Chapter 16- Tetrapod limb development

1. 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

2. Human development 11 weeks 5 weeks Hands 7 weeks 11weeks 5 weeks Feet

3. 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 Proteins families involved 1. ________________ (hip-toe) _______________ 2. _________________ (thumb-pinky) _______________ _________ 3. _________________ (knuckle-palm)

4. Recall from Chapter 11- Hox genes dictate _______________ axis development Fig. 11.36- Hox genes are organized in a _______________ that concurs with _________________ structures This is referred to as the ____________

5. Also recall that ____________ has a profound effect on development by activating _____gene expression Recall amphibian development (Ch. 10) Fig. 10.41 Structure of retinoic acid (not in textbook) RA RA RA receptor Retinoic acid activates mammalian ____genes Hox gene Retinoic acid bind a _______, then the complex binds promoter of a ________ Lacks all distal _______ Wild-type mouse embryo RA-treated mouse embryo

6. Another unusual observation- Experiment- Amputate tadpole tail, then soak the stump in ___ Result- Several _____ develop How does a _____develop? Limb development initiates from __________ of _____________ of _____________ mesoderm and from ___________ Limb bud _______ Somatic layer of ___________ mesoderm Fig. 14.1- mesoderm lineages

7. 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 __________________ Observations- Normal Fig. 16.5 • If Place FGF-soaked bead near forelimb bud, generate a _____ ______ • If Place FGF-soaked bead near hindlimb bud, generate a __________________ FGF bead inserted • If Place FGF-soaked bead between hindlimb and forelimb buds, generate a ___________ Thus, FGF _______ to make a limb, but tbx5 and tbx4 instruct ______ of limb

8. 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

9. Generating the ______________ axis Forelimb AER experiments Limb devel. ceases The _______ ____________ (AER) is a critical structure AER removed AER duplicated Duplicate wing AER replaced with leg mesoderm chimera • AER is required for: • ______________, • _______________ axis formation, • ______ axes specification AER replaced with other mesoderm Limb devel. ceases AER replaced by FGF bead normal Fig. 16.8

10. Generating the _____________________ axis 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) 9 10 11 12 13 Fig. 16.1 Fig. 16.14 Ulna, Radius Scalpula Digits Humerous Metacarpels Example- If KO Hoxa-11 and Hoxd–11 in mouse, no _________________ Example- Humans with defective HOX-13 display ________________

11. Specifying the anterior-posterior limb axis the proximal-distal axis What dictates thumb-pinky orientation?? 1. A group of cell located near ____________ region of limb bud- called __________________________ (ZPA) directs polarity 2. These cells in the ZPA express __________________ (Shh) Evidence …then observe second set of __________ Extra set If place Shh bead in anterior limb bud… Normal 3. Sonic hedgehog (Shh) activates __________ genes Hoxd genes specify _____________ orientation Fig. 6.15

12. 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.