Tisler M et al. (2016), Cilia are required for asymmetric nodal inducti...

ECB-ART-44880

BMC Dev Biol 2016 Aug 23;161:28. doi: 10.1186/s12861-016-0128-7.

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Cilia are required for asymmetric nodal induction in the sea urchin embryo.

Tisler M , Wetzel F , Mantino S , Kremnyov S , Thumberger T , Schweickert A , Blum M , Vick P .

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BACKGROUND: Left-right (LR) organ asymmetries are a common feature of metazoan animals. In many cases, laterality is established by a conserved asymmetric Nodal signaling cascade during embryogenesis. In most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer (LRO), a ciliated epithelium present during gastrula/neurula stages. Conservation of LRO and flow beyond the vertebrates has not been reported yet. RESULTS: Here we study sea urchin embryos, which use nodal to establish larval LR asymmetry as well. Cilia were found in the archenteron of embryos undergoing gastrulation. Expression of foxj1 and dnah9 suggested that archenteron cilia were motile. Cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. High-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. Removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. Our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos. CONCLUSIONS: Based on these results we argue that the anterior archenteron represents a bona fide LRO and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes.

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Species referenced: Echinodermata
Genes referenced: foxj1 LOC100887844 LOC115919910 LOC581088 nodall pitx2 pkd2 pole tubgcp2
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	Fig. 1. Polarized cilia at the sea urchin archenteron. a–e P. lividus (a–c) and S. pallidus embryos (d–e’) were analyzed by IF for the presence of cilia at the archenteron. Optical sections showed ectodermal (a’; a”) and archenteron cilia (b’–c) at mid gastrula stages. Late (e; e’) but not early S. pallidus gastrula stage embryos revealed cilia in the archenteron and at the archenteron tip (d, d’). Cilia were stained with an antibody against acetylated-α-tubulin (red, a’–c) or anti-α-tubulin (green, d–e’), nuclei were stained with DAPI (d–e’), and cell boundaries were visualized by phalloidin-green (c) or phalloidin-red (d–e’). f, g SEM analysis of P. lividus ectodermal and archenteron cilia. Fractured embryos allowed the visualization of monocilia on archenteron cells (g). Cilia are highlighted by an arrowhead (f–h’) Posterior polarization of cilia on cells which invaginated into the archenteron. Cilia are colored in yellow and individual cells alternating in green and purple. The ciliary base is marked by a red semicircle, the center of the cell is indicated by a yellow dot. Schematic drawings adapted from Blum et al. 2014 [3]
	Fig. 2. Motile cilia marker genes dnah9 and foxj1 are expressed throughout sea urchin gastrulation. Whole mount in situ hybridization of early (EG), mid (MG) and late (LG) gastrula stage P. lividus embryos, as well as prism stages (k’) for mRNA expression of dnah9 (a–d), foxj1 (e–h) and pkd2 (i–l). Schematic representation of staining in mid-gastrula embryos is highlighted in drawings in (d, h and l). White arrowheads highlight vegetal blastopore and archenteron tip expression areas. Schematic drawings adapted from Blum et al. 2014 [3]
	Fig. 3. Deciliation impairs LR asymmetric nodal cascade induction. SEM (a, b) and IF (c, d) analyses of cilia in control untreated embryos (a, c) and specimens exposed to a 60–90 s osmotic shock (b, d). Note that cilia were almost completely absent following high salt treatment. Cilia in (c) and (d) were stained with an antibody against anti-acetylated-α-tubulin (purple), nuclei with DAPI, and cell boundaries visualized by phalloidin-green. e, g Unilateral induction of asymmetric nodal cascade genes in control embryos. f, h Bilateral ectodermal nodal and pitx2 expression following deciliation of early to mid-gastrula stage embryos. Black arrowheads highlight expression in the archenteron and ectoderm; white arrowheads indicate lack of expression. i Quantification of expression pattern from (e–h). Unilateral pitx2 expression in coelomic pouches of pluteus stage control specimen (j) and bilateral expression in deciliated embryos (k). Bilateral expression of pitx2 after deciliation is independent of MAPK/p38 inhibition through SB203580 (l–m). o Quantification of expression patterns. Posterior/vegetal is to the top in (a, b) and to the bottom in (c–g)
	Fig. 4. The ciliated Left-Right Organizer: a synapomorphy of the deuterostomes. a–c Schematic drawings of sea urchin (a), amphioxus (b) and Xenopus (c) gastrula/neurula stage embryos (anterior to the left). In all cases the archenteron harbors cells of mesodermal fate (SM, red), which in frog and sea urchin are ciliated and function as LROs. The position of mesodermal LROs is indicated by an arrowhead. d nodal is evolutionary conserved among metazoans except for the ecdysozoa. Deuterostomes share a ciliated LRO as a common synapomorphy. Drawings adapted in parts from Blum et al. 2014 [3]

References [+] :

Auclair, Cilia regeneration in the sea urchin embryo: evidence for a pool of ciliary proteins. 1966, Pubmed, Echinobase