AN Limb Developement

Bone & Cartilage:

Mesenchymal cells in posterior lateral plate mesoderm -> skeletal, Cartilage, dermis

Muscle:

Mesenchymal cells from somites (Paraxial Mesoderm)

Epidermis:

bud’s overlying ectoderm

Dermis:

Lateral plate mesoderm

Nerves:

Neural Crest cells

Pigment cells:

Neural Crest cells -> Migrate to skin and differentiate into melanocytes for pigmentation

Muscle precursor cells migrate from adjacent somites into the limb bud, establishing a mesenchymal cell population that proliferates under the ectoderm to create the limb bud.

-> establish limb muscle precursor cells

- Hgf acting on C-met for Epithelial to mesenchymal transition

-  Pax3 expression to define the migrating cells that develop into limp muscle, c-Met receptor expression on the future muscle cells so that they can react on the HGF signaling from the bud mesenchyme (guiding signal), FGF signaling too, and a similar mechanism is the SDF-1 signal gradient in the limp that is received by CXCR4 on the migrating cells and of course cell adhesion signaling

Limb position is influenced by an antagonistic interaction between retinoic acid (RA) and fibroblast growth factor 8 (Fgf8). RA restricts Fgf8 expression, permitting the forelimb field's initiation, while Fgf8, expressed just anterior and posterior to the forelimb field, inhibits limb formation if it is overly expressed. RA thus helps promote forelimb development by repressing Fgf8, allowing specific limb formation sites along the body axis.

The difference between forelimbs and hindlimbs is determined by the expression of specific transcription factors: —> Tbx5 specifies forelimbs, —> while Tbx4, Pitx1, and Islet1 specify hindlimbs.

Hox genes determine positional information specifies where limb bud formes

• Position der Somiten (Wirbel = Paraxial mesoderm -> aus diesen entstehen auch Muskeln) legen HOX Gene expression fest

• FGF8 begrenzt von 2 Seiten RA signal welches die Bereiche permissiv für die spezifischen Signale macht —> vorderen extremitäten durch Tbx5 und hintere durch Tbx4, Pitx1, Islet1 expression entsteht (vereinfacht)

• Epithelial to Mesenchyme transition

• FGF8 <-> FGF10 positive feedback loop (mit Wnt) zur Bildung von Extremitätenknospe (Proliferation und Wachstum)

- Begins in limb fields through expression of specific Transcription factors

- Tbx5 in limb fields of forelimbs  - anterior portion

- Islet1, Tbx4, Pitx1 expressed in presumptive hindlimbs – posterior portion of lateral plate mesoderm (downstream FGF10)

- Induction by either expressing Tbx4/TBx5 to induce ectopic limb bud

- K/o of Tbx5 -> no limbs instead put Tbx4

- Differential Hox gene pattern

4 Phases:

1) Making Mesoderm permissive for forelimb formation by RA (Anterior & posterior Fgf8 expression-inhibits forelimb bud formation, RA in somitic regions repress Fgf8 expression -> RA restricts Fgf8 expression from presumptive limb region)

2) Specifying forelimb & hindlimb

TFs Tbx5 (anterior portion -> forelimbs), Tbx4, Islet1,Pitx1 (posterior laeral plate mesoderm -> hindlimbs)

3) Inducing epithelial to mesenchymal Transition 

epithelial mesoderm of somatopleure, Tbx5 is a major regulator of EMT in the forelimb field

4) Establishing 2 (+)-feedback loops für limb bud formation

Tbx5 (forelimb) & Islet1 (hindlimb) drive mesenchymal cells to express Fgf10. Growth mainained by 2 positive feedback loops: Fgf10 induces Wnt signaling further upregulating Fgf10. Fgf10 stimulates Fgf8 in ectoderm reinforcing Fgf10    

1) apical ectodermal ridge (AER) is a thickening of the ectoderm at the apex of the developing limb bud = multipurpose signaling center is induced by Fgf10 - patternin along all axes

1. Maintaining mesenchymal proliferation for proximal-distal limb growth (prevents cartilage formation)

2. Supporting anterior-posterior axis formation (thumb to pinkie).

3. Interacting with dorsal-ventral signals to guide differentiation.

2) experiments on chick embryos:

1. removing the AER at different stages of development halted the formation of limb structures beyond that point - role in sustaining distal limb growth

2. adding extra AER onto AER - supernumerary limb structures

3. Placing leg mesenchyme under a wing AER resulted in hindlimb structures.

4. Replacing limb mesenchyme with nonlimb mesenchyme caused AER regression and stopped limb growth.

3) FGFs (e.g., Fgf8, Fgf4, Fgf9, Fgf17): Maintain mesenchymal proliferation and distal limb outgrowth and limb flattening. Fgf8 beads can substitute for the AER in inducing growth. (removing multiple Fgfs -severe skeletal malformations)

Wnt signaling: Orients mesenchymal cell polarity and promotes division, influencing limb bud shape and outgrowth.

1) Zone of Polarizing Activity (ZPA) - group of mesodermal cells in most posterior region of the progress zone of limb bud. patterns cell fates along anterior-posterior axis, crucially influencing digit formation and identity.

2) The ZPA was discovered through transplantation experiments in the 1960s, where placing posterior limb tissue on the anterior side of a chick limb bud resulted in mirror-image duplications of the digits, demonstrating the ZPA's role in axis polarization.

3) primary signaling molecule of the ZPA is Sonic hedgehog (Shh). Experiments showed that expressing Shh in non-ZPA cells could recreate the ZPA's effects, confirming its sufficiency for polarizing activity. Transplanted cells with active Shh could induce digit duplication, just as ZPA tissue does.

-          SHH enhancer inhibited in anterior region -> inhibitor mutation results in double digits too where normally the thumb would form (Shh inhibits Gli3R production = no receptor for Gli3)

-          Digit 1: Shh-independent Digit 2: Shh concentration Digit 3: Shh time of expression and concentration Digits 4–5: Shh time of expression

—> Turing Model digit skeletogenisis

dorso-ventral axis of the limb, which distinguishes dorsal features (e.g., knuckles, nails) from ventral features (e.g., pads, soles), is primarily determined by the ectoderm surrounding the limb bud    

Wnt7a and BMP signaling pathways. Wnt7a, expressed in the dorsal ectoderm, induces the expression of Lmx1b in the underlying dorsal mesenchyme - specifying dorsal cell fates

• No wnt7a -> ventral footpads on both paw sides

Conversely, BMP(from mesoderm) signaling in the ventral mesenchyme promotes the expression of Engrailed-1 (En1), which specifies ventral limb identity.

• BMP KO -> no En1 -> Wnt7a both dorsal & ventral -> dorsalized

BMP/Engrailed = Ventral und Wnt7a/Lmx1b = dorsal

Proximal-Distal (PD): AER (FGFs & WNT)

Antero-Posterior (AP): ZPA (SHH)

Dorsal-Ventral (DV): non AER ectoderm (BMPs)

-> Interlinked signaling loops ensure coordinated limb growth & termination.

• SHH (ZPA) ↔ FGFs (AER) positive feedback loop

• SHH → Gremlin1 —I BMP → FGF8 (AER maintenance)

• Wnt7a (dorsal) regulates Shh (A-P) and maintains AER (P-D)

• BMPs terminate patterning by shutting down AER, ZPA, and Wnt7a

-> regulated by feedback mechanisms: FGF & Shh positive and negative feedback

• AER-FGFs & ZPA-SHH positive feedback

• SHH induces Gremlin -> maintains FGF8 expression in AER

• Growth -> FGF levels suppress Gremlin (BMP inhibitor) -> BMP inhibits FGF production

• reduced FGF -> AER & ZPA cease

• specify limb segment identity along PD & AP axes, Regulate key signaling pathways (FGF, WNT, SHH). expression under influence from flank (proximal) and AER (distal), Hoxb8 expression forms a ZPA

• Phases of Hox gene expression:

  ELCR (Early Limb Control Region) → Broad proximal-distal expression.

  Smaller-number Hox genes (e.g., Hoxd17) in larger regions

• GCR (Global Control Region) & POST (Posterior Restriction) Regions → Refined posterior-anterior pattern.

• Hoxd13 activates Shh → Enhancer elements refine limb fate.

• Expression pattern inversion: Hoxd13 expands its domain

Reaction diffusion mechanism: 2 homogeneously distributed substances produce nonuniform pattern

• local autoactivation of morphogen A (Activator) & lateral inhibition by morphogen I (Inhibitor)

• Activator stimulates inhibitor production (ubiquitously expressed)

• Inhibitor diffuses quickly and inhibits autocatalysis of Activator (self-upregulation) but fast diffusion leads to activator peak (Differentiation) -> if inhibitor in region but no activator -> direct inhibition

Proximal-distal limb specification:

AER divides in inhibitory domain (precartilage condensation repressed) & active zone (cartilage forming)

Self-organizing digit skeletogenesis:

• BMP-Sox9-Wnt Turing network

• BMP activator of Sox9

• Wnt inhibitor

• functions under control of FGF and distal Hox genes