Development Of The Limb

Question 1

What are the 3 stages of limb development?

Answer 1

4 weeks: Undifferentiated mesenchyme.
6 weeks: Formation of skeletal elements.
11 weeks: Sculpting & rotation of limb.

Question 2

Limb bud outgrowth takes place at a particular area known as what and, that forms 3 regions from proximal to distal known as?

Answer 2

Apical ectodermal ridge (outgrowth requires epithelial-mesenchymal interactions).
Stylopod - Zeugopod - Autopod (proximal to distal).

Question 3

Where does the limb skeletal elements arise from?

Answer 3

Lateral plate mesoderm.
Limb myogenic cells arise from somite, delaminate and migrate to their destination.
Cartilage condenses in between dorsal & ventral muscle mass.

Question 4

The upper and lower limbs appear on what embryonic days?

Answer 4

Upper limb: day 24 - forms at boundary between cervical & thoracic vertebrates.
Lower limb: day 28.
Controlled by Hox genes.

Made up of ectodermal jacket with mesenchymal core.

Question 5

What signalling molecule is required for initiation of limb bud outgrowth?

Answer 5

FGF.

Absence of FGF - limbs don’t develop.

Question 6

What happens to limb outgrowth if the AER is removed early and late?

Answer 6

Since the AER is found in distal part of limb, with FGF signalling:
Remove early: limb truncate (only humerus present), due to decreased proliferation & increased cell death.
Remove late: have humerus, ulna & radius. No hand plate.

Question 7

Which FGF is critically expressed in the AER?

Answer 7

FGF8.

Question 8

What is the FGF8-FGF10 signalling loop?

Answer 8

In early limb mesenchyme, FGF8 signals to FGF10 to maintain its signalling. Leading to outgrowth of limb.

Question 9

How is the patterning of proximo-distal axis established?

Answer 9

Proximal part signals retinoic acid that forms humerus and distal part signals FGF that forms the hand. Via a signalling loop.

Hox genes expressed in nested domains along proximo-distal axis. So limb buds develop at boundary of Hox gene expression.

Question 10

What does loss of Hox11 complex lead to?

Answer 10

Lose Zeugopod, no ulna & radius.

Question 11

What does the zona polarisation activity (ZPA) control?

Answer 11

Digit formation via anterior-posterior patterning.
2 ZPA.

ZPA releases a morphogen, diffusing across limb bud forming a concentration gradient.
High concentration (cells nearest to ZPA), give posterior digits & low concentration gives anterior digits.

Question 12

What does an anterior ZPA graft set up?

Answer 12

Counter-gradient of morphogen, inducing the mirror image duplication.

Question 13

What signalling molecule is localised to the ZPA?

Answer 13

Shh localised in posterior mesenchyme of limb bud.

Hence Shh is the morphogen released by ZPA.

Question 14

What is the function of Shh signalling in ZPA?

Answer 14

Digits 3-5 arise from ZPA sequentially on dose and time and depends on Shh.
Digit 1 forms in absence of Shh.

Hence Shh is required for limb outgrowth.

Question 15

What is the Shh/gremlin/FGF signalling loop?

Answer 15

FGF8, Shh & gremlin signals to each other (interplay).
Gremlin inhibits BMP.
BMP inhibits FGF8.

Question 16

How is dorsal-ventral patterning established?

Answer 16

Dorsal expresses Wnt7a and ventral expresses engrailed, needed to maintain Shh.

Question 17

What signalling molecule determines the position and identity of limb?

Answer 17

Expression of Tbx5 forms forelimb & expression of Tbx4 (regulated by Pitx1) forms hindlimb.

Mutations in Tbx5 leads to Holt-Oram (heart/hand) syndrome.
Mutations in Tbx4 leads to Small Patella syndrome.

Question 18

How are the digits sculpted?

Answer 18

Prominent cell death in interdigital areas via apoptosis. The interdigital areas express BMP signalling, because digits express Noggin (BMP inhibitor).

Also seen in the elbow.

Question 19

What is syndactyly?

Answer 19

Persistence of soft tissue webs between digits associated with reduced death.
Due to Hoxd13 mutation - Where FGF inhibits BMP.

Question 20

How do the skeletal elements develop?

Answer 20

1. Condensation (Sox9).
2. Matrix production & perichondrium formation (Sox9).
3. Differentiation & cartilage zones established (ihh).
4. Type 10 collage.
5. Vascularisation & bone collar formation (Runx2).
6. After birth: secondary growth centre.

Question 21

What does the epiphyseal growth plate contain?

Answer 21

Reserve, proliferating & hypertrophic regions.

Question 22

What does the metaphysis contain?

Answer 22

Mineralised cartilage & trabecular bone.

Question 23

What are the features of achondroplasia?

Answer 23

Autosomal dominant & increases with paternal age.
80% de novo mutations.
Most common form of dwarfism.
Sudden death 1st year.

Features:
Short limbs, frontal bossing, midface hypoplasia, lumbar lordosis.

Question 24

What is thought to cause achondroplasia, hypochondroplasia & thanatophoric dysplasia (type 1 & 2)?

Answer 24

FGF18 signalling decreases proliferation in FGFR3 expressing chondrocytes.
Activating mutations in FGFR3 decreases chondrocyte proliferation further.
Excessive decrease in proliferation limits number of chondrocytes available to become hypertrophic and participate in bone growth.

Question 25

Why does a python have no limbs?

Answer 25

No AER.

Question 26

What on the 3 categories that congenital anomalies of the limb fall into?

Answer 26

1. Reduction defects:
   Meromelia = part
   Amelia = whole limb missing.
   Phocomelia = Section of limb missing with distal and proximal intact.
2. Duplication defects:
   Supernumerary limb elements are present (Polydactyly - presence of extra digits).
3. Dysplasia:
   Includes syndactyly (fusion of digits).
   Gigantism (excessive growth of part of limb).

Question 27

What is the ZPA regulatory sequence involved in?

Answer 27

Regulating Shh expression.

Question 28

What causes split-hand-split foot syndrome?

Answer 28

p63 mutation and localised disruption in AER.

Question 29

What are amniotic bands?

Answer 29

Abnormal embryonic membranes, that can constricts fingers, limbs and other body parts.
Affects 0.8% pregnancies.
Occurs spontaneously or in association with trauma to abdomen. There is association with early chorion villous sampling.

Causes Club Foot deformity.

Question 30

How does thalidomide cause defects which include amelia and phocomelia, as well as hypoplasia of thumb & syndactyly between thumb and index finger?

Answer 30

Causes decreased angiogenesis, increased oxidative stress & decreased growth factor signalling.