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Limb Development Flashcards

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1
Q

describe the structure of the early limb bud

A

consists of an ectodermal epithelial jacket surrounded by a mesenchymal core of loosely packed mesenchymal cells

dorsal tip of limb bud has a specialised structure - apical ectodermal ridge/AER
- composed of pseudostratified epithelial cells

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2
Q

the apical ectodermal ridge (AER) is a structure at the dorsal tip of the limb bud - where does it arise from? significance?

A

apical ectodermal ridge/ AER - composed of pseudostratified epithelial cells

arises from the distal ectoderm following the physical movement of epithelial cells - physically marks the boundary between dorsal and ventral surfaces

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3
Q

What is the lateral plate mesoderm’s role in limb initiation?

A

lateral plate mesoderm specifies the region for limb formation by accumulating mesodermal cells at specific sites along the body axis - guided by intersecting gene expression domains

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4
Q

what are the intersecting gene domains that mark where lateral plate mesodermal cells migrate to for forming limb buds? (along different axis)

A

rostro-caudal axis:
- Hox genes (e.g., Hoxb8, Hoxc6, Hoxc8) define where along the body the limb bud forms = positional identity of forelimbs & hindlimbs
- Tbx4, Tbx5, Pitx1 = forelimb vs hindlimb identities (& hindlimb traits from Pitx1)

DV axis
- Wnt7a from dorsal ectoderm/ En-1 from ventral ectoderm repressing dorsal genes

PD signals
- RA from somites initiates limb field formation, induces FGF10 in lateral plate mesoderm
- FGF10 induces bud growth with mesenchymal proliferation & positive feedback loop with FGF8 in AER

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5
Q

How do the AER, ZPA, and Wnt7a interact in limb patterning?

A

AER promotes PD growth via FGF signalling

ZPA (posterior limb bud mesenchyme) produces Shh = establishes AP patterning & controls digit identity.

Dorsal ectoderm releases Wnt7a = promotes dorsal identity for DV patterning

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6
Q

What do Hox genes primarily control in limb development in limb bud initiation?

A

Hox genes specify positional identity of limbs along rostro-caudal axis
- define where forelimbs and hindlimbs will form with colinearity in Hox gene expression
- e.g. Hoxb8/c6 mark forelimb region; Hoxc9 specifies hindlimb region

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7
Q

How do Hox genes and Tbx 4 & 5 work to specify forelimb-hindlimb identity?

A

Hox genes establish forelimb/hindlimb positional identity along body axis

Tbx4 & 5 specify the type of limb (forelimb or hindlimb) in those regions
- Tbx5 = forelimb
- Tbx4 = hindlimb
- Pitx1 = hindlimb characteristics

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8
Q

How do Wnt7a and En-1 control dorso-ventral (DV) patterning?

A

Wnt7a from dorsal ectoderm promotes dorsal patterning
En-1 from ventral ectoderm represses dorsal gene expression in ventral domain (repressive TF)

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9
Q

What is the significance of retinoic acid (RA) in limb initiation?

A

RA induces Meis expression for proximal limb formation early on

later on RA induces Hand2 - leads to FGF10 expression = important for ZPA formation and mesodermal proliferation for limb PD outgrowth

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10
Q

What would happen in the absence of RA (e.g., in Raldh2-/- embryos)?

A

RA deficiency = failure of forelimb formation
- genes like Hand2, Tbx5, and Meis cannot be expressed = leads to absence of limb buds

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11
Q

What is the function of FGF10 in limb bud initiation?

A

FGF10 induces the mesodermal cell proliferation in the lateral mesoderm - initiates limb bud PD outgrowth
- absence of FGF10 = no limb buds form

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12
Q

How does the FGF10-FGF8 feedback loop function in limb development?

A

FGF10 signalling initially induced by RA signalling from somites & Tbx4 and 5

FGF10 in mesoderm signals to ectoderm - induces FGF8 in AER
- FGF8 maintains FGF10 in mesoderm
- positive feedback loop where FGF8 & 10 sustain each other = promote limb PD outgrowth
- FGF10 important for mesodermal proliferation
- FGF8 promotes cell proliferation in progress zone underlying AER

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13
Q

What happens if the AER is removed during limb development?

A

no AER = no distal limb growth
- no FGF8 signal, no sustained FGF10 signals
- no mesodermal proliferation at distal limb bud

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14
Q

What occurs when FGF10-soaked beads are implanted into the flank region of embryos?

A

formation of supernumerary limbs in the flank region - shows the flank is competent to form limbs when exposed to specific signals like FGF10

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15
Q

How does Tbx5 affect forelimb development?

A

Tbx5 crucial for forelimb development and AER formation
- no Tbx5 = no forelimb development/structures

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16
Q

What is the evolutionary significance of Tbx4 and Tbx5?

A

both evolved from a common ancestral Tbx4/5 gene - divergence helps maintain forelimb vs hindlimb identity

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17
Q

What is the primary role of Tbx4 in limb development?

A

needed for hindlimb development - mainly expressed in hindlimb mesenchyme
- no Tbx4 = defective hindlimb development

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18
Q

How does Pitx1 contribute to hindlimb identity?

A

Pitx1 is expressed in hindlimb mesenchyme - critical for the specification of hindlimb characteristics
- no Pitx1 = abnormal hindlimb identity, affected development

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19
Q

How do Tbx4 and Tbx5 functionally compare in limb development?

A

Tbx4 and Tbx5 are interchangeable in driving initial limb outgrowth - both can induce FGF10 in mesoderm
- Tbx5 specific to forelimbs; Tbx4 is specific to hindlimbs
- Tbx5 KO mice can be rescued by expressing Tbx4 in the forelimb region

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20
Q

What happens in Tbx5 KO mice when Pitx1 is expressed in the forelimb?

A

Tbx5 KO mice with Pitx1 expression in the forelimb develop hindlimb-like features in the forelimb
- shows Pitx1 imparts hindlimb characteristics but doesn’t convert the forelimb into a hindlimb

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21
Q

What role do Msx1 and Msx2 play in limb development?

A

Msx1/2 - marker TFs expressed in the AER
- support AER formation and function of distal limb outgrowth

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22
Q

Why do snakes fail to develop full limbs despite the presence of early limb bud-like structures?

A

snakes form rudimentary limb buds but:
- lack Msx1/2 TF expression = markers for AER; maintain AER formation and function for distal limb outgrowth
- disrupted Wnt7a and Shh signalling
prevents limb development along all three axes - snakes lose limbs

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23
Q

Describe the feedback loop involving Wnt7a, Shh, and FGF signalling in limb development. Why is this loop important?

A

Wnt7a (dorsal ectoderm) induces Shh expression in the posterior mesenchyme (ZPA)

Shh promotes FGF8 (AER) - FGF8 promotes + sustains FGF10 in mesenchyme, creating a positive feedback loop between FGF8 & 10

positive feedback loop ensures sustained limb outgrowth and coordinated patterning along the three axes - if disrupted = limb loss (like in snakes)

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24
Q

what determines DV limb identity/polarity?

Study These Flashcards
A

Wnt7a expressed in dorsal ectoderm - induces Lmx1 in underlying mesenchyme to establish dorsal identity

En-1 induced in ventral ectoderm - repressive TF against Radical Fringe & Wnt7a (dorsal-specific genes) - repression maintains DV polarity

25
Al-Qattan Palmar Duplication Syndrome in humans is seen with the loss of Wnt7a? what are some expected characteristics of this syndrome?
loss of dorsal genes - double ventral phenotype occurs - loss of dorsal features like nails - palms appear on both sides of limbs
26
How is Shh expression in the ZPA linked to DV patterning?
Wnt7a in dorsal ectoderm induces Shh in ZPA - Shh then induces Hand2 to allow for Wnt7a function in dorsal ectoderm
27
What is the role of En-1 in DV patterning?
repressive TF expressed in ventral ectoderm - prevents Wnt7a and Radical Fringe expression in ventral territories = ensures DV polarity
28
What is Radical Fringe, and how does it help define the DV boundary?
Radical Fringe - Notch signalling modulator - RFng expression induced dorsally by Lmx1 (from Wnt7a) & repressed ventrally by En-1 - reinforces DV boundary
29
what does the Progress Zone model of PD patterning propose? why is this a limited model?
mesoderm cell fate for limb formation is **determined by time spent in the progress zone** under AER influence - longer time = more distal identity however, this model doesn't explain positional plasticity of cells - early limb cells can still adopt distal fates when transplanted
30
What does the Early Specification Model of PD patterning suggest? why is it limited?
limb cells are pre-patterned early; position is fixed once cells leave the progress zone however, there's no evidence of distinct gene territories supporting this pre-patterned cell fate & doesn't explain positional plasticity
31
What is the Two-Signal Model of PD limb patterning?
PD identity is determined by opposing gradients of two signals: - RA from proximal flank/ somites = proximal signal (for stylopod) - FGF8 from distal AER = induces Hoxa11 (for zeugopod) and Hoxa13 (for autopod)
32
How does RA influence PD patterning? (two-signal model)
RA produced by somites/ proximal body flank - induces Meis1 as a proximal marker & promotes proximal fates (like the stylopod)
33
How does FGF8 from the AER affect PD identity? (two-signal model)
FGF8 from AER: - represses Meis1 expression - induces expression of an enzyme that degrades RA = lower conc. of proximal signal in distal limb bud - induces expression of Hoxa11 (for zeugopod) and Hoxa13 (autopod) - sustains progress zone, keeping cells undifferentiated and proliferative
34
What roles do FGFs play in PD limb development?
FGF10 - in mesenchyme; induces FGF8 in AER, crucial for limb initiation FGF8 - in AER; maintains outgrowth, supports PZ later FGFs (4,9,17) - promote digit development (autopod fate)
35
what region of the limb bud controls anterior-posterior (AP) patterning? how is it involved in limb development?
ZPA (in posterior mesenchyme) controls AP patterning by secreting Shh - Shh acts as a morphogen to **specify digit identity** based on its 1) concentration gradient and 2) duration of exposure
36
What happens if ZPA tissue is grafted to the anterior limb bud? What does it prove?
anterior grafting of ZPA = mirror-image duplication of more posterior digits shows ZPA emits positional info - Shh from ZPA is enough to specify posterior digit identity
37
How does Shh act as a morphogen in limb AP patterning?
Shh diffuses from ZPA - forms a graded signal across limb bud (posterior to anterior) - conc grad & exposure time determines digit identity - high Shh, prolonged exposure = more posterior digits form (e.g. digit 5/ pinkie) - low Shh, little exposure = more anterior digits form (e.g. digit 1/ thumb) spatial-temporal gradient provides positional identity to limb mesenchyme
38
How is Shh expression initiated and regulated in the limb bud?
RA in lateral plate mesoderm/somites induces Shh expression in posterior limb bud - RA induces dHand = activates Shh transcription - FGF8 from AER maintains Shh via feedback loop (FGF8 > Gremlin (BMP intermediate > Shh) - Hoxb8 restricts Shh expression to the posterior mesenchyme - Wnt7a from the dorsal ectoderm maintains ZPA activity
39
What is the role of Gli3 in AP digit patterning, and how does Shh interact with it?
Gli3 - TF processed into either Gli3R or Gli3a depending on presence of Shh - presence of Shh - Gli3 becomes Gli3A - Gli3A accumulates in posterior limb - establishes a **Gli3A: Gli3R** gradient between **posterior: anterior** regions (high Shh = Gli3A in posterior regions; low Shh = Gli3R in anterior regions) **Gli3R restricts digit formation in anterior region** - loss of Shh = high Gli3R = loss of all digits except digit 1
40
what happens with digit formation if Shh is knocked out?
Shh lost = only digit 1 forms, as it is Shh-independent other digits are lost due to widespread Gli3R-mediated repression
41
what happens with loss of Gli3 expression? what does this tell us about Gli3 importance/function?
Gli3 lost = too many digits form (polydactyly) without defined identities Gli3 limits digit number and is needed for correct patterning
42
How do BMPs contribute to digit identity and separation?
BMPs (2,4,7) expressed in interdigital mesoderm - act downstream of Shh BMPs reinforce digit identities (especially posterior digit identity) & trigger apoptosis to separate digits
43
BMPs are expressed in interdigital mesoderm - they're important for posterior digit identity & triggering interdigital apoptosis. what happens if: a) BMP is blocked by an antagonist - e.g. Noggin b) interdigital tissue is removed
a) BMP blocked = no interdigital apoptosis or posterior-specifying activity = digit fusions (syndactyly) and anterior digit transformations b) removing interdigital tissue = identity of all digits shifts anteriorly (noticeable in more posterior digits)
44
How do digits physically form during development?
mesenchymal condensation forms digital rays within autopod **BMP-mediated interdigital apoptosis** sculpts the spaces between future digits; **Noggin (BMP antagonist)** spares digital regions from apoptosis necrotic zones in the anterior, posterior, and interdigital regions help shape the final limb structure
45
What molecular mechanisms shape limb morphology diversity among tetrapods (e.g., ducks vs horses)?
Shh duration - shorter exposure = fewer digits BMP activity - increased apoptosis = loss of digits (horse hoof has 2 digits) Ptch1 expression (Shh receptor) - reduced Ptch expression = fewer digits
46
What is endochondral ossification? why is it important?
endochondral ossification = process by which mesenchymal tissue differentiates > cartilage > bone - important for long bone formation - post-natal bone growth via epiphyseal growth plates
47
What are the main steps of endochondral ossification?
1. mesenchymal condensation = mesenchymal cells condense & secrete cartilage ECM 2. chondrocyte differentiation = condensed mesenchymal cells become chondrocytes - form cartilage template 3. chondrocyte hypertrophy & apoptosis = central chondrocytes enlarge, begin matrix mineralisation, then die 4. matrix mineralisation = cartilage matrix calcifies, forming a scaffold for bones 5. vascularisation = VEGF signals induce blood vessel invasion 6. bone formation = osteoblasts replace cartilage with bone matrix 7. epiphyseal growth plates = cartilage remains at bone ends to allow longitudinal growth
48
What marks the initial site of ossification in long bones?
diaphysis (central bony shaft) is the primary site of ossification - where mineralisation, vascular invasion & bony deposition first begin epiphyses (bony ends) retain cartilage as growth plates
49
What is the role of the epiphyseal growth plate?
regulate longitudinal bone growth during development - are cartilage zones with zones of proliferating and maturing chondrocytes ossify and growth stops at approx. age 20
50
What molecular signals regulate chondrocyte differentiation and ossification?
Sox9 = for chondrocyte differentiation Indian Hedghog = co-ordinates chondrocyte proliferation, cartilage maturation, and vascular invasion VEGF = signals vascular invasion of hypertrophic cartilage BMP = chondrocyte maturation & cartilage ECM production FGFs = regulate chondrocyte proliferation
51
What is the function of Sox9 in skeletal development? what happens when it is mutated?
Sox9 = TF for chondrocyte differentiation - important for cartilage development mutations lead to campomelic dysplasia = condition of underdeveloped cartilage and skeletal abnormalities
52
How does VEGF contribute to endochondral ossification?
VEGF = **promotes vascular invasion of hypertrophic cartilage** - osteoclasts & -blast precursors enter and remodel matrix into bone
53
How do BMPs and FGFs influence ossification?
BMPs = stimulate chondrocyte maturation & cartilage matrix production FGFs = regulate chondrocyte proliferation
54
What clinical disorders are associated with defects in endochondral ossification?
Chondrodysplasias = from abnormal cartilage development or vascularisation (e.g., VEGF issues) Campomelic dysplasia = Sox9 mutations; leads to defective cartilage and skeletal deformities Achondroplasia = FGFR3 mutations, affects chondrocyte proliferation; results in short stature and shortened long bones
55
What limb defect is typically caused by thalidomide exposure during development?
phocomelia - when proximal limb elements are absent & distal elements may still form
56
How does thalidomide affect FGF signalling during limb development?
thalidomide inhibits NF-κB - necessary for FGF10 expression in mesenchyme - disrupts FGF10-FGF8 positive feedback loop - impairs limb bud outgrowth & mesenchymal proliferation (especially proximally)
57
Why does thalidomide cause more severe loss of proximal limb elements than distal ones?
induces early defects before FGF8(AER)-FGF10 feedback loop is fully established inhibits SALL4 - TF crucial for anterior and proximal identity Shh-regulated posterior elements are lest affected
58
Why might posterior and distal limb elements still form in thalidomide-exposed embryos?
- posterior elements are patterned by Shh - distal elements are supported by residual AER signalling these regions are less dependent on SALL4, the primary target disrupted by thalidomide-bound cereblon - anterior & proximal elements (with less Shh influence & more FGF8-FGF10 loop reliance) are more affected
59

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