Arakawa A et al. (2007), The secreted EGF-Discoidin factor xDel1 is esse...

XB-ART-35817

Dev Biol 2007 Jun 01;3061:160-9. doi: 10.1016/j.ydbio.2007.03.008.

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The secreted EGF-Discoidin factor xDel1 is essential for dorsal development of the Xenopus embryo.

Arakawa A , Matsuo-Takasaki M , Takai A , Inomata H , Matsumura M , Ikeya M , Takahashi K , Miyachi Y , Sasai N , Sasai Y .

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We show here that a secreted EGF-Discoidin-domain protein, Xenopus Del1 (xDel1), is an essential factor for dorsal development in the early Xenopus embryo. Knockdown of the xDel1 function causes obvious ventralization of the embryo. Conversely, overexpression of xDel1 expands dorsal-marker expression and suppresses ventral-marker expression in the gastrula embryo. Forced expression of xDel1 dorsalizes ventral marginal zone explants, whereas it weakly induces neural differentiation but not mesodermal differentiation in animal caps. The dorsalizing activity of xDel1 is dependent on the Discoidin domains and not on the RGD motif (which is implicated in its angiogenic activity) or EGF repeats. Luciferase assays show that xDel1 attenuates BMP-signaling reporter activity by interfering with the pathway downstream of the BMP receptor. Thus, xDel1 functions as a unique extracellular regulatory factor of DV patterning in early vertebrate embryogenesis.

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Species referenced: Xenopus
Genes referenced: babam2 bmp4 chrd edil3 egf gsc smad2 smad6 szl tbxt ventx1.2 wnt8a
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	Fig. 1. Spatial and temporal pattern of xDel1 expression analyzed by whole-mount in situ hybridization. (A) The 8-cell stage, lateral view. (B) Stage 9, lateral view. (C) Stage 11, vegetal view. Arrowhead, dorsal lip. (D) Stage 11, sagittal section across the dorsal lip (white arrow). Black arrow, bottle cells. Open and closed arrowheads, dorsal ectoderm and mesoderm, respectively. (E) Mid-neurula stage, dorsal view. (F) Late-neurula stage, dorsal view.
	Fig. 2. Knockdown of xDel1 causes ventralization of the Xenopus embryo. (A) External appearance at the late-tailbud stage. Top, control. Middle and bottom, xDel1-MO-injected embryos (MO-A and -B, 6.25 ng/cell each at the 4-cell stage). (B, E, H, K) Uninjected gastrula embryos. (C, F, I, L) xDel1-MO-injected embryos. (D, G, J, M) Embryos injected with 5-base-mispaired control MO. (N, O) Co-injection of xDel1-MO and xDel1 RNA (coding). Embryos were analyzed by whole-mount in situ hybridization with a probe for Chd (B–D, N), Gsc (E–G), Vent1 (H–J, O) or Szl (K–M).
	Fig. 3. Knockdown of xDel1 causes ventralization in DMZ explants. (A) Control DMZ explants (equivalent to the mid-neurula stage). (B) xDel1-MO-injected DMZ explants. (C) RT–PCR analysis of DMZ explants (equivalent to stage 11): lane 1, whole embryo; lane 2, uninjected DMZ; lane 3, xDel1-MO-injected DMZ; lane 4, DMZ injected with 5-base-mispaired MO.
	Fig. 4. Overexpression of xDel1 induces dorsal markers and suppresses ventral ones. Whole-mount in situ hybridization analysis with a probe for Chd (A, B), Gsc (C, D), Vent1 (E, F) or Szl (G, H). (A, C, E, G) Control. (B, D, F, H) Injection of xDel1 RNA (400 pg/cell, 4-cell stage) into the right blastomeres.
	Fig. 5. Dorsalization of MZ explants by xDel1. (A–C) External appearance of VMZ explants (equivalent to the mid-neurula stage). Embryos (4-cell stage) were radially injected with control (A), xDel1 (B) or Chd (100 pg/cell; C) RNA, and VMZ explants were prepared at stage 10.5. (D) RT–PCR analysis of Gsc, Chd, Wnt8 (lateral–ventral) and Vent1 (ventral) expression in the VMZ explants (stage 11); lane 1. whole embryo; lane 2, control VMZ; lane 3, xDel1-injected VMZ; lane 4, Chd-injected VMZ. (E) Animal cap assay. RT–PCR analysis of the mesodermal markers Xbra and Gsc: lane 1, whole embryo; lane 2, control animal caps; lane 3, xDel1-injected animal caps; lane 4, CA-Smad2-injected animal caps.
	Fig. 6. The RGD motif is not required for the dorsalizing activity of xDel1. (A) Schematic of mutant xDel1 constructs (xDel1-E2 type; see Materials and methods). EGF, EGF repeat; RGD, RGD motif; Discoidin, Discoidin domain. (B) RT–PCR analysis of RNA-injected VMZ explants for dorsalizaton assay (late neurula stage-equivalent): lane 1, whole embryo; lane 2, control DMZ; lane 3, control VMZ; lane 4, wild-type xDel1-injected VMZ; lane 5, xDel1-RGE mutant-injected VMZ; lane 6, xDel1-delN mutant-injected VMZ; lane 7, xDel1-delC mutant-injected VMZ.
	Fig. 7. BRE-reporter activity is attenuated by xDel1. Animal caps injected with reporters were excised at stage 9 and harvested for the luciferase assay at stage 11. (A) The inhibitory effect of xDel1 (200 pg/cell, radial injection into all animal cells at the 8-cell stage) on BRE-reporter activity in the animal cap (lane 2). BMP4 (2.5 pg/cell, lane 4) and Chd (50 pg/cell, lane 3) were used as positive controls for activation and inhibition, respectively. (B) Injection of xDel1 (lane 3) inhibited the activation effect by BMP4 (lane 2). BMP4 + Chd (lane 4), positive control. (C) Injection of xDel1 (lane 3; 200 pg/cell) inhibited the activation effect by CA-BMPR (lane 2; 5 pg/cell). CA-BMPR + Smad6 (800 pg/cell; lane 4), positive control; CA-BMPR + Chd (lane 5), negative control. (D) RT–PCR analysis of animal cap explants: lane 1, whole embryo at stage 15; lane 2, control animal caps; lane 3, xDel1-injected animal caps; lane 4, Chd-injected animal caps.
	Supplementary Fig. S1. (A) Domain structures of two splicing variants of xDel1. The shorter variant (xDel1-E2) is generated by alternative splicing (in-frame) within the first EGF repeat. SP, signal peptide; RGD, the integrin-binding RGD motif. (B) Quantitative PCR analysis of expression level of xDel1 mRNA in DMZ and VMZ tissues. There is no substantial difference between them. Chd, positive control as a dorsal gene; Szl, positive control as a ventral gene. Although xDel1 expression appeared slightly stronger on the dorsal side in whole-mount in situ hybridization ( Fig. 1C), it is likely that it reflected the higher cell density on the dorsal side.
	Supplementary Fig. S2. (A) The sequence design of MO-A and the control 5-base-mispaired MO. The sequences of pseudoalleles a and b are shown below. The red letters stand for the initial Met codon. (B) The sequence design of MO-B and the control 5-base-mispaired MO. (C) Western blot analysis of translates from xDel1 (pseudoallele a; tagged with V5) RNA that contained the 5′ UTR sequence. The translation of xDel1 in the animal cap (stage 9) was specifically inhibited by co-injection of MO-A, but not by that of the control MO. (D) Western blot analysis of translates from xDel1 (pseudoallele b; tagged with V5) RNA that contained the 5′ UTR sequence. The translation of xDel1 was specifically inhibited by co-injection of MO-B, but not by that of the control MO. (E) Percentages of the xDel1 MO-injection phenotypes (suppression of Chd and Gsc expression, and upregulation of Vent1 and Szl expression). Top, MO-A alone; middle, MO-B alone; bottom, both combined.
	Supplementary Fig. S3. (A) Effects of Chd-MO co-injection on xDel1-induced dorsalization in the VMZ assay. Chd (A), Gsc (B) and Myf-5 (C) were used as dorsal markers, and Szl (D) and Vent1 (E) were used as ventral markers.