Chapter 16- Tetrapod limb development

“It is one thing to differentiate the chondrocytes and osteocytes that synthesize the cartilage and bone matrices; it is another thing to produce those cells in a temporal-spacial orientation that generates a functional bone.”

“How is it that the fingers form at one end of the limb and nowhere else?”

“How is it that the little finger forms at one edge of the limb and nowhere else?”

We will use limb development to examine the larger question of morphogenesis

5 weeks 11 weeks

5 weeks 7 weeks 11weeks

Feet

Hands

Human development

Morphogenic rules• These rules appear to be the same in all tetrapods

Example- Grafted mammalian limb bud will direct formation of chick limbs

• These rules must follow a _______________ coordinate system

Dimension

1. ________________ (hip-toe)

2. _________________ (thumb-pinky)

3. _________________ (knuckle-palm)

Proteins families involved

_______________

_______________

_________

Fig. 11.36- Hox genes are organized in a _______________ that concurs with _________________ structures

This is referred to as the ____________

Recall from Chapter 11- Hox genes dictate _______________ axis development

Also recall that ____________ has a profound effect on development by activating _____gene expression

Structure of retinoic acid (not in textbook)

Fig. 10.41

Recall amphibian development (Ch. 10)

RA

Retinoic acid activates mammalian ____genes

Wild-type mouse embryo

RA-treated mouse embryo

Lacks all distal

_______

Hox gene

Retinoic acid bind a _______, then the complex binds promoter of a ________

RA receptorRA

Another unusual observation-

Experiment- Amputate tadpole tail, then soak the stump in ___

Limb development initiates from __________ of _____________ of _____________ mesoderm and from ___________

Fig. 14.1- mesoderm lineages

Somatic layer of ___________ mesoderm

_______

How does a _____develop?

Limb bud

Result- Several _____ develop

What dictates forelimb from hindlimb?• Transcription factor ____ is expressed in mouse ________

• Transcription factor ____ is expressed in mouse _________

• Loss of one copy of _____ gene in humans results in __________________

•If Place FGF-soaked bead near forelimb bud, generate a _____ ______

•If Place FGF-soaked bead near hindlimb bud, generate a __________________

Observations-

Thus, FGF _______ to make a limb, but tbx5 and tbx4 instruct ______ of limb

•If Place FGF-soaked bead between hindlimb and forelimb buds, generate a ___________

Normal

FGF bead inserted

Fig. 16.5

Fig. 16.5F -Chimera limb from FGF bead placed between limb buds-

Fig. 16.5E Extra wing from FGF bead placed near forelimb bud

The apical ectodermal ridge (AER) is a critical structure

The _______ ____________ (AER) is a critical structure

AER removed

AER duplicated

AER replaced with leg

mesoderm

AER replaced with other mesoderm

AER is required for: 1) ______________, 2) _______________

axis formation,3) ______ axes

specification

Forelimb AER experiments

Fig. 16.8

Generating the ______________ axis

Limb devel.ceases

Duplicate wing

chimera

Limb devel.ceases

AER replaced by FGF bead

normal

Hox genes specify two things-1. Where the limb _______ (recall chapter 11)

2. Whether a cell will become a ________ (humerus), ________ (Ulna and radius) or an __________ (metacarpels and digits)

Fig. 16.1

Example- If KO Hoxa-11 and Hoxd–11 in mouse, no _________________

Fig. 16.14

Example- Humans with defective HOX-13display ________________

Generating the _____________________ axis

Ulna,RadiusHumerous

Scalpula

MetacarpelsDigits

9 10 11 12 13

What dictates thumb-pinky orientation??

1. A group of cell located near ____________ region of limb bud- called __________________________ (ZPA) directs polarity

Specifying the anterior-posterior limb axis the proximal-distal axis

2. These cells in the ZPA express __________________ (Shh)

If place Shh bead in anterior limb bud…

…then observe second set of __________

Extra set

Normal

3. Sonic hedgehog (Shh) activates __________ genes

Hoxd genes specify _____________

orientationFig. 6.15

Evidence

Specifying the _____________ limb axis

The surrounding ectoderm signal dorsal-ventral axis

•_____ activates ______expression in mesenchyme

•_____ is required to specify ______cell fate

•Lmx1 mutant humans have loss of _________, ________________•May also have “____________”

All three axes are ________________

Ectoderm secretes ____…

..which maintains ____ expression in ___..

..which activates ___ genes in limb bud..

..which specifies _____________ limb axis.