8.1 Limb Development in Chick

Question 1

The 3 Parts of a chicken forelimb and their respective borns

Answer 1

• Stylopod - humorous or femur
• Zeugopod - Radius ulna, tibia fibula
• Autopod -carpals and digits

Question 2

Direction of things in chick limb

Answer 2

distal = digits
proximal = body
poster = pinky
antieror = thumb
dorsal = knuckles
ventral = hand

Question 3

Limb bud-Apical Ectodermal Ridge (AER) formation

Answer 3

1. Induction of Precursors
2. Migration of Precursors
3. Compaction of the precursor cells to form
   tall ridge

• Ecctodermal cells and mesodermal are in limb bud.
• Ectodermal cells are in the AER which comprises of compacted cells resulting in pesudocolumuna cells (not actaully collumar cells)
• The mesoderm involved is the lateral plate meso that gives rise to bones cartilage.

Question 4

Limb bud of Chick Embryo components

Answer 4

• AER at the tip
• under Are is the progress zone
• progress zone has undifferentiating progress cells that help in formation of various regions of limb
• zone of polarizing activity
• ZPA is always at posterior end of limb bud.

Question 5

Proximal-Distal Axis Formation

Answer 5

• AER influences the underlying limb mesoderm.
• Zone immediately next to the AER is the progress zone (distal mesenchyme).
• The cells in the progress zone proliferate and are undifferentiated.
• As the cells leave this zone they begin to differentiate.

Question 6

Experiment: Effect of AER removal on Limb

Development

Answer 6

If you lose the AER really early. Then more of the proximal structures are lost.
3 days just humurous (or nothing)
3.5 forms humorous and R/U
At 4 days even more proximal structures formed

Question 7

Multi-limbed frog

Answer 7

•Early on the limb buds have the to capacity to regulate for the lost part or added parts.
• At tadpole stage the developing limb bud is infested with trematode cysts.
• Parasitic cysts splits the limb
buds in several places resulting in extra limb formation.
• Limb bud to divide into independent pieces.
Each bud generates its own limb

Question 8

Signaling and Limb formation

Answer 8

Specific signals that induce the cells of lateral plate mesoderm at specific locations in the embryonic body axis.

Activation of Hox in lateral plate of mesoderm and result in Hox protein which induces expression of retinoic acid. This activates TbX TF. This enter nuc and influences the cells to start forming fibroblastic growth factor 10 in mesodermal cells. These will induce ectodermal cells in their neighbourhood to produce wnt which will turn on Fgf8 in the ectodermal cells that result in formation of AER and its starts budding!

Hox–>Reinoic Acid–>Txb–>Fgf10 (mesoderm)–>Wnt–>Fgf8 (ectoderm)

Question 9

How are the loca-ons of the limb buds established?

Answer 9

Forelimbs: are initiated by lateral plate mesoderm secreting fibroblast growth factor 10 adjacent to somites 16-19-at the anterior margin of Hoxc6 expression, where Hoxc6 is expressed in the absence of Hoxc8

Hindlimbs: form at the posterior margin of Hoxc8 expression, where Hoxc8 is expressed in the absence of Hoxc6

Both: In the regions where both Hoxc8 and Hoxc6 are expressed, thoracic vertebrae, with ribs, form.

Question 10

Snakes No limbs

Answer 10

An extreme example of the co-expression of Hoxc8 and Hoxc6 is seen in snakes, where these genes are co-expressed along the entire animal, and all the vertebrae bear ribs, and there are no limbs!!

Rudimentary limbs: just a small
femur forms.

No sonic hedgehog expression in mesenchyme, thus no apical
ectodermal ridge!!

Question 11

Review of Signaling in limb formation

Answer 11

• Specific Hox genes regulate the synthesis of retinoic acid in areas of lateral plate mesoderm.
• Retinoic acid induces the transcription of Tbx genes.
• Tbx are transcription factors that activate the synthesis of Fgf10 in the mesodermal cells.
• Fgf10 activates the Wnt signaling pathway which induces Fgf8 expression in the ectodermal cells.
• The positive feedback between the Fgf10 in the mesodermal cells and Fgf8 in the ectodermal cells initiates limb outgrowth.

Question 12

Specification of Limb type: hind or fore?

Answer 12

Tbx4 – Hind Limbs
Tbx5 – Fore Limbs
FGF beads placed in the region where there are both Tbx4 and 5 expressed then Fgf would induce Tbx4 posteriorly and Tbx5 anteriorly leading to the formation of a chimeric limb.

Now trasplant FGF between hind and fore limb you get chimeric limb that’s half wing half leg. FgF beads is the molecule that inducing the mesoderm cells and therefore inducing ectodermal to form AER. If you put in between it forms the AER there and since it has a gradient from both Tbx4/5 it forms chimeric limb.

Question 13

Experiment: Influence of AER
What Happens if:
-AER removed
-AER removed late
-AER removed and FGF bead added?
-extra AER
-Les mesenchyme added under wing
-limb replaced with non limb mesenchyme

Answer 13

AER removed early-No limb development (may form only honours)

AER removed later - Proximal structures form

AER removed early+ FGF beads-Normal limb form

An extra AER is graꢂed onto the existing limb bud, supernumery structures form at the distal end.

Leg mesenchyme is replaced with wing mesenchyme, underneath wing AER then distal hind limb structures develop. ( We get leg bc the mesenchyme has Tbx4 expressing in it., the Tbx5 has been removed.)

Limb mesenchyme replaced with nonlimb mesenchyme beneath AER then the limb development ceases.