Peres JN and Durston AJ (2006), Role of X-Delta-2 in the early neural developme...

XB-ART-870

Dev Dyn 2006 Mar 01;2353:802-10. doi: 10.1002/dvdy.20677.

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Role of X-Delta-2 in the early neural development of Xenopus laevis.

Peres JN , Durston AJ .

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The Drosophila Delta gene and its vertebrate homologues are ligands for the Notch receptor and are involved in a variety of developmental processes, including neurogenesis, boundary formation, and axon guidance. This study deals with the ectodermal expression and function of X-Delta-2 during early Xenopus laevis development. X-Delta-2 is expressed, from early neurula stages on, throughout the central nervous system (CNS; forebrain, eyes, midbrain, hindbrain, and spinal cord) and in the majority of the cranial placodes. Loss of function experiments using a morpholino knockdown approach revealed that X-Delta-2 is necessary for hindbrain segmentation and the correct specification of the anterior CNS. X-Delta-2 also seems to be important in the determination of the size of the eyes. Furthermore, our results suggest that X-Delta-2 is involved in the migration of the cranial placodes cells, as well the migration of the cranial neural crest cells.

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Species referenced: Xenopus laevis
Genes referenced: dlc dll1 dll4 dlx2 egr2 emx2 en2 gbx2 hoxb3 myod1 neurog2 nkx2-1 notch1 nrp1 pax6 snai1 snai2
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	Figure 1. Evolution of X-Delta-2 anterior neural expression. A-G: Albino embryos from stage 14 (A) to 22 (G) shown from the anterior side with dorsal to the top. H-J: Lateral view of stage 25, 27, and 36 albino embryos; anterior to the left. The first appearance of X-Delta-2 expression in a certain region is marked in the figure, except in G and H, where all the regions are marked. Arrowheads in A indicate X-Delta-2 expression in the neural plate border, and the arrow indicates the basal plate border. Arrows in B indicate the expression in the neural tube. The arrow in H indicates the otic vesicle. Arrowheads in K indicate forebrain-midbrain and midbrain-hindbrain boundary. Arrows in L indicate the X-Delta-2 expression in the middle of the hindbrain. In M, white arrows indicate the expression in the forebrain, black arrows in the tectum of the midbrain, and the arrowheads the expression in the hindbrain. The dotted line in M represents the level of the cut shown in N. Arrowheads in N mark the rhombomere boundaries. Dorsal and ventral are shown by the arrow. E, eye; epVII, facial epibranchial placode; epIX, glossopharyngeal epibranchial placode; epX2/X3, fused second and third vagal epibranchial placodes; FB, forebrain; HB, hindbrain; MB, midbrain; OP, olfactory placode; pAD, anterior dorsal lateral line placode; pDL, dorsolateral placodal area; pM, middle lateral line placode; pP, posterior lateral line placode; pPr, profundal placode; pPrV, profundal-trigeminal placode area; pV, trigeminal placode.
	Figure 2. X-Delta-2 loss of function affects the central nervous system. A prime : X-Delta-2 morpholino (MO) was injected in the two left blastomeres at the four-cell stage, and the phenotype was analyzed in embryos at stage 48. B prime -D prime : The expression of X-Delta-2 at stage 21 (B prime ) and Nrp-1 at stage 21 (C prime ) and stage 25 (D prime ) were also analyzed. A-D: Noninjected (Nic) embryos are also shown. Embryos at stage 48 are shown from the dorsal side with anterior to the top (A), and embryos at stage 21 and stage 25 are shown from the anterior side with dorsal to the top (B-D). Brackets and dotted circles in A prime indicate the olfactory pits and the otic vesicle, respectively. The asterisk indicates the injected side.
	Figure 3. X-Delta-2 role in the establishment of the identity of the forebrain. A prime -E prime : Embryos injected with X-Delta-2 morpholino (MO) in the left-hand side were analyzed for the expression of the forebrain marker Pax-6 (A prime ,B prime ), the telencephalon marker Emx-2 (C prime ,D prime ), and the ventral forebrain marker Nkx2.1 (E prime ). Pax-6 and Emx-2 were analyzed at stage 21 (A,A prime and C,C prime , respectively), and at stage 25 (B,B prime and D,D prime , respectively), whereas Nkx2.1 was only analyzed at stage 25 (E,E prime ). Noninjected (Nic) controls are shown for each marker (A-E). All embryos are shown from the anterior side with dorsal to the top. The arrow in B prime indicates the loss of Pax-6 expression in the olfactory placode. Dotted lines in E and E prime indicate the midline. Asterisk indicates the injected side.
	Figure 4. Rescue of Emx-2 expression in the telencephalon. A-D: Noninjected controls (A) and embryos injected on the left-hand side with X-Delta-2 RNA (B), X-Delta-2 morpholino (MO; C), or a combination of X-Delta-2 RNA and X-Delta-2 MO (D) were analyzed for the expression of Emx-2 at stage 21. Embryos are shown from the anterior side, with the dorsal side to the top. D: Overexpression of X-Delta-2 RNA was able to rescue the telencephalon expression of Emx-2 in X-Delta-2 MO-injected embryos. B: Emx-2 expression is slightly expanded when X-Delta-2 RNA is overexpressed. Asterisk indicates the injected side of the embryo.
	Figure 5. X-Delta-2 is involved in the segmentation and patterning of the hindbrain. A prime -C prime : X-Delta-2 morpholino (MO) was injected in the left-hand side, and the embryos were analyzed for the expression of different hindbrain markers at stage 21. A-C: Noninjected (Nic) embryos are also shown for all the markers. The expression of Engrailed-2 in the midbrain-hindbrain boundary (En in A,A prime ), Krox-20 in rhombomeres (r) 3 and 5 (A,A prime ), Gbx-2 in the r1 (bracket in B,B prime ), and Hoxb-3 (C,C prime ) in r5 were analyzed. The MyoD expression shows loss of segmentation in the injected side, confirming the loss of X-Delta-2 function. D: The morphology of the brain is shown using the neural antibody 2-G9 in stage 48 embryos. A-D: Embryos at stage 21 are shown from the anterior side with dorsal to the top (A-C), and the brain of a stage 48 embryo is shown from the dorsal side with anterior to the left (D). E: The distance between the posterior border of the Engrailed-2 stripe and the anterior border of the r3 Krox-20 stripe (r1-r2 region) and the distance between the anterior border of the r3 Krox-20 stripe and the posterior border of the r5 Krox-20 stripe (r3-r5 region) was measured in stage 21 (n = 6) and stage 25 (n = 5) embryos. E: The hindbrain regions measured are indicated by brackets in a stage 21 embryo stained for Engrailed-2 and Krox-20. E: Both sides were measured, and the ratio left-hand side/right-hand side (LHS/RHS; Nic/Nic or Delta MO/NIC) was plotted on a graph. Values above 1.0 indicate an increase in the length of the region on the left (injected) side, and values below 1.0 indicate a decrease. In both stages, the r1-r2 region is increased on the X-Delta-2 MO-injected side and the r3-r5 region is decreased. The arrow in D indicates the forebrain, and arrowheads indicate the rhombomere boundaries in the hindbrain. The asterisk indicates the injected side.
	Figure 6. A-D prime : X-Delta-2 role in the neurogenic placodes. Noninjected (Nic) controls (A-D) and embryos injected on the left-hand side with X-Delta-2 morpholino (MO; A prime -D prime ) were analyzed for the expression of N-tubulin (A,A prime ) X-Ngnr-1 at stage 21 (B,B prime ) and stage 25 (C,C prime ), and for the expression of X-Delta-2 at stage 25 (D,D prime ). Embryos at stage 21 are shown from the anterior side with dorsal up, and the ones at stage 25 are shown from the lateral side with dorsal to the top, Nic with anterior to the right side, and injected embryos with anterior to the left side. Arrows in A prime and B prime indicate the N-tubulin and X-Ngnr-1 expression, respectively, in the profundal-trigeminal placodal area (pPrV). pAD, anterodorsal lateral line placode; pM, middle lateral line placode. Asterisk indicates the injected side. Note that the stage 25 Nic inC and D for X-Ngnr-1 and X-Delta-2 are the noninjected side of the same embryo shown in C prime and D prime , respectively.
	Figure 7. A-E prime : X-Delta-2 loss of function affects cranial neural crest cell migration. Noninjected (Nic) controls (A-E) and embryos injected on the left-hand side with X-Delta-2 morpholino (MO; A prime -E prime ) were analyzed for the expression of cranial neural crest markers or stained with Alcian blue to highlight the cartilage. A,A prime ,B,B prime show stage 21 embryos from the anterior side with dorsal to the top; C,C prime ,D,D prime show stage 25 embryos from the lateral side with dorsal to the top; and in E,E prime show stage 48 embryos from the ventral side with anterior to the top. Cranial neural crest cells fail to migrate as shown by the expression of X-Slug (A prime ), Xsnail (B prime ), and X-dll4 (C prime ,D prime ). The cartilage staining shows that the gills, derived from the branchial arches, do not form in the X-Delta-2 MO-injected side (arrow in E prime ). The asterisk indicates the injected side. Nic for X-dll4 in C and D is the noninjected side of the same embryos shown in C prime ,D prime , respectively.
	dlc (putative ortholog of delta-like protein C precursor (SwissPro) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, anterior view, dorsal up.
	dlc (putative ortholog of delta-like protein C precursor (SwissPro) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, anterior view, dorsal up.
	dlc (putative ortholog of delta-like protein C precursor (SwissPro)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up.
	dlc (putative ortholog of delta-like protein C precursor (SwissPro)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, anterior view, dorsal up.
	dlc (putative ortholog of delta-like protein C precursor (SwissPro)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, anterior view, dorsal up. Key: pDL = ; HB= hindbrain; pPrV=
	dlc (putative ortholog of delta-like protein C precursor (SwissPro)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, anterior view, dorsal up.
	dlc (putative ortholog of delta-like protein C precursor (SwissPro)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
	dlc (putative ortholog of delta-like protein C precursor (SwissPro)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, lateral view, anterior left, dorsal up.
	dlc (putative ortholog of delta-like protein C precursor (SwissPro)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, dorsal view, anterior left, head and upper trunk region.


	dlc (putative ortholog of delta-like protein C precursor (SwissPro)) gene expression, as assayed by in situ hybridization, in NF stage 48 Xenopus laevis embryo brain dissected, dorsal view, anterior left.
	nrp1 (neuropilin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, anterior view, dorsal up.
	nrp1 (neuropilin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, anterior view, dorsal up.
	emx2 (empty spiracles homeobox 2 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, anterior view, dorsal up.
	dlc (putative ortholog of delta-like protein C precursor (SwissPro)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, anterior view, dorsal up.