Diversification and Molecular Evolution of ATOH8, a Gene Encoding a bHLH Transcription Factor

ATOH8 is a bHLH domain transcription factor implicated in the development of the nervous system, kidney, pancreas, retina and muscle. In the present study, we collected sequence of ATOH8 orthologues from 18 vertebrate species and 24 invertebrate species. The reconstruction of ATOH8 phylogeny and sequence analysis showed that this gene underwent notable divergences during evolution. For those vertebrate species investigated, we analyzed the gene structure and regulatory elements of ATOH8. We found that the bHLH domain of vertebrate ATOH8 was highly conserved. Mammals retained some specific amino acids in contrast to the non-mammalian orthologues. Mammals also developed another potential isoform, verified by a human expressed sequence tag (EST). Comparative genomic analyses of the regulatory elements revealed a replacement of the ancestral TATA box by CpG-islands in the eutherian mammals and an evolutionary tendency for TATA box reduction in vertebrates in general. We furthermore identified the region of the effective promoter of human ATOH8 which could drive the expression of EGFP reporter in the chicken embryo. In the opossum, both the coding region and regulatory elements of ATOH8 have some special features, such as the unique extended C-terminus encoded by the third exon and absence of both CpG islands and TATA elements in the regulatory region. Our gene mapping data showed that in human, ATOH8 was hosted in one chromosome which is a fusion product of two orthologous chromosomes in non-human primates. This unique chromosomal environment of human ATOH8 probably subjects its expression to the regulation at chromosomal level. We deduce that the great interspecific differences found in both ATOH8 gene sequence and its regulatory elements might be significant for the fine regulation of its spatiotemporal expression and roles of ATOH8, thus orchestrating its function in different tissues and organisms

Wnt11 Is Required for Oriented Migration of Dermogenic Progenitor Cells from the Dorsomedial Lip of the Avian Dermomyotome

<div><p>The embryonic origin of the dermis in vertebrates can be traced back to the dermomyotome of the somites, the lateral plate mesoderm and the neural crest. The dermal precursors directly overlying the neural tube display a unique dense arrangement and are the first to induce skin appendage formation in vertebrate embryos. These dermal precursor cells have been shown to derive from the dorsomedial lip of the dermomyotome (DML). Based on its expression pattern in the DML, Wnt11 is a candidate regulator of dorsal dermis formation. Using EGFP-based cell labelling and time-lapse imaging, we show that the <i>Wnt11</i> expressing DML is the source of the dense dorsal dermis. Loss-of-function studies in chicken embryos show that <i>Wnt11</i> is indeed essential for the formation of dense dermis competent to support cutaneous appendage formation. Our findings show that dermogenic progenitors cannot leave the DML to form dense dorsal dermis following <i>Wnt11</i> silencing. No alterations were noticeable in the patterning or in the epithelial state of the dermomyotome including the DML. Furthermore, we show that <i>Wnt11</i> expression is regulated in a manner similar to the previously described early dermal marker <i>cDermo-1</i>. The analysis of <i>Wnt11</i> mutant mice exhibits an underdeveloped dorsal dermis and strongly supports our gene silencing data in chicken embryos. We conclude that Wnt11 is required for dense dermis and subsequent cutaneous appendage formation, by influencing the cell fate decision of the cells in the DML.</p></div

Effects of <i>Wnt11</i> silencing on dermomyotome.

<p>A, C, E, G and I. Chicken embryos electroporated with <i>Wnt11</i> RNAi at stage HH14-17 and after a reincubation period of 24 hours. B. The embryo in A (HH20) hybridized for <i>En-1,</i> shows that the central dermomyotomal compartment marked with <i>En-1</i> was unaffected in the electroporated area (the space between white arrows in B). D. The photo represents the embryo in C after ISH for <i>Sim-1</i> probe. The lateral dermomyotomal compartment marked with <i>Sim-1</i> (white arrows in D indicate the electroporated area) did not show any change in its expression after <i>Wnt11</i> silencing. F. ISH for <i>Paraxis</i> of the embryo presented in photo E. <i>Paraxis</i> transcripts seems not to be altered following <i>Wnt11</i> silencing in the DML (area between white arrows in F). H. The dermomyotomal marker <i>Pax3</i>, in contrast, is significantly upregulated at the site of <i>Wnt11</i> RNAi transfection (area between white arrows in H). K. The cross-section through the embryo in H in the manipulated area shows a strong upregulation of <i>Pax3</i> in the DML, while the DM remains normal when compared to the control side. I. Electroporated embryo at stage HH14-17 with <i>Wnt11</i> RNAi and after 24 hours reincubation (HH19). J. Hybridized embryo from photo I for <i>Snail1</i> probe. At stage HH20 the EMT has already started, and the <i>Snail1</i> expression can be seen in the myotome, dermomyotome, sclerotome and in the space above the neural tube (dermal progenitor cells). Whole-mount ISH of the embryo electroporated with <i>Wnt11</i> RNAi shows a decreased <i>Snail1</i> expression (the region in the bracket), while the white arrows point towards the normal expression of <i>Snail1</i> above the neural tube (dermogenic progenitors) at untreated levels. L. Section through the embryo in J in the affected region shows a decreased <i>Snail1</i> expression in the DML (black arrowhead) and above the neural tube (black arrow). Scale bar: 100 μm.</p

Murine dermis volume and hair follicles number comparison between <i>Wnt11</i> knock-out and wild-type mice.

<p>The diagram represents a graphical comparison of two averages with standard deviation bars of mice skin volume in sections of E18.5 mice wild-type and <i>Wnt11</i> knock-out mice. The results showed that the dermis volume in the sections of <i>Wnt11</i> knock-out mice was approximately 45% lesser when compared to the wild-type mice sections. The graphical comparison was performed in 20 sections after HE staining, 10 slides representing 3 mice for each genotype. The photos of the analysed sections were acquired at 40x magnification. For the graph representing the hair follicles number, the hair placodes on the back skin of the hybridized embryos for <i>mDermo-1</i> were counted in identical squares drawn on photos obtained using standardized magnification parameters. For this experiment, 6 <i>Wnt11</i> knock-out and 6 wild-type embryos were used. The diagram representing the graphical comparison of the two averages with standard deviation bars of hair follicles placodes number in mice embryos E14.5 hybridized for <i>mDermo-1</i>, shows a decrease of 35% in the number of hair follicles placodes in the mutant mice.</p

Relative expression of dorsal skin development-related genes in <i>Wnt11</i> knock-out mice and wild-type.

<p>A. At stage E12.5, <i>Msx1</i> shows much less expression in mutant mice than wild-type mice. (***:P<0,001). B. At stage E16.5, <i>Wnt11</i> expression in <i>Wnt11</i> knock-out mice is undetectable compared to wild-type mice. <i>Fgf10</i> has less expression in the homozygous mice. <i>Twist1</i>, <i>Twist2</i> and <i>Krt5</i> expression does not show significant differences between wild-type and homozygous animals.</p

Atonal homolog 8/Math6 regulates differentiation and maintenance of skeletal muscle

Atonal Homolog 8 (Atoh8) belongs to a large superfamily of transcriptional regulators called basic helix-loop-helix (bHLH) transcription factors. Atoh8 (murine homolog "Math6") has been shown to be involved in organogenesis during murine embryonic development. We have previously identified the expression of Atoh8 during skeletal myogenesis in chicken where we described its involvement in hypaxial myotome formation suggesting a regulatory role of Atoh8 in skeletal muscle development. Within the current study, we analyzed the effect of the loss of function of Atoh8 in murine primary myoblasts and during differentiation of pluripotent stem cells into myotubes, and the effect of its gain of function in C2C12 cells. Based on the observed results, we conclude that Atoh8 regulates myoblast proliferation via modulating myostatin signaling. Further, our data revealed a reduced muscle mass, strength and fiber size with significant changes to the muscle fiber type suggesting atrophy in skeletal muscle of Atoh8 mutants. We further report that Atoh8 knockout mice suffer from a condition similar to ambient hypoxia which may be the primary cause of the phenotype. Altogether, this study shows the significance of Atoh8 not only in myogenesis but also in the maintenance of skeletal muscle

Decrease of dermal markers after <i>Wnt11</i> RNAi.

<p>A. Embryo showing the region of transfection after electroporation with EGFP-<i>Wnt11</i> RNAi. The embryo has been electroporated at stage HH14-17 and reincubated for 20 hours. B. The embryo in A after 2.5 days reincubation time following manipulation (HH28). Starting with stage 24, <i>cDermo-1</i> expression pattern can be found in the subectodermal mesenchyme of the trunk, and at stage 26 it is very strong along the dorsal midline. Following additional 2.5 days of reincubation after EGFP signal documentation for embryo in A, <i>cDermo-1</i> is remarkably reduced along the dorsal midline in the transfected region (area between white arrows in B). C. Higher magnification of the embryo in B. D. Electroporated embryo with <i>Wnt11</i> silencing construct at stage HH14-17 and photographed 20 hours later, at stage HH20. E. After longer reincubation periods following electroporation (4.5 days of the embryo in D), the embryo shows a retarded feather bud development as seen after <i>cDermo-1</i> ISH, which is expressed in this stage (HH31) in the mesenchyme of the nascent feather buds (the white arrowheads in E and F mark the missing row of feather buds on the manipulated side in D). F. Higher magnification of the embryo in E. G. Embryo showing the region of transfection after electroporation with EGFP-<i>Wnt11</i> RNAi at stage HH14-17 and photographed 24 hours later, at stage HH20. H. After 8 days reincubation for embryo in G after EGFP documentation (HH38) and hybridisation with a <i>Shh</i> probe we have noticed a retarded feather bud development as seen with <i>cDermo-1</i>. <i>In situ</i> hybridisation for <i>Shh</i> also reveals a changed morphology of the eventually formed feather buds, which are smaller and more flattened, with lower expression of <i>Shh</i> on the manipulated side (white arrow and arrowheads in H). The white square presents in a higher magnification the altered feather buds formation (yellow arrow). Scale bar: 100 μm.</p

<i>Wnt11</i> expression in the DML is important for recruitment of dorsal dermal progenitors.

<p>A. EGFP-transfected DML of chicken embryo at stage HH18-19, 20 hours after electroporation. B. The embryo in A after 3 days of reincubation following electroporation. Note the presence of EGFP positive cells in the myotome and region of the future dorsal dermis (dotted circles). The white dotted lines squares delineate the somites. C. Cross-section of the embryo in B. EGFP-positive cells can be seen to be migrating into the subectodermal space overlying the spinal cord (white arrows). D. <i>Wnt11</i> RNAi on chicken embryo. DML of EFGP-<i>Wnt11</i> RNAi construct transfected chicken embryos at HH18-19, 20 hours after electroporation. E. After 3 days of reincubation following electroporation, the EGFP-<i>Wnt11</i> RNAi expressing cells are only to be found in the myotome in a disorganized manner, whereas EGFP-<i>Wnt11</i> RNAi positive cells are missing in the dorsal dermis anlage. F. In cross-section of the embryo in E, only very few cells (nearly undetectable) migrating from DML are present into the subectodermal space (white arrow) overlying the spinal cord (white line). White lines were traced along the neural tube for a better orientation. G. Targeting of the DML at stage HH14-17 by EGFP-<i>Wnt11</i> RNAi constructs after 24 hours reincubation and the corresponding <i>Wnt11</i> silencing as seen by ISH (area between black arrows in H). I. There is no evidence of increased cell death at the sites of EGFP-<i>Wnt11</i> construct transfection as seen by TUNEL staining. J. TUNEL staining of an electroporated embryo with scrambled DNA was used as control for our TUNEL assay. K. Represents the untreated control (Control 2) for TUNEL assay. Photos in I, J, K show the DML of the coressponding sections after TUNEL assay. Black arrows in I, J, K point towards Tunel-positive cells. NT: neural tube. DML: dorso-medial lip. Scale bar: 100 μm.</p

BMP2 signaling controls <i>Wnt11</i> expression in DML.

<p>A, B. BMP2 beads implanted into somites of chicken embryos (black arrow in A) lead to an upregulation of <i>Wnt11</i> as seen by ISH in vibratome sections (black arrow in B; section level indicated by red line in A). C, D. In contrast, grafting of Noggin cells (BMP antagonist) lead to a downregulation of <i>Wnt11</i> transcripts on the operated side (C) also visible in vibratome sections of the same embryo (black arrow in D; section level indicated by red line in C). Scale bar: 100 μm.</p

Dorsal dermis is decreased in dermis thickness and number of hair follicles in the <i>Wnt11</i> knock-out mice.

<p>A, C, E. Homozygous <i>Wnt11</i> knock-out embryos. B, D, F. Wild-type embryos. Embryo in A represents a homozygous <i>Wnt11</i> knock-out embryo of embryonic day E 14.5. In the dorsal mid-line region of this embryo a large area devoid of hair placodes is detectable. The white square shows a decrease number of hair placodes in comparison to the WT embryo of similar developmental stage (B). Section through the embryo in A shows that the <i>cDermo-1</i> signal is diminished with corresponding decrease in dermal thickness in the mutant (black arrow in C) as compared with a similar section of the same magnification of a WT embryo (black arrow in D). Hematoxylin-Eosin staining in paraffin sections of knock-out mice of E 18.5 shows a remarkable decrease in dermis thickness (45%), as well as a reduced number of hair follicles (35%). (E) In comparison with a Hematoxylin-Eosin treated section of a WT mouse of the same developmental stage (F). Double-headed arrows in E and F show the remarkable difference in dermis thickness between the mutant mouse embryo as compared to the normal thickness of dermis in a wild-type embryo (photographs were taken at the same magnification). The sections were taken at the trunk regions of the mice. The analysis was restricted to the same anatomical area of the dorsal skin for all embryos investigated. Scale bar: 100 μm.</p