Additional file 1: of New genomic data and analyses challenge the traditional vision of animal epithelium evolution

Figure S1A. Comparison of p120 sequences. Residues involved in interaction with E-cadherin are boxed in red. Most of them are conserved. Figure S1B. Comparison of β-catenin sequences. A single β-catenin gene copy was identified in every studied species except for calcareous sponges that exhibit a duplication. All residues essential for E-cadherin interaction are boxed in pink and are highly conserved except for the R386 and N387 residues (replaced by L and T, respectively) in two hexactinellids and a more anecdotal change from A656 to S in placozoans. Residues boxed in blue are involved in α-catenin binding and in orange for the DTDL PDZ binding motif. Figure S1C. Analyses of α-catenins and vinculins sequences. Sequences of α-catenins and vinculins were aligned based on the structural domains helix0 to helix5 in Mus musculus α-catenin and vinculin. Helices are boxed and the numbers at the end of each sequence indicate the range encompassed in the alignment. Secondary structure prediction by JNet (Jalview option) identified six helices in all sponge α-catenin sequences except for A. queenslandica (missing the 4 first helices) and A. vastus (missing helix0). All species analyzed in this study have one copy of α-catenin and one copy of vinculin well-separated in Bayesian tree with high support (pp = 1) (bottom). Figure S2. Structure of Par3 proteins in metazoans. Par3 exhibits a conserved N-terminal domain (CR1), three central PDZ domains, and a C-terminal region containing multiple protein binding sites including the aPKC-binding motif. Figure S5. Domain composition of PatJ (D. melanogaster), INADL and MUPP1 (M. musculus) and Multiple PDZ containing protein (MPDZ) (O. lobularis, S. ciliatum, A. queenslandica and O. minuta). Note that only O. lobularis exhibits an MPDZ with a well-detected L27 domain (Evalue = 8.5 10− 4) as bilaterians. A. queenslandica and S. ciliatum MPDZ have a low-scoring L27 domain (shaded in grey) according to the HMM profile search. There is no recognizable similarity to the L27 domain in the N- terminal region of O. minuta MPDZ. Tables S1. and S2. Information on the domain structures of the Lethal giant larvae (LGL) and Scribble (Src) proteins in various metazoans. Spreadsheet containing Tables S3. and S4. The spreadsheet contains information on the characteristics of the new private databases and public databases (nature = genome/transcriptome) and links for new sequences used in this study. Spreadsheet containing Tables S5. and S6. The spreadsheet contains information on the accession numbers or contig/scaffold references where candidate genes were identified. In bold accession numbers of sequences annotated from our new transcriptomic and genomic sponge datasets. Links for new sequences used in this study are provided. (PDF 2610 kb

恐ろしい「第二の誕生」 : W. B. イェイツ"The Second Coming"を読む

<div><p>Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of <i>Crb1</i> and <i>Crb2</i> genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of <i>CRB1</i> function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.</p></div

Retinal development is impaired in <i>Crb1Crb2</i> cKO.

<p>(A–E) Histological sections from E13.5 to P5 control (left), <i>Crb1^+/−Crb2</i> cKO (middle) and <i>Crb1Crb2</i> cKO (right). From E13.5 onwards, disruption of the outer limiting membrane (A right, black arrowhead) accompanied with ectopic localization of cells extended in <i>Crb1Crb2</i> cKO developing retinas (A,B right). At E17.5 and P1, in contrast to control retinas no proper ganglion cell layer was formed (C,D). The separation of the outer nuclear/photoreceptor layer formed around P5, which never happened in the <i>Crb1Crb2</i> cKO retinas (E). <i>Crb1^+/−Crb2</i> cKO retinas showed the first disruption in the outer limiting membrane at the periphery at E15.5 (B middle, black arrowhead), which progressively extended to the centre accompanied with rosette formation (B–E middle). Electron microscopic pictures from E17.5 littermate control (F) and <i>Crb1Crb2</i> cKO (G) retinas. Control retinas showed an organized outer limiting membrane with adherens junctions (white arrowheads), retinal pigment epithelium and retinal nuclei alignments. <i>Crb1Crb2</i> cKO retinas showed absence of layer organization and adherens junctions. GCL, ganglion cell layer; INL, inner nuclear layer; NBL, neuroblast layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium. Scale bar, 100 µm (A–E); 5 µm (F,G).</p

The number of late born cell types is increased in <i>Crb1Crb2</i> cKO retinas.

<p>The distribution of early (A–C) and late born (D–F) cell types in the three layers was quantified as a percentage of each cell type in outer, inner and ganglion cell nuclear layer in the control (white bars), and top and bottom half of nuclear layer (tNL and bNL) and ganglion cell layer in the <i>Crb1Crb2</i> cKO (black bars) retinas at P14 (3–4 different animals/genotype). The distribution of early-born ganglion cells (Brn3b), cone photoreceptors (cone arrestin) and cholinergic amacrine cells (choline acetyltransferase, ChAT) was slightly affected in contrast to late-born rod photoreceptors (rhodopsin), Müller cells (Sox9) and bipolar cells (Chx10), which were to a larger extent wrongly distributed in the two nuclear layers. (G,H) The number of cells for each cell types was quantified at P14 (G) and the rods at P10 (H) in 3–4 retinas of control and <i>Crb1Crb2</i> cKO, and represented by the mean ± s.e.m. The number of early born cells was not affected whereas the number of late born cells was increased in <i>Crb1Crb2</i> cKO compared to control retinas at P14 and the rods at P10. bNL, bottom nuclear layer; Calb, calbindin positive horizontal cells; CAR, cone arrestin; GCL, ganglion cell layer; GlyT1, glycinergic amacrine cells; INL, inner nuclear layer; IPL, Inner plexiform layer; ONL, outer nuclear layer; RHO, Rhodopsin; tNL, top nuclear layer. **P<0.01; ***P<0.001.</p

CRB1 and CRB2 acts on the proliferative signalling pathways.

<p>Transcript levels measured by quantitative PCR at E17.5 (A) and P3 (C) in 3–6 control and <i>Crb1Crb2</i> cKO retinas showed changes in Notch1, YAP, sonic hedgehogs and cell cycle genes at E17,5 whereas at P1 these genes were not significantly changed except Hey1 and Smoothened. Quantification of protein levels of control and <i>Crb1Crb2</i> cKO retinal lysates (N = 3–5 for each Western blot and Western blots were repeated 2–4 times) at E17.5 (B) and P1 (D). Protein levels of YAP and pYAP were reduced at E17.5 and P1 whereas P120-catenin was increased and β-catenin and Kaiso unchanged at E17.5. Data are presented as mean ± s.e.m *P<0.05; **P<0.01.</p

<i>In vivo</i> retinal imaging in <i>Crb1Crb2</i> cKO mice.

<p>12M old control <i>Crb1^+/−Crb2^F/+</i> cKO (A–C) and 1M (D–F), 3M (G–I) and 6M (J–L) old <i>Crb1Crb2</i> cKO mice were subjected to scanning laser ophthalmoscopy (A,D,G,J) and vertical spectral domain optical coherence tomography (B, E, H, K). C, F, I and L are magnifications of the boxes in B, E, H, and K respectively. At 1M, a disorganized retina with an abnormally thick ganglion cell layer, one plexiform and one nuclear layer was observed (E, asterisk in F). At 3M (G–I) and 6M (J–L), an obvious loss in the retinal thickness was ascertained as well as several fundus alterations (G,J). Abbreviations: AF, autofluorescence; d, dorsal; FA, Fluorescein angiography; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS/OS, inner segment/outer segment border; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; RF, Red-free; v, ventral.</p