Peres JN et al. (2006), Interaction between X-Delta-2 and Hox genes reg...

XB-ART-34575

Mech Dev 2006 Apr 01;1234:321-33. doi: 10.1016/j.mod.2006.03.001.

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Interaction between X-Delta-2 and Hox genes regulates segmentation and patterning of the anteroposterior axis.

Peres JN , McNulty CL , Durston AJ .

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In vertebrates, the paraxial mesoderm already exhibits a complex Hox gene pattern by the time that segmentation occurs and somites are formed. The anterior boundaries of the Hox genes are always maintained at the same somite number, suggesting coordination between somite formation and Hox expression. To study this interaction, we used morpholinos to knockdown either the somitogenesis gene X-Delta-2 or the complete Hox paralogous group 1 (PG1) in Xenopus laevis. When X-Delta-2 is knocked down, Hox genes from different paralogous groups are downregulated from the beginning of their expression at gastrula stages. This effect is not via the canonical Notch pathway, as it is independent of the Notch effector Su(H). We also reveal for the first time a clear role for Hox genes in somitogenesis, as loss of PG1 gene function results in the perturbation of somite formation and downregulation of the X-Delta-2 expression in the PSM. This effect on X-Delta-2 expression is also observed during neurula stages, before the somites are formed. These results show that somitogenesis and patterning of the anteroposterior axis are closely linked via a feedback loop involving Hox genes and X-Delta-2, suggesting the existence of a coordination mechanism between somite formation and anteroposterior patterning. Such a mechanism is likely to be functional during gastrulation, before the formation of the first pair of somites, as suggested by the early X-Delta-2 regulation of the Hox genes.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 cad cdx4 dlc dll1 egr2 en2 fgf4 hes4 hes7.1 hes7.2 hoxb4 hoxb9 hoxc6 hoxc9-like hoxd1 myod1 notch1 tbxt
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	Fig. 3. X-Delta-2 and Hox genes are expressed in an overlapping domain during gastrulation. The expression patterns of X-Delta-2 (A,C,E) and Hoxd1 (B,D,F) were analysed by whole-mount in situ hybridisation. The expression of both genes at stage 101/2 (A,B), stage 11 (C,D) and stage 12 (E,F) shows an overlap in their domains of expression. Embryos at stage 101/2 (A,B) and 11 (C,D) are shown from the vegetal side with dorsal to the top, and those at stage 12 (E,F) from the dorsal side with anterior to the top.
	dlc (putative ortholog of delta-like protein C precursor) gene expression in Xenopus laevis embryos, NF stage 11, as assayed by in situ hybridization, vegetal view, dorsal up.
	Fig. 1. X-delta-2 loss of function perturbs somite formation and affects the expression of segmentation genes. Embryos were injected with two different concentrations of X-delta-2 MO (C and D) or with a dominant negative form of X-delta-2, X-delta-2tr (B). Embryos were cultivated until stage 28 and the somites were analysed by whole-mount in situ hybridisation using MyoD as a marker. Embryos are shown from the lateral side with anterior to the left. A higher magnification of the somite area indicated by the dashed square is shown in the lower right corner. Krox-20 expression in the hindbrain and engrailed expression in the midbrain–hindbrain boundary is still present when X-delta-2 is down regulated (B–D). The expression of two segmentation genes ESR-4 (E,F) and Hairy-2a (G,H) is also downregulated in the X-Delta-2 MO injected side (F and H, respectively). Embryos at neurula stage are shown from the dorsal side, anterior up (E,F) and at early tailbud stage from the dorsal side, anterior to the left (G,H). Arrowhead indicates the injected side of the embryo.
	Fig. 2. Hox gene expression during somitogenesis is affected by the downregulation of X-Delta-2. Non-injected controls (Nic; A,C,E) and embryos injected in the left-hand side (LHS) with X-Delta-2 morpholino (MO) (B,D,F) were analysed for Hoxb4 (A,B), Hoxc6 (C,D), and Hoxb9 (E,F) expression at tailbud stage (stage 21). Embryos are shown from the dorsal side with anterior to the top. All three Hox genes were downregulated but whilst Hoxb4 (B: 97%, n=31) and Hoxb9 (F: 100%, n=22) expression was severely downregulated along the AP axis, the posterior expression of Hoxc6 (D: 91%, n=23) was not affected. The arrowhead indicates the injected side of the embryo. Dotted line indicates the midline.
	Fig. 4. X-Delta-2 is necessary for Hox gene expression during early development, prior to somite formation. X-Delta-2 morpholino (MO) was injected in the LHS of the embryo and the expression of Hoxb4 (A-D), Hoxc6 (E-H) and Hoxb9 (I-L) analysed by whole-mount in situ hybridisation at gastrula and neurula stages. Gastrula stage embryos (A,B,E,F,I,J) are shown form the vegetal side with dorsal to the top and neurula stage embryos (C,D,G,H,K,L) from the dorsal side with anterior to the top. At gastrula stage almost no expression of Hox genes was observed in the injected side (B: Hoxb4, 90%, n=21; F: Hoxc6, 94%, n=37; J: Hoxb9, 100%, n=30). At neurula stage Hoxb4 expression was shifted posteriorly (D: 79%, n=24), Hoxc6 expression was only slightly downregulated (H: 80%, n=25) and Hoxb9 expression was almost completely absent (L: 87%, n=88). The arrowhead indicates the injected side of the embryo.
	Fig. 5. Specificity of X-Delta-2 morpholinos. Two non-overlapping morpholinos were injected in the LHS of the embryos and the Hoxc6 expression analysed at gastrula stage. The Hoxc6 expression is shown for NIC (A), control MO (B: 100%, n=21), MO1 (C: 88%, n=26) and MO2 (D: 100%, n=35). Overexpression of X-Delta-2 mRNA (E: 100%, n=26) and rescue of X-Delta-2 MO2 with X-Delta-2 mRNA (F: 66% rescued, n=30) are also shown. Embryos are shown from the vegetal side with dorsal to the top. Both MOs knocked down Hoxc6 expression in the LHS and co-injection of X-Delta-2 mRNA is able to rescue the Hoxc6 expression. Arrowheads indicate the injected side of the embryo.
	Fig. 6. Activation of the Delta-Notch pathway does not affect Hox expression. To activate Notch signalling we overexpressed X-Delta-2 (B,E,H,K,N) or Notch-ICD (C,F,I,L,O) in the LHS of the embryos and analysed them at neurula stage for the expression of ESR4 (A–C), at gastrula stage for the expression of Hoxd1 (D–F) and Hoxc6 (G–I) and at tailbud stage for Hoxb4 (J–L) and Hoxc6 (M–O) expression. Embryos at neurula and tailbud stage are shown from the dorsal side, anterior up; embryos at gastrula stage from the vegetal side, dorsal up. The activation of the Notch pathway, by overexpressing either X-Delta-2 or Notch-ICD perturbs the expression of ESR4 (B: 97%, n=35; C: 95%, n=44), but does not affect the expression of either Hoxd1 (E: 90%, n=40; F: 100%, n=23) or Hoxc6 (H: 100%, n=20; I: 100%, n=21) at gastrula stage, or of Hoxb4 (K: 96%, n=26; L: 90%, n=22) nor Hoxc6 (N: 100%, n=29; O: 90%, n=20) at tailbud stage. The arrowhead indicates the injected side of the embryo.
	Fig. 7. Expression of a dominant negative Su(H) does not affect Hox expression. Non-injected controls (Nic; A,C,E,G) and embryos injected in the left-hand side with dominant negative Su(H) [Su(H)DBM; B,D,F,H] were analysed at neurula stage for the expression of ESR4 (A,B: 85%, n=35) and X-Delta-2 (C,D: 86%, n=37), and at gastrula stage for the expression of Hoxd1 (E,F: 100%, n=21) and Hoxc6 (G,H: 100%, n=23) by whole-mount in situ hybridisation. Embryos at neurula stage are shown from the dorsal side, anterior up, and gastrula embryos from the vegetal side, dorsal up. Su(H)DBM works as a dominant negative affecting the downstream genes of the Notch pathway, ESR4 (B) and inhibiting the X-Delta-2 downregulation in the somitomere region (D) but does not show any effect on Hox expression (F,H). The arrowhead indicates the injected side of the embryo.
	Fig. 8. X-Delta-2 MO effect on upstream regulators of Hox genes. Non-injected controls (Nic; A,C,E,G) and embryos injected on the left-hand side with X-Delta-2 MO (B,D,F,H) were analysed at gastrula stage for the expression of Xbra (A,B: 89%, n=27) and BMP-4 (C,D: 68%, n=31), and at neurula stage for the expression of Xcad-3 (E,F: 75%, n=28) and FGF-4 (G,H: 90%, n=32) by whole-mount in situ hybridisation. X-Delta-2 MO downregulates the expression of BMP-4 and Xcad-3, without affecting the expression of Xbra and FGF-4. Embryos at gastrula stage are shown from the vegetal side, dorsal up (A–D), and at neurula stage from the dorsal side, anterior up (E,F) or posterior side, dorsal up (G,H). The arrowhead indicates the injected side of the embryo.
	Fig. 9. Hox PG1 genes are necessary for somitogenesis and X-Delta-2 expression. Morpholinos for each of the Hox PG1 genes (Hoxa1, -b1, and -d1) were injected in combination into the LHS of the embryo (PG1 MO, C,F). Non-injected controls (Nic; A,D) and embryos injected with control MO (B: 100%, n=31; E: 100%, n=23) are also shown. The expression of the muscle precursor marker MyoD (A-C) and of X-Delta-2 (D–F) was analysed at late tailbud stage (stage 25). Embryos are shown from a lateral view, with the anterior side to the left. Loss of PG1 function perturbs somite formation (C: 76%, n=47) and downregulates X-Delta-2 expression in the somitomeric region (F: 79%, n=29). Arrow in (F) indicates X-Delta-2 expression in the somitomeric region.
	Fig. 10. mHoxb1 rescues X-Delta-2 expression in PG1 knockdown embryos. Non-injected control (A) and embryos injected in the LHS with control MO (B: 100%, n=22), PG1 MOs (C: 81%, n=32) or a combination of PG1 MOs and mHoxb1 (D: 91% rescued, n=23), were analysed for the expression of X-Delta-2 at neurula stage. Embryos are shown from the dorsal side, with the anterior side to the top. X-Delta-2 expression in the somitomeric region is rescued by co-injecting mHoxb1. The arrowhead indicates the injected side of the embryo.
	Fig. 11. mHoxb1 fails to rescue somitogenesis. Non-injected control (A) and embryos injected in the LHS with mHoxb1 mRNA (B: 100%, n=27), PG1 MOs (C: 81%, n=32) or a combination of PG1 MOs and mHoxb1 (D: 91% rescued, n=23), were analysed for the expression of MyoD (somites), Krox-20 (rhombomeres 3 and 5) and engrailed-2 (midbrain–hindbrain boundary) at early tailbud stage. Embryos are shown from a lateral view with anterior side to the left. The co-injection of mHoxb1 fails to rescue the somite, as shown by the continuous MyoD expression. Krox-20 expression shows that both PG1 MO and mHoxb1 overexpression is effective.
	hes7.2 ( hairy and enhancer of split 7, gene 2) gene expression in Xenopus laevis embryos, NF stage 14, asassayed by in situ hybridization, posterior-dorsal view, anterior up.