Hoxa13 regulates expression of common Hox target genes involved in cartilage development to coordinate the expansion of the autopodal anlage.

To elucidate the role of Hox genes in limb cartilage development, we identified the target genes of HOXA11 and HOXA13 by ChIP-Seq. The ChIP DNA fragment contained evolutionarily conserved sequences and multiple highly conserved HOX binding sites. A substantial portion of the HOXA11 ChIP fragment overlapped with the HOXA13 ChIP fragment indicating that both factors share common targets. Deletion of the target regions neighboring Bmp2 or Tshz2 reduced their expression in the autopod suggesting that they function as the limb bud-specific enhancers. We identified the Hox downstream genes as exhibiting expression changes in the Hoxa13 knock out (KO) and Hoxd11-13 deletion double mutant (Hox13 dKO) autopod by Genechip analysis. The Hox downstream genes neighboring the ChIP fragment were defined as the direct targets of Hox. We analyzed the spatial expression pattern of the Hox target genes that encode two different categories of transcription factors during autopod development and Hox13dKO limb bud. (a) Bcl11a, encoding a repressor of cartilage differentiation, was expressed in the E11.5 autopod and was substantially reduced in the Hox13dKO. (b) The transcription factors Aff3, Bnc2, Nfib and Runx1t1 were expressed in the zeugopodal cartilage but not in the autopod due to the repressive or relatively weak transcriptional activity of Hox13 at E11.5. Interestingly, the expression of these genes was later observed in the autopodal cartilage at E12.5. These results indicate that Hox13 transiently suspends the cartilage differentiation in the autopodal anlage via multiple pathways until establishing the paddle-shaped structure required to generate five digits.

Etv1 and Ewsr1 cooperatively regulate limb mesenchymal Fgf10 expression in response to apical ectodermal ridge-derived fibroblast growth factor signal.

Fibroblast growth factors (FGFs) expressed in the apical ectodermal ridge (AER) and FGF10 expressed in the underlying mesoderm are essential for limb bud outgrowth. Their expression is maintained through a positive feedback loop. We identified the cis-regulatory element and trans-acting factors involved in the AER-FGF-dependent transactivation of Fgf10. Etv1 and Ewsr1 stimulated transcription from the Fgf10 promoter in the sub-AER mesenchyme of mouse and chick limb buds in a conserved AGAAAR cluster-dependent manner. We found that both Etv1 and Ewsr1 were necessary for Fgf10 expression and elongation of the limb bud. In addition, Etv1 and AER-FGF synergistically stimulated Fgf10 promoter activity in an Ewsr1-dependent manner. We also found that Etv1 and Ewsr1 bound to the segment of DNA containing the AGAAAR cluster in vivo and in vitro. Moreover, Etv1 directly bound to the AGAAAR sequence in vitro. Our results suggest that Etv1 and Ewsr1 transactivate Fgf10 directly and cooperatively in response to AER-FGFs.

Leucophores are similar to xanthophores in their specification and differentiation processes in medaka.

Animal body color is generated primarily by neural crest-derived pigment cells in the skin. Mammals and birds have only melanocytes on the surface of their bodies; however, fish have a variety of pigment cell types or chromatophores, including melanophores, xanthophores, and iridophores. The medaka has a unique chromatophore type called the leucophore. The genetic basis of chromatophore diversity remains poorly understood. Here, we report that three loci in medaka, namely, leucophore free (lf), lf-2, and white leucophore (wl), which affect leucophore and xanthophore differentiation, encode solute carrier family 2, member 15b (slc2a15b), paired box gene 7a (pax7a), and solute carrier family 2 facilitated glucose transporter, member 11b (slc2a11b), respectively. Because lf-2, a loss-of-function mutant for pax7a, causes defects in the formation of xanthophore and leucophore precursor cells, pax7a is critical for the development of the chromatophores. This genetic evidence implies that leucophores are similar to xanthophores, although it was previously thought that leucophores were related to iridophores, as these chromatophores have purine-dependent light reflection. Our identification of slc2a15b and slc2a11b as genes critical for the differentiation of leucophores and xanthophores in medaka led to a further finding that the existence of these two genes in the genome coincides with the presence of xanthophores in nonmammalian vertebrates: birds have yellow-pigmented irises with xanthophore-like intracellular organelles. Our findings provide clues for revealing diverse evolutionary mechanisms of pigment cell formation in animals.

Wnt and BMP signaling cooperate with Hox in the control of Six2 expression in limb tendon precursor.

The number and shape of limb tendons vary along the proximodistal axis, and the autopod contains more tendons than the zeugopod. The transcription factor Six2 is expressed in the developing tendons, and its expression can be traced back to a group of limb mesenchymal cells that are thought to be tendon precursor cells. We tried to elucidate the mechanism controlling position-specific tendon pattern formation using Six2 as a tendon marker. Six2 expression was always found in cells between the limb cartilage and ectoderm. Administration of BMP-2 or BMP antagonist Noggin to the limb bud, respectively repressed or facilitated Six2 expression. Removal of the ectoderm or administration of the Wnt antagonist sFRP-2 abolished Six2 expression and ectopic Wnt expression induced ectopic Six2 expression. Taken together, Six2 expression is induced in the cells located at the point where cartilage-derived Noggin and ectoderm-derived Wnt signals meet. Misexpression of the autopod-specific Hoxa-13 or Hoxd-13 induced ectopic expression of Six2 in the zeugopodal mesenchymal cells of the chick limb bud. Six2 expression in the dorsal autopodal mesenchyme was not detected in Hoxa-13(-/-);HoxD(del/del) mice, indicating that autopod-specific Hox is required for the regulation of Six2 expression. Misexpression of Wnt in the autopod induced ectopic Six2 expression in the autopod. On the other hand, Wnt misexpression alone never induced Six2 expression in the zeugopod, yet co-misexpression of Hoxa-13 and Wnt in the zeugopod enhanced ectopic Six2 expression. Our results indicate that autopodal Hox genes regulate Six2 expression in the autopodal tendon precursor in cooperation with the factors from cartilage and ectoderm.

FGF9 monomer-dimer equilibrium regulates extracellular matrix affinity and tissue diffusion.

The spontaneous dominant mouse mutant, Elbow knee synostosis (Eks), shows elbow and knee joint synosotsis, and premature fusion of cranial sutures. Here we identify a missense mutation in the Fgf9 gene that is responsible for the Eks mutation. Through investigation of the pathogenic mechanisms of joint and suture synostosis in Eks mice, we identify a key molecular mechanism that regulates FGF9 signaling in developing tissues. We show that the Eks mutation prevents homodimerization of the FGF9 protein and that monomeric FGF9 binds to heparin with a lower affinity than dimeric FGF9. These biochemical defects result in increased diffusion of the altered FGF9 protein (FGF9(Eks)) through developing tissues, leading to ectopic FGF9 signaling and repression of joint and suture development. We propose a mechanism in which the range of FGF9 signaling in developing tissues is limited by its ability to homodimerize and its affinity for extracellular matrix heparan sulfate proteoglycans.