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Different regulation of limb development by p63 transcript variants.

Manabu Kawata1, Yuki Taniguchi1, Daisuke Mori1,2

  • 1Sensory & Motor System Medicine, Faculty of Medicine, The University of Tokyo. 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.

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p63 variants, Delta Np63gamma and TAp63gamma, are crucial for limb development by regulating key genes in the apical ectodermal ridge (AER). Their distinct roles in controlling FGFs and Jag2 highlight complex molecular networks in limb formation.

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Area of Science:

  • Developmental Biology
  • Molecular Genetics
  • Transcriptional Regulation

Background:

  • The apical ectodermal ridge (AER) is vital for limb development, controlling outgrowth and patterning via secreted molecules like FGFs.
  • The p63 transcription factor is essential for limb formation, but its variants' specific roles and regulatory mechanisms in the AER remain unclear.
  • The Jag2-Notch pathway negatively regulates AER function and limb development.

Purpose of the Study:

  • To investigate the expression patterns and functional roles of p63 variants in mouse limb development.
  • To elucidate the molecular mechanisms by which p63 variants regulate AER signaling.
  • To identify the direct transcriptional targets of p63 variants in limb development.

Main Methods:

  • Quantification of p63 variant expression in mouse limbs across different embryonic stages.
  • Fluorescence-activated cell sorting (FACS) analysis of p63 variant expression in AER and mesenchymal cells.
  • Generation of AER-specific p63 knockout mice (Msx2-Cre;p63Δ/fl) to study limb malformations.
  • Analysis of AER-related gene expression in knockout mice and p63-knockdown induced pluripotent stem cells.
  • Chromatin immunoprecipitation (ChIP) and promoter analyses to identify transcriptional targets.

Main Results:

  • Delta Np63γ was highly expressed throughout limb development, while TAp63γ expression increased in later stages.
  • All p63 variants were abundant in AER cells but scarce in mesenchymal cells.
  • AER-specific deletion of p63 led to limb malformations, particularly in distal elements, and decreased expression of AER-related genes.
  • Fgf8 and Fgf4 were identified as direct transcriptional targets of Delta Np63γ, while Jag2 was a target of TAp63γ.
  • Overexpression of TAp63γ worsened the limb malformation phenotype in knockout mice.

Conclusions:

  • Delta Np63γ and TAp63γ play distinct and critical roles in limb development by transcriptionally regulating different target genes within the AER.
  • Delta Np63γ primarily regulates FGF signaling, essential for AER maintenance and proliferation.
  • TAp63γ regulates Jag2, contributing to the negative feedback of AER function, with its role becoming more prominent in later development.