Essential requirement for zebrafish anosmin-1a in the migration of the posterior lateral line primordium.

International audienceKallmann syndrome (KS) is a human genetic disease that impairs both cell migration and axon elongation. The KAL-1 gene underlying the X-linked form of KS, encodes an extracellular matrix protein, anosmin-1, which mediates cell adhesion and axon growth and guidance in vitro. We investigated the requirement for kal1a and kal1b, the two orthologues of the KAL-1 gene in zebrafish, in the journey of the posterior lateral line primordium (PLLP). First, we established that while the accumulation of kal1a and kal1b transcripts was restricted to the posterior region of the migrating primordium and newly deposited neuromasts, the encoded proteins, anosmin-1a and anosmin-1b, respectively, were accumulated in the PLLP, in differentiated neuromasts and in a thin strip extending along the trail path of the PLLP. We also show that morpholino knockdown of kal1a, but not kal1b, severely impairs PLLP migration. However, while the PLLP of kal1a morphants displays highly abnormal morphology, proper expression of the cxcr4b gene suggests that kal1a does not play a role in PLLP differentiation. Conversely, wild-type levels of kal1a transcripts are detected in the PLLP of cxcr4b or sdf1a morphant embryos, strongly suggesting that kal1a transcription is independent of CXCR4b/SDF1a signalling. Last, moderate depletion of both anosmin-1a and SDF1a markedly affects PLLP migration providing strong evidence that anosmin-1a acts as an essential co-factor in SDF1a-mediated signalling pathways. Our findings, which demonstrate, for the first time, an essential requirement for anosmin-1a in PLLP migration, also strongly suggest that this protein plays a key role for proper activation of the CXCR4b/SDF1a and/or CXCR7/SDF1a signalling pathway in PLLP migration

Evidence for Involvement of Wnt Signalling in Body Polarities, Cell Proliferation, and the Neuro-Sensory System in an Adult Ctenophore

International audienceSignalling through the Wnt family of secreted proteins originated in a common metazoan ancestor and greatly influenced the evolution of animal body plans. In bilaterians, Wnt signalling plays multiple fundamental roles during embryonic development and in adult tissues, notably in axial patterning, neural development and stem cell regulation. Studies in various cnidarian species have particularly highlighted the evolutionarily conserved role of the Wnt/b-catenin pathway in specification and patterning of the primary embryonic axis. However in another key non-bilaterian phylum, Ctenophora, Wnts are not involved in early establishment of the body axis during embryogenesis. We analysed the expression in the adult of the ctenophore Pleurobrachia pileus of 11 orthologues of Wnt signalling genes including all ctenophore Wnt ligands and Fz receptors and several members of the intracellular b-catenin pathway machinery. All genes are strongly expressed around the mouth margin at the oral pole, evoking the Wnt oral centre of cnidarians. This observation is consistent with primary axis polarisation by the Wnts being a universal metazoan feature, secondarily lost in ctenophores during early development but retained in the adult. In addition, local expression of Wnt signalling genes was seen in various anatomical structures of the body including in the locomotory comb rows, where their complex deployment suggests control by the Wnts of local comb polarity. Other important contexts of Wnt involvement which probably evolved before the ctenophore/ cnidarian/bilaterian split include proliferating stem cells and progenitors irrespective of cell types, and developing as well as differentiated neuro-sensory structures

Oral rings of gene expression (details) and evidence for intense cell proliferation around the mouth.

<p>The four genes encoding Wnt ligands have graded extensions of their expression domains starting from the mouth margin, with <i>PpiWnt9</i> (A) expression restricted to a very thin band, whereas <i>PpiWntX</i> (B) and <i>PpiWntA</i> (C) are expressed in a distinctly wider belt around the mouth, and <i>PpiWnt6</i> transcripts are detected in an even broader domain (D). Details of expression at the oral pole are also shown for two other genes, <i>PpiFzB</i> (E), <i>PpiTcf</i> (F), and for the stem cell marker gene <i>PpiPiwi1</i> (G). Most of these genes are also expressed in the two paragastric canals (white stars in A–D, G, J), elements of the gastro-vascular system that run parallel to the pharynx, from the stomach to the mouth area. The mouth border is a region of intense cell proliferation, as evidenced by EdU DNA label incorporation after a 2 h pulse (H) (I: higher magnification of the area boxed in H) and by immunolabelling of mitotic chromosomes using an anti-phospho-Histone H3 antibody (J). The arrowhead points to the mouth margin (mo) in all pictures. Scale bars: (A–H, J) 100 µm; (I) 10 µm.</p

Gene expression in the tentacle root.

<p>(A) Schematic representation of the <i>P. pileus</i> tentacle root in internal view, notably showing the three characteristic longitudinal ridges containing the stem cells of colloblasts (lateral ridges, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084363#pone.0084363-Ali1" target="_blank">[32]</a>) and of muscle cells (median ridge, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084363#pone.0084363-Dayraud1" target="_blank">[34]</a>). (B) Whole-mount <i>in situ</i> hybridisation in dissected tentacle roots for four Wnt signalling genes. Orientation as in (A). (C) Schematic view of the tentacle root in transverse section (at about midlenght). The arrows indicate the direction of cell movement and differentiation along the colloblast (lateral light blue arrows) and muscle (median light pink arrow) cellular lineages. Colour code: blue: ectodermal epithelium; green: endodermal epithelium; red: mesogleal component of the tentacle root (muscle and nervous system); purple: body mesoglea; black: lumen of the tentacle sheat; black with white dots: lumen of the tentacular gastro-vascular canals. EXT: external side (i.e. towards body periphery); INT: internal side (i.e. towards pharynx). (D) Gene expression in transverse cryosections of the tentacle root. Orientation as in (C). Scale bars: 100 µm.</p

Gene expression in the aboral neuro-sensory complex and architecture of the polar field nervous system.

<p>(A) Summary of the morphology of the aboral neuro-sensory complex (polar field represented only on one side). Abbreviations: b: balancers; cg: ciliated groove; co: connection between Z-bodies; cz: central zone; ep: epithelial papilla; lb: lamellate bodies; mz: marginal zone; pfm: polar field muscle; Zb: Z-body. General distribution of transcripts within the aboral neuro-sensory complex is shown for <i>PpiDvl1</i> in (B), where the white dotted line delineates the apical organ and the black dotted line represents the polar field outline. The two distinct zones making up the polar field are labelled as mz (marginal zone) and cz (central zone). Detailed views of <i>in situ</i> hybridisation in the central area of the aboral neuro-sensory complex are given for <i>PpiWnt6</i> (C) and <i>PpiWntX</i> (D). Both genes are strongly expressed in the proximal extremities of the polar field marginal zones (asterisks). In addition, within the apical organ, <i>PpiWnt6</i> (C) is expressed in the balancers (b), in the epithelial floor (flo), and in the lithocytes (not visible because not on focus), and <i>PpiWntX</i> is expressed in two peripheral transverse spots in the tentacular plane (arrowheads in D). In the polar fields outside from their most proximal area, examples are shown in (E–G) (corresponding to the boxed area in B) for each of the three observed categories of gene expression patterns: a continuous band along the inner border of the marginal zone (E); same territory but with additional weaker expression in the rest of the marginal zone (F); or discontinuous spots along the inner border of the marginal zone, this situation being observed only for <i>Ppiβ-cat</i> (G). These spots correspond to the anti-tyrosylated-α-tubulin immunoreactive Z-bodies (white arrowheads in H), as shown by comparison of the distribution of cell nuclei in Dapi counter-staining of <i>Ppiβ-cat in situ</i> hybridisation (G’) and of anti-tyrosylated-α-tubulin immunostaining (H). The distribution of anti-serotonin (5-HT) immunoreactive neurons in the polar field is shown in (I) and (K), the latter corresponding to a detailed view of the area boxed in (I) with additional anti-tyrosylated-α-tubulin staining. The inner region of the polar field marginal zone furthermore contain a continuous row of catecholaminergic elongated cells, strongly immunoreactive with antibodies directed against norepinephrin (NE) (J, L, M), L-Dopa (N), tyrosine hydroxylase (TH) (O), and Vmat2 (P) (L–P: higher magnification views of area boxed in J). The white arrowhead in (K) and (L) points to a Z-body. The neuro-sensory architecture of the polar field is summarised in (Q). Scale bars: (B) 50 µm; (C–J) 20 µm; (K–P) 10 µm.</p

Summary of gene expression patterns in the aboral neuro-sensory complex.

<p>Summary of gene expression patterns in the aboral neuro-sensory complex.</p

Organisation and cellular dynamics of the comb.

<p>(A) Scanning electron microscopy view of two successive combs along a comb row. The dotted line materialises the outline of the comb basal cushion (bc), the cilia (ci) emerging along its midline. (B) Schematic transverse section of a basal cushion according to the double-arrowed line in (A) (after <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084363#pone.0084363-HernandezNicaise2" target="_blank">[70]</a>). Polster cell bodies are represented in white, the covering epithelium in yellow, and nerve cells in purple (bc: basal cushion; ci: cilia). (C) Expression in a basal cushion of the stem cell marker <i>PpiPiwi1</i>. (D) Expression of the early ciliogenesis marker <i>PpiBBS-5</i>. (E) Expression of the later ciliogenesis marker <i>PpiIFT-88</i>. (F) Expression of <i>β-tubulin</i>. (G) Expression of <i>PpiSox6</i>, a marker of mature polster cells of the basal cushion oral half. Transverse sections of the basal cushion after <i>in situ</i> hybridisation are shown for <i>Ppiβ-tub</i> in (H) and for <i>PpiSox6</i> in (I); bc: basal cushion; ci: cilia; see (B) for interpretation of these pictures. (J) Distribution of EdU labelled nuclei after a pulse of 20 h and a chase of 5 days, showing that the youngest polster cells are those located near both comb extremities as well as those situated on the border all around the basal cushion. The detailed view in (K) shows that in addition to differentiated nuclei, some undifferentiated nuclei concentrated at the extremity have retained the label (putative stem cells; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084363#pone.0084363-Ali1" target="_blank">[32]</a>). (L–N) Model of cellular dynamics within the basal cushion derived from these data. Stem cells and proliferating progenitors are segregated at both extremities (L) then differentiating polster cells travel (purple arrows in M, N) below the covering epidermis to reach their final destination either close to the extremity (growth in length) or all around the border (growth in width). In (C–G) and (J) the dotted line materialises the basal cushion midline and the aboral pole is on top. Scale bars: 100 µm in all pictures except (K) (10 µm).</p

General morphology of the ctenophore <i>Pleurobrachia pileus</i>.

<p>The mouth (mo) and the aboral neuro-sensory complex (nsc) are located respectively at the oral and aboral pole of the single body axis. The eight comb rows (cr) are locomotory structures, each made of regularly repeated swimming paddles or combs. The body is biradially symmetrical notably due to the presence of a single pair of tentacles (tcl), each bearing numerous lateral branchings or tentillae (tt). The tentacle root (tr), enclosed in the tentacle sheath, is responsible for constant regeneration of tentacle tissues. The pharynx (ph) and some canals of the endodermal gastro-vascular system are also visible by transparency. Scale bar: 1 cm.</p

Oral ring of gene expression for Wnt signalling genes and <i>Piwi</i> (general views).

<p>The pictures show whole adult specimens of <i>P. pileus</i> after gene <i>in situ</i> hybridisation. Legends in the first picture as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084363#pone-0084363-g001" target="_blank">Figure 1</a>; mouth margin indicated by black arrow; oral pole at the bottom in all pictures. Scale bars: 500 µm.</p