling

kit from Sigma, using ROX as the internal standard dye.

Thermal cycling and fluorescence detection was by a

MX4000 (Stratagene Inc., USA). Semi-quantitative data

were obtained by using measurements three cycles after

reactions had risen above the base line, and where clearly in

exponential increase. Ef1a was used as a loading control,

and all values are presented as a ratio of target to ef1a

signal.

Embryo culture and microinjection and notochord dissection

Culture and microinjection were performed as de-

scribed (Sive et al., 2000). Notochords were removed

from stage 28 (tailbud) embryos as follows: (1) Using a

scalpel blade, make two dorsal to ventral bisections half

way along the axis to give a ca. 2 mm section. The

notochord is clearly visible in section. (2) Cut beneath the

notochord to remove the bulk of the endoderm and

mesoderm ventral to it. (3) Cut dorsally to the notochord

to remove most of the neural tube. (4) The cells lateral to

the notochord (mainly ectoderm) can now be removed

easily using forceps.

Whole mount in situ hybridisation

FLASH was cloned into vector pGEMT-easy (Promega),

and this was linearised using NcoI. A DIG-labelled in situ

probe was transcribed from this template using SP6 poly-

merase. A DIG-labelled Hoxb4 probe was transcribed as

previously described (Godsave et al., 1994). Probe purifi-

cation and subsequent in situ analysis were performed as

described (Sive et al., 2000).

TUNEL analysis of stage 28 Xenopus notochords

This was done as previously described for whole mount

TUNEL analysis in Xenopus embryos (Hensey and Gautier,

1997).

Cycloheximide and dexamethasone treatments of

Hoxb4–GR injected embryos

These were performed as described (Gammill and Sive,

1997). Embryos were incubated with cycloheximide for 30

min before the addition of dexamethasone. RNA was

extracted from embryos 2 h after dexamethasone treatment,

by which point the untreated control embryos had reached

stage 25.

Antisense MO oligo treatment of stage 28 Xenopus

notochords

The notochords were cultured for 1 h in 0.1 � Marc’s

Modified Ringers (MMR; Sive et al., 2000; Ubbels et al.,

1983) at 18jC. Antisense or control oligos were added to a

total concentration of 3 nmol/ml. The sequence of the

morpholino oligos were: anti-Hoxb4 5V TGA TCA AAA

ACG AAC TCA TTC TCA T 3V, anti-FLASH 5VCTC CCC

TCA GAT TCATGC TC 3Vand ‘control’ 5VTGATCT TTT

TGC TTG ACT AAG TCA T 3V. They were synthesised by

Gene Tools LLC (USA).

R. Morgan et al. / Developmental Biology 265 (2004) 105–112112

Acknowledgments

The authors would like to thank St. George’s Hospital

Medical School for their support.

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