AN Limp Developement

-          derived from the posterior lateral plate mesoderm -> skeletal, Cartilage, dermis

-          Paraxial Mesoderm (somites) -> muscle

-          bud’s overlying ectoderm -> epidermis

-          Neural Crest cells and neural tube -> nerves

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

-          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

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.

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.

• 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)

-          Tbx5 marks forelimb buds, while Islet1, Tbx4, and Pitx1 marks hindlimb buds (downstream of these TF (DNA binding domains) is FGF10 for growth)

-          Different HOX Gene expression

-          The apical ectodermal ridge (AER) is a thickening of the ectoderm at the apex of the developing limb bud, a multipurpose signaling center

-          Transplantation experiments. -> removed: limp development ceases, grafted onto another limp bud: multiple limps … / skeletal Elements form one After another

-          FGF8 for limp growth / elongation we know because normal development when FGF bead is replaced for AER, also FGF expression or KO studies

-          The cells found within the most posterior region of the progress zone constitute the zone of polarizing activity (ZPA), since it patterns cell fates along the anteriorposterior axis. The zone is defined by SHH signaling

-          When a ZPA is grafted to anterior limb bud mesoderm, duplicated digits emerge as a mirror image of the normal digits

-          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

Back to Palm Axis

BMP/Engrailed = Ventral und Wnt7a/Lmx1b = dorsal

Patterning along the dorso-ventral axis of the limb is governed by the Wnt7a and BMP signaling pathways. Wnt7a, expressed in the dorsal ectoderm, induces the expression of Lmx1b in the dorsal mesenchyme, which specifies dorsal cell fates. Conversely, BMP signaling in the ventral mesenchyme promotes the expression of Engrailed-1 (En1), which specifies ventral limb identity. In Wnt7a-deficient mice, dorsal structures are absent, resulting in ventralized limbs with ventral characteristics on both surfaces, which highlights Wnt7a’s crucial role in dorsal patterning​.

Engrailed limits the expression of Wnt7a on the ventral side.

-          Shh from the ZPA supports AER maintenance, coordinating anterior-posterior with proximal-distal growth.

-          Shh —> Gremlin —I BMP (to regulate FGF8)

-          FGFs from the AER influence Shh expression in the ZPA, ensuring proper growth and patterning along the thumb-to-pinky and shoulder-to-fingertip axes.

-          Wnt7a in the dorsal ectoderm also helps maintain the AER, integrating dorsal-ventral with other axis signals.

-          Shh Expression ist von der Präsenz einer intakten AER abhängig und wird durch FGF Protein erhalten

-          Feedback inhibition (Shh from ZPA)

-          Gradients from AER (FGF) or ZPA (Shh)

-          Temporal and special (cellular response) regulation

-          Apoptosis to form the limp shape but also limit the size of limp bud

-          Determination of limp segments, regional identity

-          contribute to A-P and P-D patterning by regulating the expression of other signaling molecules, such as FGFs, WNTs and Shh (mainly HOXd region)

-          Hoxb8 expression forms a ZPA and dictates further signaling from these

-          BMP and Wnt morphogens differentially regulate Sox9 expression, which function under the tuning control of FGF and distal Hox genes, + Shh provides polarization distal mesenchyme

Different diffusibility of activator and inhibitor

In limb development, this model explains skeletal patterning along the limb's proximal-distal axis. Cells in the limb bud mesenchyme interact using morphogens—activators that promote differentiation and inhibitors that prevent it, diffusing at different rates. This setup allows alternating regions of high activator and low inhibitor concentrations, forming cartilage at specific intervals and creating a patterned skeleton structure.