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Figure 1. Developmental expression of IA1. A-P: Dorsal views (A,B), anterior views (C,D), ventral views (E,F) and lateral views (G-P) of embryos at the indicated stages. A-C,G: IA1 is expressed in the neural plate primary neurons and in scattered cells in the anteroventral region of embryos (arrowhead). D: Xash1 is expressed at stage 12.5-13 in the anteroventral cells (arrowhead). E: Late neurula embryo double stained with IA1 and CG probes. IA1 (dark blue) punctated staining (arrowhead) is detected in front of the cement gland (light blue). F: Early tail bud embryo double stained with IA1 and Nkx-2.5 showing that IA1-positive cells are found in the developing heart field. H-O: High magnification views of the head region of early tail bud embryos stained with the indicated probes. Like IA1, Myt1, Phox2, Hand2, Hox11L2, Etr-1, CRMP-4, and TH are expressed in the anteroventral cells (arrowheads). P: HNK-1 antibody staining in early tadpole. Staining in anteroventral cells (arrowheads) is indicated. Q: Two-color double in situ hybridization of IA1 (dark blue) and epidermal keratin (light blue). Note that IA1 expressing cells are found directly below the epidermal keratin expression layer. R: One-color double in situ hybridization of IA1 (dark punctated staining) and vestigial like-4 (light uniform staining in the deep layer of the ectoderm). Expression of IA1 can be seen in cells within the deep layer (arrowheads) of the ectoderm as well as in cells adhering to the underside of the deep layer (arrow). S: Transversal section of a tail bud stage embryo showing IA1 expression in the dorsal pancreas. T: Embryo in P bisected at the level of the hindbrain showing IA1 staining in the pancreas and in the adrenal medulla. U,Y: Transverse sections through the hindbrain region. Note that IA1 is transiently expressed in differentiating neurons of the neural tube and that IA1 transcripts, like that of Delta1, are located immediately adjacent to the Ngnr1-positive cells. ElrC and CRMP4 are found more peripherally. Tg, trigeminal ganglia; an, anterior neural plate; am, adrenal medulla; dl, deep layer of the ectoderm; dp, dorsal pancreas; fm, forebrain and midbrain neurons; sl, superficial layer of the ectoderm; ov, otic vesicle.
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Figure 2. Differentiation of anteroventral cells is regulated by BMP and Notch. Anterior (A-D,I,J,L,N,O), lateral (E-H), or dorsal (K,M,P) views of neurula to early tail bud embryos are shown. A,B: Stage 13 embryo stained with BMP4 alone (A) or double stained with Phox2a (arrowhead) and BMP4 (B). C,D: Embryos injected with tBR mRNA show a reduction of Phox2a and tyrosine hydroxylase (TH) expression in the anteroventral region. E-H: Induction of Phox2a and TH by Xash1 is reduced by coinjection of tBr. I: Delta1 is transiently expressed in the anteroventral cells (arrowhead). J,K: Embryos injected with Notch1 ICD or Su(H)Ank show a decrease of IA1 expression in the neural plate primary neurons and anteroventral cells (arrowheads). L,M: Embryos injected with Delta1stu or Su(H)DBM mRNA show an increase of IA1 expression in the neural plate primary neurons, trigeminal placodes, and anteroventral cells (arrowheads).
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Figure 3. Xash1 controls the formation of the anteroventral neurons. Dorsolateral (A-F) and anterior (G-L) views of late neurula to early tail bud embryos are shown. A-F: Overexpression of Xash1 induces ectopic expression of IA1, Phox2a, Hand2, Hox11L2, TH, and CRMP-4 in the ectoderm. G-L: Embryos injected with Xash1 MOs showing a reduction of IA1, Phox2a, Hand2, Hox11L2, TH, and CRMP-4 expression in anteroventral cells.
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Figure 4. Phox2a and Hand2 contribute to the formation of anteroventral cells. Lateral (A,B,E,I-N) or anterior (C,D,F-H) views of neurula to early tail bud embryos are shown. A,E: Overexpression of Phox2a moderately induces IA1 expression but not TH in the ectoderm. B,F: Overexpression of Hand2 weakly induces IA1 expression but strongly up-regulates TH expression in the anteroventral region of embryos (arrowheads). C,D,G,H: Phox2 or Hand2 inactivation leads to a reduction of IA1 and TH expression in the anteroventral region of embryos. I-N: Coinjection of the Phox2a MO or Hand2 MO slightly reduces IA1 and strongly inhibits TH induction.
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Figure 5. IA1 knock-down affects the formation of anteroventral cells while neurogenesis in the neural plate is unaffected. Anterior (A-F,H) or dorsal (G) views of neurula to early tail bud embryos are shown. A-E: IA1 MO injection decreases the expression of MyT1, Phox2a, Hand2, CRMP-4, and TH in anteroventral cells. F: Delta-1 expression in anteroventral cells (arrowhead) is unaffected by injection of the IA1 MO. G: Normal expression of Myt1 in the neural plate of an IA1 MO injected embryo. H: Injection of a second IA1 MO also affects TH expression in anteroventral cells.
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