Glavic A et al. (2004), Interplay between Notch signaling and the homeo...

XB-ART-4249

Development 2004 Jan 01;1312:347-59. doi: 10.1242/dev.00945.

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Interplay between Notch signaling and the homeoprotein Xiro1 is required for neural crest induction in Xenopus embryos.

Glavic A , Silva F , Aybar MJ , Bastidas F , Mayor R .

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The neural crest is a population of cells that originates at the interface between the neural plate and non-neural ectoderm. Here, we have analyzed the role that Notch and the homeoprotein Xiro1 play in the specification of the neural crest. We show that Xiro1, Notch and the Notch target gene Hairy2A are all expressed in the neural crest territory, whereas the Notch ligands Delta 1 and Serrate are expressed in the cells that surround the prospective crest cells. We have used inducible dominant-negative and activator constructs of both Notch signaling components and Xiro1 to analyze the role of these factors in neural crest specification without interfering with mesodermal or neural plate development. Activation of Xiro1 or Notch signaling led to an enlargement of the neural crest territory, whereas blocking their activity inhibited the expression of neural crest markers. It is known that BMPs are involved in the induction of the neural crest and, thus, we assessed whether these two elements might influence the expression of Bmp4. Activation of Xiro1 and of Notch signaling upregulated Hairy2A and inhibited Bmp4 transcription during neural crest specification. These results, in conjunction with data from rescue experiments, allow us to propose a model wherein Xiro1 lies upstream of the cascade regulating Delta 1 transcription. At the early gastrula stage, the coordinated action of Xiro1, as a positive regulator, and Snail, as a repressor, restricts the expression of Delta 1 at the border of the neural crest territory. At the late gastrula stage, Delta 1 interacts with Notch to activate Hairy2A in the region of the neural fold. Subsequently, Hairy2A acts as a repressor of Bmp4 transcription, ensuring that levels of Bmp4 optimal for the specification of the neural plate border are attained in this region. Finally, the activity of additional signals (WNTs, FGF and retinoic acid) in this newly defined domain induces the production of neural crest cells. These data also highlight the different roles played by BMP in neural crest specification in chick and Xenopus or zebrafish embryos.

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Species referenced: Xenopus
Genes referenced: bmp4 dll1 foxd3 hes4 irx1 irx3 msx1 myc notch1 snai1 snai2 sox2 srrt tubb2b

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	Fig. 1. Comparison of Xiro1, Delta1, Serrate, Notch, Hairy2A and Xslug expression. Embryos were fixed at late gastrula (stage 12.5-13) or mid-neurula stage (stage 18-19), and double or single in situ hybridization was performed for each gene. The stages and probes analyzed are indicated in each figure. Anterior is towards the top, and the sections are shown with the dorsal side towards the top. d, dorsal; v, ventral; orange arrowhead, neural crest. (A-E) Comparison of Xiro1 and Xslug expression at the late gastrula stage. (A) Initial visualization of a double in situ hybridization of Xiro1 (green). Xiro1 has a dorsal or neural domain of expression, and transcripts are also found in the preplacode domain outside of the neural plate (star). (B) Section of an embryo stained as in A. (C) Visualization of Xslug in purple in the same embryo as shown in A, where Xiro1 expression is in green. As the dorsal and placodal domain (star) are continuous, Xiro1 overlaps with the neural crest territory (orange arrowhead). (D) Section of the embryo shown in C. (E) Higher magnification of the box highlighted in C. The continuity between the neural domain and the preplacode domain (star) is visible. (F-J) In situ hybridization for Delta1 and Slug. (F,G) Late gastrula embryos were sectioned and divided in two groups. One group was stained for Delta1 (F) and the other for Xslug (G). Note that Slug expression, in the neural crest (orange arrowhead), coincides with the gap in the expression of Delta1 at the neural folds. (H,I) Double in situ hybridization for Delta1 and Xslug; note that the cells expressing Xslug (arrowhead) are surrounded by cells expressing Delta1 (arrows). (I) Higher magnification of the box highlighted in H. (J) Section of embryos along the plane indicated in I; note that the cells expressing Xslug are surrounded by Delta1 expression in the deep layer of the ectoderm. (K) Double in situ hybridization of Serrate (purple) and Xslug (green), note that Xslug expressing cells (orange arrowhead) are surrounded by Serrate expressing cells (arrows) in the anterior neural crest region. (L,M) Expression of Notch can be seen in the neural plate and it overlaps with Xslug expression at the border of the neural plate (arrowheads). (N-P) Expression of Hairy2A (N,O: green in left panel and purple in the right panel of N, and green in O) can be detected in the entire neural fold, including the domain were Xslug is expressed (orange arrowhead). The bracket indicates the region in which Slug and Hairy2A expression overlap. The arrow (N,O) indicates the expression of Hairy2A in the prospective posterior neural crest. (P) The expression of both genes persists at the late neurula stage. A summary of the expression of all these genes is represented as a whole mount in Q, and in a section in R.
	Fig. 2. Activation of Notch signaling on mesoderm, neural plate and neural crest development. Two-cell stage embryos were injected with NICDGR mRNA in one blastomere, treated with dexomethasone either directly after the injection (A,C,E) or at stage 12 (B,D,F), and cultured until stage 25 (A,B) or stage 18-19 (C-F). Subsequently, the expression of distinct markers was analyzed. The side of the injection is indicated with an arrowhead. (A,B) Immunostaining of somites with the 12/101 antiserum. Note the expansion of the somite in the injected side following early activation (A), and the normal morphology after late activation (B). Following activation at stage 2, 78% of embryos demonstrated somite expansion (n=70), whereas activation at stage 12 did not produce any expansion of the somites (0%; n=87). (C,D) In situ hybridization to visualize Sox2 transcripts in the neural plate. Activation at stage 2 (C) leads to an expansion of the neural plate (65% of embryos with expanded neural plate; n=83), whereas activation at stage 12 (D) produces no effect on the neural plate (100% normal; n=92). (E,F) In situ hybridization to visualize Xslug transcripts in the cephalic neural crest. Activation at stage 2 (E) produces an inhibition in the expression of this neural crest marker (58% of inhibition; n=102), whereas activation at stage 12 (F) produces an expansion of Xslug expression (expanded in 75% of embryos; n=152).
	Fig. 3. Notch signaling is required for neural crest specification. Two-cell embryos were injected in one blastomere with 0.7 ng of NICDGR (A,E,I,M,Q), 0.7 ng of Su(H)ankGR (B,F,J,N,R), 1 ng of DeltaStu (C,G,K,O,S) or 0.25 ng of Su(H)DBMGR (D,H,L,P,T) mRNA, and the inducible constructs were activated at stage 12. NICDGR and Su(H)ankGR activate Notch signalling, and DeltaStu and Su(H)DBMGR inhibit Notch signalling. The expression of Xslug, Foxd3, Bmp4, Xmsx1 and Hairy2A was analyzed at stage 17 or 18 by in situ hybridization, and the injected sides were visualized by alkaline phosphatase-mediated FITC immunodetection. The injected side is labeled with an arrow and all embryos are presented dorsally with the anterior to the top. (A,B,E,F) Note the expansion of Xslug (A,B) and Foxd3 (E,F) expression on the injected side after activation of Notch signaling. (C,D,G,H) Note the inhibition in Xslug (C,D) and Foxd3 (G,H) expression on the injected side, after inhibition of Notch signaling. (I-L) The domain of expression of Bmp4 is highlighted in the neural folds by the brackets. Scale bar: 80 μm. Note the reduced expression domain after Notch activation (I,J), and the expansion and increase in the intensity of Bmp4 expression on the injected side after Notch inhibition (K,L). (M-P) Expression of Xmsx1. Note the expansion in the Xmsx1 expression domain after Notch activation (M,N) and the reduction of Xmsx1 expression on the injected side after Notch inhibition (O,P). (Q-T) Hairy2A expression. Note the expansion in Hairy2A expression in the injected side after Notch activation (Q,R) and the decrease in Hairy2A expression on the injected side after Notch inhibition (S,T). Each experiment was performed at least twice with a minimum of 45 embryos. The effect seen in each experiment was observed in at least 70% of embryos.
	Fig. 4. The Notch target gene Hairy2A produces an expansion of the neural crest population. Eight-cell embryos were injected in two blastomeres with 1 ng Hairy2A mRNA, the arrowhead indicates the injected side, which was visualized by alkaline phosphatase-mediated FITC inmunodetection. (A) N-tubulin expression is clearly reduced. (B) Immunodetection of the somite antigen 12/101 analyzed at stage 25. Note that there is no difference in the staining between the injected and uninjected side. (C) The domain of Xslug expression is expanded on the injected side, whereas Bmp4 is dramatically repressed (D,E). (E) Corresponds to a higher magnification of D. (F) Xmsx1 expression is increased on the injected side. Each experiment was performed at least twice with a minimum of 35 embryos. The effect seen in each experiment was observed in at least 70% of embryos.
	Fig. 5. Xiro1 participates in the induction of neural crest cells. Two-cell embryos were injected in one blastomere with 1 ng of the inducible forms of a repressor of Xiro1 (HDGREnR) (A,D,G,J), an activator form of Xiro1 (HDGRE1A) (B,E,H,K), or with a dominant-negative form of Xiro1 (HDGR) (C,F,I,L). The embryos were treated with dexomethasone at stage 12, and the expression of Xslug, Bmp4, Xmsx1 and Hairy2A was analyzed by in situ hybridization. The injected side was visualized by Myc inmunostaining, or alkaline phosphatase-mediated FITC inmunostaining, and is indicated with an arrowhead. (A-C) Xslug expression. (A) An expansion of the Xslug expressing neural crest domain is observed. (B,C) Xslug expression is reduced on the injected side. (D-F) Bmp4 expression. (D) A repression of Bmp4 in the neural fold domain is indicated by the bracket. (E,F) The levels of Bmp4 transcripts are augmented on the injected side and an expansion in the expression domain is also observed. Note that in F, the expression indicated by two small brackets on the uninjected side is transformed into a single big bracket on the injected side. Scale bar: 85 μm. (G-I) Xmsx1 expression. (G) Note the expanded Xmsx1 expression domain. (H,I) A reduction in the expression of Xmsx1 can be seen in the neural fold region. (J-L) Hairy2A expression. (J) An expanded domain of Hairy2A expression is observed in the neural fold, whereas Hairy2A expression is inhibited by the injection of HDGRE1A and HDGR (K,L). Each experiment was performed at least twice with a minimum of 42 embryos. The effect seen in each experiment was observed in at least 65% of embryos.
	Fig. 6. Xiro1 is upstream of Notch signaling in the specification of the neural crest. Embryos were injected with 1 ng HDGR (A) mRNA, and co-injected with 1 ng Hairy2A (B) or 1 ng Su(H)ankGR (C) mRNA. A second set of experiments was performed by injecting two-cell embryos in one blastomere with 0.25 ng Su(H)DBMGR (D) mRNA, and co-injecting 1 ng Hairy2A (E) or 0.7 ng Xmsx1GR (F) mRNA. Finally, a third set of experiments was performed by injecting one blastomere of a two-cell embryo with 1 ng of NICDGR (G) mRNA, and co-injecting 0.7 ng of dnXmsxGR (H). The embryos were treated with dexomethasone at stage 12, and the expression of Xslug was analyzed by in situ hybridization between stage 17 and 19. The injected side was visualized by alkaline phosphatase-mediated FITC inmunodetection and is indicated by an arrowhead. (A) Xslug expression was inhibited by HDGR. (B) The inhibition of Xiro1 activity was rescued by co-injection of Hairy2A, reaching 89% recovery of Xslug expression (n=56). (C) A similar reversion of Xiro1 inhibition was obtained by activating Notch signaling, 93% rescue of Xslug expression was observed (n=47). (D) Xslug expression was inhibited by Su(H)DBMGR. (E) The inhibition of the Notch signaling could be rescued by co-expression of Hairy2A (92% rescue; n=43). (F) The effect of inhibiting Notch signaling could be rescued by co-expression of Xmsx1 (97% rescue; n=39). (G) Expansion of Xslug expression by injecting 1 ng NICDGR. (H) The effect of NICDGR was rescued by blocking msx1 activity with dnXmsxGR, (92% rescue; n=45), whereas the effect of NICDGR was not rescued by the co-injection of GFP mRNA (I; 0% rescue; n=25).
	Fig. 7. Delta1 expression is upregulated by Xiro1 and down regulated by Snail. (A,B) Two-cell embryos were injected in one blastomere with 1 ng HDGREnR (A) or with 1 ng HDGR (B) mRNA. The embryos were treated with dexomethasone at stage 12 and the expression of Delta1 was analyzed by in situ hybridization at stage 17. The injected side was visualized by alkaline phosphatase-mediated FITC immunodetection. (A) HDGREnR produces a moderate expansion of Delta1 expression in the neural crest region (arrowhead), whereas HDGR leads to a complete inhibition of Delta1 expression in the crest region (B; arrowhead). (C-E) Animal caps taken from stage 9 embryos were cultured until the equivalent of stage 18, and the expression of Delta1 was analyzed by in situ hybridization. (C) In control animal caps, no expression of Delta1 could be detected. (D,E) In animal caps taken from embryos injected with 1 ng Xiro3 mRNA, Delta1 expression was observed in 87% of the caps (n=57). (E) Higher magnification of the animal cap shown in D. (F) RT-PCR to analyze Delta1 and H4 mRNA. Arrowheads in D,E indicate Delta1-expressing cells. Left panel, control embryo and PCR in the absence of reverse transcriptase; right panel, mRNA taken from a control animal cap, a cap injected with 1 ng Xiro3 mRNA, or a cap co-injected with 1 ng Xiro3 mRNA and 0.7 ng Xsnail mRNA. (G) Quantification of data shown above in F. Note the increase in Delta1 mRNA produced by Xiro3, and the complete inhibition produced by Xsnail. (H-J) Two-cell embryos were injected in one blastomere with 0.7 ng dnSnail mRNA. The embryos were treated with dexomethasone at stage 12, and the expression of Delta1 was analyzed by in situ hybridization at stage 17. The injected side was visualized by alkaline phosphatase-mediated FITC inmunodetection. (H) Delta1 expression is upregulated in the neural crest region. (I,J) Higher magnification of the neural crest region indicated by the box in H, where the staining was stronger on the injected (J) than on the uninjected side (I). Arrow, uninjected side; arrowhead, injected side. Each experiment was performed at least twice with a minimum of 52 embryos. The effect seen in each experiment was observed in at least 65% of embryos.