Morov AR et al. (2016), Acquisition of the dorsal structures in chordat...

ECB-ART-44759

Open Biol 2016 Jun 01;66:. doi: 10.1098/rsob.160062.

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Acquisition of the dorsal structures in chordate amphioxus.

Morov AR , Ukizintambara T , Sabirov RM , Yasui K .

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Acquisition of dorsal structures, such as notochord and hollow nerve cord, is likely to have had a profound influence upon vertebrate evolution. Dorsal formation in chordate development thus has been intensively studied in vertebrates and ascidians. However, the present understanding does not explain how chordates acquired dorsal structures. Here we show that amphioxus retains a key clue to answer this question. In amphioxus embryos, maternal nodal mRNA distributes asymmetrically in accordance with the remodelling of the cortical cytoskeleton in the fertilized egg, and subsequently lefty is first expressed in a patch of blastomeres across the equator where wnt8 is expressed circularly and which will become the margin of the blastopore. The lefty domain co-expresses zygotic nodal by the initial gastrula stage on the one side of the blastopore margin and induces the expression of goosecoid, not-like, chordin and brachyury1 genes in this region, as in the oral ectoderm of sea urchin embryos, which provides a basis for the formation of the dorsal structures. The striking similarity in the gene regulations and their respective expression domains when comparing dorsal formation in amphioxus and the determination of the oral ectoderm in sea urchin embryos suggests that chordates derived from an ambulacrarian-type blastula with dorsoventral inversion.

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Species referenced: Echinodermata
Genes referenced: actr2 bmp2 chrd lefty1 LOC100887844 LOC115919910 LOC590297 LOC593805 nodall parp2 pole tubgcp2 wnt8a

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	Figure 1. Sperm entry site and location of male pronucleus. (a(i–iv)) Four examples of sperm entry sites (arrowheads) at 1–2 min after insemination. (b(i–iv)) Varied locations of male pronucleus (arrowheads) at 5 min after insemination. (c) Histograms for sperm entry site (n = 48) and location of male pronucleus (n = 76) collectively showing each in a half circle; both show the number observed on/in sector divided by 10° in rendering image of 10 sections with 0.5 µm interval. Scale bar, 100 µm.
	Figure 2. Maternal and zygotic expression of nodal and lefty genes. Animal pole to the top for all but a(iii–iv), c(i), d(i), e(i), c(v), d(v) and e(v). (a(i–ix)) Maternal expression of nodal gene showing gradual asymmetrical pattern (arrowheads). (a(x)) Initial zygotic expression of nodal in lefty expression domain (arrowhead). (b(i–v)) Onset of lefty zygotic expression at 32-cell stage on the one side (arrowhead). (c(i), d(i)) Expression domain of nodal (c) and lefty (d) at blastula stage viewed from vegetal side. (c(ii–iv), d(ii–iv)) Lateral view (anterior to the left) of nodal (c) and lefty (d) expression from initial gastrula to late gastrula stage. Note attenuation of both gene expressions at very margin of blastopore (arrowheads). (c(v), d(v)) Dorsal view of nodal (c) and lefty (d) expression at late gastrula stage. (e(i–v)) Expression of lefty in SB505124-treated embryos. Note changes in expression pattern with strong expression spots in blastula (arrowheads in e(i)), shrunken blastopore and archenteron (arrowheads in e(iv)), and mid-dorsal deformation (arrowhead in e(v)). (e(i)) vegetal view, (e(ii–iv)) lateral view and (e(v)) dorsal view. Bl, blastula; iG, initial gastrula; lG, late gastrula; mG, mid-gastrula. Scale bar, 100 µm.
	Figure 3. Expression of lefty and wnt8 genes in single embryos. (a,b) Lateral view (animal pole to the top) of double WISH with lefty (red arrowhead) and wnt8 (black arrowhead) probes at late blastula stage. Double colour precipitates (a) and single colour precipitates (b). (c,d) Future dorsovegetal view of the same samples as (a) and (b), respectively. Note apex pointing to vegetal pole (arrowheads). (e) Lateral view (animal pole to the top) of double WISH with lefty (red arrowhead) and wnt8 (black arrowhead) at initial gastrula stage. (f) Blastopore view (dorsal to the right) of the same sample as (e). Note a wide mid-dorsal domain of lefty (arrowheads). (g,h) Scanning electron microscopic (SEM) montages showing expression domains of lefty (light blue) and wnt8 (purple) at initial and mid-gastrula stage. Original SEM micrograph films from the late Dr R. Hirakow. bp, blastopore; pg, archenteron. Scale bar, 100 µm.
	Figure 4. Distribution pattern of active Arp2/3 complex in unfertilized and fertilized egg and its co-localization with microtubules and actin filaments. Partial rendering lateral images near maximum diameter in all but (d(iv–vi)), which are viewed vegetally near vegetal pole. (a) Eggs are oriented by the aid of polar body (pb) and/or vegetal tuft-like structure (tu). (b(i–vi)) Change in location of cortical pArp2 immunopositive signals after sperm fusion from ubiquitous in cortical region in unfertilized egg (b(i)) to on the one side of egg (b(vi)) passing through animal pole (b(iii,iv)) (arrowheads). (c(i–iii)) Co-localization of F-actin and pArp2 immunopositive signals in late fertilized egg. (d(i–vi)) Co-localization of microtubules and pArp2 immunopositive signals in late fertilized egg. Note a tuft-like structure at the vegetal pole in immunostain for pArp2 and for tubulin (d(ii)). Scale bar, 100 µm.
	Figure 5. Distribution patterns of maternal nodal mRNA and Arp2/3 complex. (a(i)–h(i)) Maternal nodal mRNA (red) and pArp2 immunopositive signals (green) are co-localized from one-cell to blastula stage shown as partial rendering images near maximum diameter. Animal pole to the top in (a(i)), (d(i)) and (h(i)). (a(ii)–h(ii)) Relative fluorescent intensity curve at section denoted by white arrow that indicates direction of x-axis. Red for nodal and green for pArp2. Note a tuft-like immunopositive for anti-pArp2 antibody in a cell at 16- and 64-cell stage (arrowheads) (e(i),g(i)). (i(i–iii)) Expression pattern of nodal in CK666-treated blastula to late gastrula stage. (j(i–iii)) Expression pattern of lefty in CK666-treated blastula to late gastrula stage. Note expression at median furrow (arrowheads) and a half of embryo in both genes. (k(i–iii)) Co-localization of pArp2 immunopositive signals and nodal mRNA in CK666-treated blastula. Bl, blastula; bp, blastopore; iG, initial gastrula; lG, late gastrula. Scale bar, 100 µm.
	Figure 6. Disappearance of downstream target gene expressions in embryos treated with Nodal signalling inhibitor (SB505124). All lateral view (anterior to the left) except late blastula stage. Initial expressions are denoted by red arrowheads. (a(i–ix)) Zygotic expression of goosecoid in untreated (a(i–v)) and short-term-treated (a(vi–ix)) embryos. (b(i–ix)) Zygotic expression of chordin in untreated (b(i–v)) and short-term-treated (b(vi–ix)) embryos. (c(i–ix)) Zygotic expression of not-like in untreated (c(i–v)) and short-term-treated (c(vi–ix)) embryos. (d(i–ix)) Zygotic expression of brachyury1 in untreated (d(i–v)) and short-term-treated (d(vi–ix)) embryos. (e) Transverse section of untreated neurula showing differentiating dorsal structures. (f) Transverse section of short-term-treated neurula showing lack of dorsal structures. Note shrunken blastopore and archenteron (arrowheads), expanded expression of brancyury1 in archenteron (arrowhead in d(viii)) and retained non-mid-dorsal expression in treated embryos (arrowheads in a(ix) and d(ix)). ch, notochord; ep, epidermis; g, gut; iG, initial gastrula; lBl, late blastula; lG, late gastrula; mG, mid-gastrula; N, neurula; np, neural plate; s, somite. Scale bar, 100 µm for all, but 50 µm for (e) and (f).
	Figure 7. Expression pattern of bmp2/4 and wnt8 in embryos untreated and treated with inhibitors. (a(i–vii)) Maternal and zygotic (red arrowheads) expression of bmp2/4 in untreated embryos. (a(vi)) Double WISH for bmp2/4 (red arrowheads) and lefty (white arrowhead) showing the direction of bmp2/4 gradient. Blastopore view. (a(vii)) Blastopore view of early neurula showing clear contrast of expression between paraxial mesoderm and axial structures (red arrowheads) in its magnification. (b(i–iii)) Expression pattern of SB505124-short-term-treated embryos showing enhanced and ectopic expression (arrowhead in b(iii)). (c(i)) Co-localization of bmp2/4 mRNA (white arrowhead) and anti-pSmad1 immunopositive signals (yellow arrowhead) at early gastrula (partial rendering image in left lateral view). (c(ii–iii)) Opposed distributions of bmp2/4 mRNA and anti-pSmad1 immunopositive signals at mid-gastrula (c(ii): partial rendering image in left lateral view) and early neurula (c(iii): partial rendering image viewed from blastopore). Note nuclear accumulation of pSmad1 in ventral cells (yellow arrowheads). Green signals in archenteron are non-specific. (d(i–iii)) Expression of wnt8 in untreated embryos (lateral view). (e(i–iii)) Expression of wnt8 shifted towards vegetal pole (arrowheads in e(i–ii)) and its expansion in archenteron (arrowheads in e(iii)) in SB505124-short-term-treated embryos (lateral view). (f(i–iii)) Expression pattern of wnt8 in CK666-treated embryos showing bifurcated expression (arrowheads in f(i)) and double gastrulation (arrowheads in f(iii)). Bl, late blastula; ch, notochord; iG, initial gastrula; lG, late gastrula; mG, mid-gastrula; N, neurula; np, neural plate; pg, archenteron; pm, paraxial mesoderm. Scale bar, 100 µm.
	Figure 8. Phylogenetic relationship between chordate dorsal structures and ambulacrarian oral ectoderm. Shared molecular patterning of ambulacrarian oral ectoderm and amphioxus dorsal specification consistent with dorsoventral inversion hypothesis occurred in chordate lineage. The last common ancestor of deuterostomes is parsimoniously supposed to have passed through a coeloblastula with asymmetrical nodal–lefty expression, but it remains unknown whether this blastula type is ancestral or a derived character. Ambulacrarian pattern is adapted from [25,66,67].

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