Ossipova O et al. (2018), Regulation of neural crest development by the f...

XB-ART-54817

Genesis 2018 Jun 01;566-7:e23108. doi: 10.1002/dvg.23108.

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Regulation of neural crest development by the formin family protein Daam1.

Ossipova O , Kerney R , Saint-Jeannet JP , Sokol SY .

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The neural crest (NC) multipotent progenitor cells form at the neural plate border and migrate to diverse locations in the embryo to differentiate into many cell types. NC is specified by several embryonic pathways, however the role of noncanonical Wnt signaling in this process remains poorly defined. Daam1 is a formin family protein that is present in embryonic ectoderm at the time of NC formation and can mediate noncanonical Wnt signaling. Our interference experiments indicated that Daam1 is required for NC gene activation. To further study the function of Daam1 in NC development we used a transgenic reporter Xenopus line, in which GFP transcription is driven by sox10 upstream regulatory sequences. The activation of the sox10:GFP reporter in a subset of NC cells was suppressed after Daam1 depletion and in embryos expressing N-Daam1, a dominant interfering construct. Moreover, N-Daam1 blocked reporter activation in neuralized ectodermal explants in response to Wnt11, but not Wnt8 or Wnt3a, confirming that the downstream pathways are different. In complementary experiments, a constitutively active Daam1 fragment expanded the NC territory, but this gain-of-function activity was eliminated in a construct with a point mutation in the FH2 domain that is critical for actin polymerization. These observations suggest a new role of Daam1 and actin remodeling in NC specification.

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Species referenced: Xenopus laevis
Genes referenced: chrd daam1 fmn1 foxd3 h2bc21 myod1 nrp1 pax3 snai2 sox10 sox2 sox8 twist1 wnt11 wnt11b wnt3a wnt8a
GO keywords: actin polymerization or depolymerization [+]
Lines/Strains:

Xla.Tg(sox10:GFP)Jpsj

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	FIGURE 2 Spatial and temporal domains of the sox10:GF P reporter expression during development. GFP reporter expression in Tg(sox10:GFP) embryos at different stages. a, b, stages 19, 23 . White arrowhead in b marks hindbrain expression, the arrow points to migrating NC cells. c, stage 25. Major cranial migratory streams are indicated by arrows, white arrowhead points to the otic vesicle, yellow arrowhead to the hindbrain domain. d, stage 33/34. GFP fluorescence is visible in the otic vesicle (white arrowhead), and three branchial arches (arrows).Yellow arrowheads point to the expression in the eye (a positive control GFP driven by the gamma-crystallin promoter, see Methods) and the hindbrain. (c, d) Lateral view is shown, anterior is to the left. (e) stage 33/34, dorsal view. Strong GFP signal remains in the hindbrain,weaker signals are detected in the forebrain and the otic vesicle.
	FIGURE 3 Characterization of GFP-positive cells in Tg(sox10:GFP) embryos. a, Tg(sox10:GFP) embryo ( stage 25) with GFP expression in the hindbrain and major migratory streams (dashed lines in a ). Approximate position of the section in b is shown by a solid line. b, Cross-section at the hindbrain level of a transgenic embryo double immunostained for GFP and FoxD3. Single (GFP) channel is shown. Nt, neuraltube; not, notochord; en, endoderm. c, High magnification of the boxed area in b shows that the majority of GFP-expressing cells are FoxD3-positive. Dashed lines indicate tissue boundaries.
	FIGURE 4 Wnt signaling activates sox10:GFP in neuralized ectoderm. a, Experimental scheme for induction of sox10:GFP in neuralized ectoderm. bâ€“g, Ectoderm explants were prepared at st age 10 from Tg(sox10:GFP) embryos injected with chordin RNA (150 pg, c) alone, or in combination with wnt8 (0. 8 ng, d), wnt11a (1 ng, e),wnt5a(1 ng, f), and wnt11b (1 ng, g) RNAs as indicated. The explants were cultured until the equivalent of stage 26 and imaged (b-g) or lysed for RT-PCR (h). h, GFP reporter is activated in transgenic ectoderm explants by chordin and wnt8 RNAs. i, Frequencies of GFP-positive caps (%) . Total number of explants is shown above each bar.
	FIGURE 5 Sox10:GFP activation is prevented by N-Daam1 and Daam1 MO. aâ€“c, Embryos were unilaterally injected with control MO (40ng, a), Daam1 MO (40 ng, b) or with N-Daam1 RNA (3 ng , c) as indicated. RFP-H2B RNA is a lineage tracer (1 00 pg, red). Dorsal views are shown, anterior is to the top. Arrows in b and c point to lack of reporter activation on the injected side. d, Frequencies of embryos with inhibited sox10:GFP expression. The number of embryos analyzed is indicated on top of each bar.
	FIGURE 6 Selective inhibition of Wnt signaling by N-Daam1 in neuralized explants. Ectoderm explants were dissected at stage 9â€“10 from Tg(sox 10:GFP) uninjected embryos, and from those injected with indicated RNAs. The explant s were cultured until the time when control embryos reached stage 28 and were imaged (aâ€“h). Chordin RNA was used either alone (150 pg, b) or in combination with different wntRNAs. N-Daam1 (850 pg) inhibits the response to wnt11a (2 ng, e, f), wnt11b (2ng,g,h),but not to wnt8 (700 pg, c, d) or wnt3a (525 pg, i and data not shown). i, Frequencies of GFP-positive explants (%) are representative of two different experiments. Total number of animal caps is shown above each bar.
	FIGURE 7 C-Daam1 with an intact FH2 domain promotes NC specification. C-Daam1 and C-Daam1 F H2 mutant domain structures are shown on the top. a, b, d, e, in situ hybridization of stage 15/16 embryos with indicated antisense RNA probes. Anterior views are shown. Light blue is the lineage tracer (nbGal). Arrowheads point to expanded foxd3 and sox8 in C- Daam1 RNA-injected embryos (10â€“20 pg, a, b).c, C-Daam1 up regulates NC gene expression in neuralized ectoderm. Ectoderm explants were prepared from embryos injected with C-Daam1 RNA (10â€“20 pg) or wnt8 RNA (0 .5 ng) and chordin RNA (150 pg) as indicated, cultured to stage 16 and processed for RT-PCR analysis. The explants coexpressing chord in and C-Daam1 upregulate pax-3 and sox8 but do not activate myod.d,e,C-Daam1-FH2* RNA (10â€“ 20pg) does not promote NC gene expression. f, Frequencies of embryos with altered NC marker expression. Total number of explants is shown on top of each bar.
	FIGURE 1 Daam1 is required for premigratory NC development. Eight-to-sixteen-cell embryos were unilaterally injected into animal blastomeres with Daam 1 MO (20 ng), control MO (CoMO, 20 ng) or N-Daam1 RNA (10â€“2 0 pg) as indicated. aâ€“c, eâ€“g, In situ hybridization of stage 14/16 embryos with different antisense RNA probes. The arrows (b, f, g) point to the injected side with inhibited NC marker. Light blue is the lineage tracer (nbGal). Anterior views are shown. NC territory did not change in wild-type (not shown) and CoMO-injected embryos (a, e). d, Daam1 domain structure and interactions are shown. h, Frequencies of embryos with altered NC marker expression. The graphs indicate the percentage of embryos with decreased gene expression. The number of embryos analyzed is shown on top of each bar.

References [+] :

Ang, DAAM1 is a formin required for centrosome re-orientation during cell migration. 2010, Pubmed, Xenbase