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Dev Biol
2007 Oct 01;3101:113-28. doi: 10.1016/j.ydbio.2007.07.031.
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The small GTPase RhoV is an essential regulator of neural crest induction in Xenopus.
Guémar L
,
de Santa Barbara P
,
Vignal E
,
Maurel B
,
Fort P
,
Faure S
.
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In vertebrates, the Rho family of GTPases is made of 20 members which regulate a variety of cellular functions, including actin cytoskeleton dynamics, cell adhesion and motility, cell growth and survival, gene transcription and membrane trafficking. To get a comprehensive view of Rho implication in physiological epithelial-mesenchymal transition, we carried out an in situ hybridization-based screen to identify Rho members expressed in Xenopus neural crest cells, in which we previously reported RhoB expression at the migrating stage. In the present study, we identify RhoV as an early expressed neural crest marker and provide evidence that its activity is essential for neural crest cell induction. RhoV mRNA is maternally expressed and accumulates shortly after gastrulation in the neural crest forming region. Using antisense morpholino injection, we show that at neurula stages, RhoV depletion impairs expression of the neural crest markers Sox9, Slug or Twist but has no effect on Snail induction. At the tailbud stage, RhoV knockdown causes a dramatic loss of cranial neural crest derived structures. All these defects are rescued by ectopic wild-type RhoV, whose overexpression on its own expands the neural crest territory. Our findings disclose an unprecedented Rho function in pathways that control neural crest cells specification.
Fig. 2. Developmental RhoV mRNA expression. In situ hybridization of stages 10.5 and 11 (A, B) (vegetal views, dorsal towards the top). At the onset of gastrulation, RhoV is expressed in the dorsal marginal zone. Xbra expression at stage 11 is shown for comparison (C, vegetal view, dorsal towards the top). Shown in panels B′ and C′ are sagittal hemisections across the transverse lines in panels B and C, respectively (dorsal side on the right). At mid-gastrula stages, RhoV and Xbra stainings are detected in the involuting dorsal mesodermal cells (idm). At the end of gastrulation (stages 12 and 12.5, D–G), RhoV mRNA localizes at the dorsal side of the embryo above the blastopore (bl). From stage 12, a second domain of expression is detected at the lateral edges of the neural plate (black arrowheads), as compared with Snail-positive neural crest territory (H, black arrowheads). Shown are vegetal views of stage 12 (D) and stage 12.5 (E), dorsal views of stage 12.5 (F–H). RhoV/Snail (I and J) and RhoV/Sox9 (L and M) double in situ hybridization analyses. Shown in panels J and M are magnified views of the area boxed respectively in panels I and L. (K) Single Sox9 in situ hybridization is shown for comparison. At early neurula stages (13–14) (N, P), RhoV mRNA appears as an arc surrounding the entire neural plate border, covering both the lateral (black arrowheads) and the anterior (white arrowhead) neural plate border as observed for Snail (O). At stage 15, RhoV (Q) like Snail (R) is expressed in the anterior (white arrowhead) neural plate border and in the medial (red arrowhead) and lateral (black arrow) neural crests. Shown in panels Q′ and R′ are sagittal hemisections across the transverse lines in panels Q and R. (S) RhoV/Snail double in situ hybridization analysis. Like Snail, RhoV is expressed in the medial (red arrowhead) and lateral (black arrow) neural crests. (T) Control Sox9 in situ hybridization is shown for comparison. Sox9 is expressed faintly in the medial and highly in the lateral (black arrow) neural crest and in the otic placodes (green arrow). (U) RhoV/Sox9 double in situ hybridization analysis. Note that RhoV is not expressed in the otic placodes stained by Sox9. At the onset of neural tube closure (stage 19, V), expression of RhoV is down-regulated in the neural crest territory. From stage 22 (X, dorsal view, anterior towards the top; Y, lateral view, anterior towards the right), RhoV expression is no longer detected in the neural crest.
Fig. 3. Canonical Wnt signaling regulates expression of RhoV in the neural crest. (A) Two-cell stage embryos were injected in one blastomere with 100 pg of plasmid DNA encoding mouse Wnt-1. DNA was used instead of mRNA to reduce expression and avoid axis duplication. (B) Alternately, embryos were injected with 1 ng of mRNA encoding human GSK3β. At this level, expression of GSK3β has no effect on mesoderm development (data not shown). At neurula stages, embryos were analyzed by whole-mount in situ hybridization for the neural crest markers Sox9 (a), Snail (b) and RhoV (c). Expression of all neural crest markers and RhoV was laterally expanded upon canonical-Wnt signaling activation, and conversely down-regulated upon Wnt inhibition. In all panels, the lineage tracer β-galactosidase mRNA was co-injected to visualize the injected side (red staining).
Fig. 5. Early neural crest marker expression is impaired in RhoV-depleted embryos. (A) Comparison of RhoVa and RhoVb mRNA initiator regions. Shown in red are the nucleotides that differ between the two sequences. RhoV-MO was designed to target the sequence underlined in black. (B) Western blot analysis of lysates prepared from control embryos (Uninjected) or embryos expressing the RhoV-GFP fusion alone (no MO) or co-injected with 20 ng per embryo of control MO (Ctrl-MO), RhoV-MO or RhoU-MO. Arrow indicates the position of the RhoV-GFP fusion. Only RhoV-MO inhibited RhoV-GFP translation. (C) Western Blot analysis of lysates prepared from control embryos (Uninjected), or embryos expressing the RhoU-GFP fusion alone (no MO) or co-injected with 20 ng per embryo of control MO (Ctrl-MO), RhoV-MO or RhoU-MO. Arrow indicates the position of the RhoV-GFP fusion. RhoV-MO does not inhibit RhoU-GFP translation. (D) Xbra and MyoD in situ hybridization analyses. Embryos were injected in one cell of two-cell stage embryo with 20 ng of RhoV-MO or control MO. β-galactosidase mRNA was co-injected to identify the injected side (red staining). Embryos were harvested at gastrula (a–d) and neurula stages (e, f) and analyzed respectively for Xbra and MyoD stainings. Shown are vegetal (a, c) or dorso-vegetal (b, d) or lateral (e, f) views. Control non-injected side (e) and RhoV-MO-injected side (f) of the same embryo. (E) In situ hybridization of early neurula stage embryos injected in one cell of two cell-stage embryos with 20 ng of control MO (Ctrl-MO) or RhoV-MO. Dorsal views, anterior to the top. RhoV depletion inhibits or strongly reduces the expression of the early neural crest markers Sox9 (b), Slug (d), Sox10 (e) and Twist (f) but has little effect on Snail expression (g). Note that depletion of RhoV also reduces the expression of Sox9 in the otic placodes (b). Injection of Ctrl-MO has no effect on Sox9 (a) or Slug (c). (F) Western Blot analysis of lysates from control embryos (Uninjected) or embryos expressing a RhoV-GFP fusion alone (no MO) or in combination with 20 ng of control MO (Ctrl-MO) or RhoV-MO, and embryos expressing a GFP-RhoV fusion alone (no MO) or in combination with 20 ng of RhoV-MO. Arrow indicates the position of GFP-RhoV and RhoV-GFP. GFP-RhoV mRNA is insensitive to RhoV-MO. (G) The loss of Sox9 expression upon RhoV depletion is rescued by injection of mRNA (100 pg) encoding GFP-RhoV or the same amount of its closely relative GFP-RhoU. The translation of GFP-Rho fusion proteins is insensitive to RhoV-MO (see panels C and F). Note that expression of RhoB is not able to restore Sox9 expression. In all cases, the injected side is oriented to the right (red staining is from the lineage tracer β-Gal).
Fig. 7. RhoV overexpression expands the neural crest territory. (A) In situ hybridization analysis of neurula stage embryos injected at two-cell stage with 100 ng of wt-RhoV mRNA and the β-galactosidase mRNA lineage tracer. Sox9 (a), Sox10 (b), Slug (c), Snail (d) and Sox2 (e) analysis on early neurula stage embryos. Twist (f, g) analysis on late neurula stage embryos. At early neurula stage, the ectopic expression of wt-RhoV enlarges the domain stained by all neural crest markers and reduces the domain stained by the neural plate marker Sox2 as indicated by black arrows. At late neurula stage, forced expression of RhoV led to an increase in Twist signal on the injected side (g) compared to the control side (f). (B) Expression of wt-RhoV does not affect cell proliferation. Two-cell stage embryos were co-injected in one cell with 100 ng of wt-RhoV and the β-galactosidase mRNA lineage tracer (red staining). At early neurula stage, injected embryos were processed either for Sox9 in situ hybridization (a) or for anti-phospho-histone H3 staining by immunochemistry (b). β-galactosidase activity was revealed using Red-Gal (a) or X-Gal (b). A magnified view of the area boxed in panel b is shown in panel c. No significant difference was observed in the number of mitotic cells in either side of the embryo. (C) The expansion of the neural crest territory induced by RhoV does not require cell proliferation. Two-cell stage embryos were co-injected in one cell with 100 ng of wt-RhoV mRNA and β-galactosidase mRNA as a tracer. At stage 11, control (a, b) and injected embryos (c, d) were treated (b, d) or not (a, c) with HUA and fixed at stage 15. Injected embryos were processed for Sox9 in situ hybridization and control embryos for anti-phospho-histone staining H3. Sox9 expansion (c) is not affected by HUA treatment (d), whereas it induced a 90% reduction in the number of mitotic cells (compare panel b with a).
Fig. 4. The loss of RhoV differentially affects the induction of neural crest markers. Embryos were injected in one blastomere at the two-cell stage with 1 ng of T50N-RhoV mRNA and analyzed at the neurula stage for Sox9 (a), Slug (b) and Snail (c) expression. β-galactosidase mRNA was co-injected with T50N-RhoV mRNA to identify the injected side (red staining), oriented to the right in all panels. The loss of RhoV strongly reduced Sox9 (a) and Slug (b) expression on the injected side of the embryo while Snail (c) was not affected.
Fig. 5. Early neural crest marker expression is impaired in RhoV-depleted embryos. (A) Comparison of RhoVa and RhoVb mRNA initiator regions. Shown in red are the nucleotides that differ between the two sequences. RhoV-MO was designed to target the sequence underlined in black. (B) Western blot analysis of lysates prepared from control embryos (Uninjected) or embryos expressing the RhoV-GFP fusion alone (no MO) or co-injected with 20 ng per embryo of control MO (Ctrl-MO), RhoV-MO or RhoU-MO. Arrow indicates the position of the RhoV-GFP fusion. Only RhoV-MO inhibited RhoV-GFP translation. (C) Western Blot analysis of lysates prepared from control embryos (Uninjected), or embryos expressing the RhoU-GFP fusion alone (no MO) or co-injected with 20 ng per embryo of control MO (Ctrl-MO), RhoV-MO or RhoU-MO. Arrow indicates the position of the RhoV-GFP fusion. RhoV-MO does not inhibit RhoU-GFP translation. (D) Xbra and MyoD in situ hybridization analyses. Embryos were injected in one cell of two-cell stage embryo with 20 ng of RhoV-MO or control MO. β-galactosidase mRNA was co-injected to identify the injected side (red staining). Embryos were harvested at gastrula (a) and neurula stages (e, f) and analyzed respectively for Xbra and MyoD stainings. Shown are vegetal (a, c) or dorso-vegetal (b, d) or lateral (e, f) views. Control non-injected side (e) and RhoV-MO-injected side (f) of the same embryo. (E) In situ hybridization of early neurula stage embryos injected in one cell of two cell-stage embryos with 20 ng of control MO (Ctrl-MO) or RhoV-MO. Dorsal views, anterior to the top. RhoV depletion inhibits or strongly reduces the expression of the early neural crest markers Sox9 (b), Slug (d), Sox10 (e) and Twist (f) but has little effect on Snail expression (g). Note that depletion of RhoV also reduces the expression of Sox9 in the otic placodes (b). Injection of Ctrl-MO has no effect on Sox9 (a) or Slug (c). (F) Western Blot analysis of lysates from control embryos (Uninjected) or embryos expressing a RhoV-GFP fusion alone (no MO) or in combination with 20 ng of control MO (Ctrl-MO) or RhoV-MO, and embryos expressing a GFP-RhoV fusion alone (no MO) or in combination with 20 ng of RhoV-MO. Arrow indicates the position of GFP-RhoV and RhoV-GFP. GFP-RhoV mRNA is insensitive to RhoV-MO. (G) The loss of Sox9 expression upon RhoV depletion is rescued by injection of mRNA (100 pg) encoding GFP-RhoV or the same amount of its closely relative GFP-RhoU. The translation of GFP-Rho fusion proteins is insensitive to RhoV-MO (see panels C and F). Note that expression of RhoB is not able to restore Sox9 expression. In all cases, the injected side is oriented to the right (red staining is from the lineage tracer β-Gal).
