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Figure 2. Spatial and temporal analysis of Xdhcr7 expression. A: WMISH analysis of the expression of Xdhcr7 and Xshh. Staging according to Nieuwkoop and Farber (1975). a-i: WMISH analysis of Xdhcr7 expression. e', h': Transversal sections as indicated by black dashes in e and h, respectively. a-d: Vegetal view (dorsal to the top) of gastrula stage embryos showing the expression of Xdhcr7 in the dorsal blastopore lip (a, b), and in the forming dorsal midline (c, d). e,f: Dorsal view (anterior to the bottom) of neural plate (e) and early neural tube (f) stage embryos. Xdhcr7 highly stains the dorsal midline (e), and additional staining appears in the epidermis (ep) at neural tube stage (f). e': The expression of Xdhcr7 in the notochord. g, h: Lateral view (anterior to the left) of late neural tube (g) and early tailbud (h) stage embryos. Xdhcr7 is expressed in the notochord, the neural tube, the optic vesicle, the head epidermis, and the epibranchial placodes. Red arrow indicates the posterior limit of the Xdhcr7 expression domain in the neural tube. h': The expression of Xdhcr7 in the hypochord, the notochord, and the lateral ventral neural tube, but not in the floor plate (inset). i: Lateral view (anterior to the left) of the head region of a late tailbud stage embryo; Xdhcr7 expression is restricted rostrally, and mainly demarcates neural structures. j,k': WMISH analysis of Xshh expression. j', k': Transversal sections as indicated by black dashes in j and k, respectively. j: Dorsal view (anterior to the bottom) of a neural plate stage embryo. Xshh is expressed in the dorsal midline and the prechordal plate. j': The expression in the notochord. k: Lateral view (anterior to the left) of a tailbud stage embryo showing the expression of Xshh. k': The expression of Xshh in the notochord and floor plate, but not in the lateral ventral neural tube. l: Schematic illustration of the spatial distribution of Xdhcr7 and Xshh transcripts at tailbud stage. Xdhcr7 and Xshh transcripts colocalise only in the notochord and hypochord, but are expressed in neighbouring domains in the neural tube. B: RT-PCR analysis of the appearance of Xdhcr7 and Xshh transcripts during development and in adult tissues. RT-PCR primers were designed to amplify nucleotides 431 to 805 of Xdhcr7. Zygotic expression of Xdhcr7 starts at around stages 10 to 11, which coincides with the start of Xshh expression. Transcript levels of Xdhcr7 drop at around tailbud stage (st. 32), when Xshh is still highly expressed. The relative distribution of Xdhcr7 and Xshh mRNA in adult tissues is quite similar, as they are both highly expressed in the brain, eye, spinal cord, skin, kidney, lung, liver, stomach, and testis. Histone H4 was probed as RNA loading control. C: WMISH analysis of the expression of Xshh (a, d), Xsal-1 (b, e), and Xdhcr7 (c, f) in banded hedgehog (Xbhh) injected embryos. Xbhh capped RNA (500 pg) was injected (in combination with 200 pg of beta -galactosidase capped RNA as linage tracer) into one blastomere of two-cell-stage pigmented embryos. a-c: Transversal sections at the level of the eyes of stage 34 embryos. d-f: Transversal sections at the level of the trunk of stage 34 embryos. a, d: Expansion of Xshh expression in the forebrain, but not in the floor plate and notochord. b, e: Expansion of Xsal-1 expression in the brain and neural tube. c, f: Xbhh suppresses the expression of Xdhcr7 in neural structures (eye, brain, and spinal chord), but not in the notochord (c'). br, brain; cs, control side; dbl, dorsal blastopore lip; dml, dorsal midline; ebp, epibranchial placodes; ep, epidermis; ey, eye; fb, fore brain; fp, floor plate; hb, hind brain; hc, hypochord; is, injected side; lvnt, lateral ventral neural tube; mb, midbrain; nc, notochord; nt, neural tube; ot, otic vesicle; ov, optic vesicle; pp, prechordal plate; Sp. cord, spinal cord. |
