Read EM et al. (1998), Evidence for non-axial A/P patterning in the no...

XB-ART-14332

Int J Dev Biol 1998 Sep 01;426:763-74.

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Evidence for non-axial A/P patterning in the nonneural ectoderm of Xenopus and zebrafish pregastrula embryos.

Read EM , Rodaway AR , Neave B , Brandon N , Holder N , Patient RK , Walmsley ME .

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Recent studies in early Xenopus and zebrafish embryos have demonstrated that posteriorizing, non-axial signals arising from outside the organizer (or shield) contribute to A/P patterning of the neural axis, in contradiction to the classical Spemann model in which such signals were proposed to be solely organizer derived. Our studies on the early expression of the transcription factors GATA-2 and 3 in both Xenopus and zebrafish nonneural ectoderm lend support to the existence of such non-axial signaling in the A/P axis. Thus we find that the earliest expression of GATA-2 and 3 is located in nonneural ectoderm and is strongly patterned in a graded manner along the A/P axis, being high anteriorly and absent from the most posterior regions. This results by early neurula stages in three broad zones: an anterior region which is positive for both GATA-2 and 3, a middle region which is positive for GATA-2 alone and a posterior region in which neither gene is expressed. These regions correspond to head, trunk and tail ectoderm and may represent the beginnings of functional segmentation of nonneural ectoderm, as suggested in the concept of the 'ectomere'. We find that A/P patterning of GATA expression in nonneural ectoderm may occur as early as late blastula/early gastrula stages. We investigate which posteriorizing signals might contribute to such distinct non axial ectodermal patterning in the A/P axis and provide evidence that both FGF and a Wnt family member contribute towards the final A/P pattern of GATA expression in nonneural ectoderm.

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Species referenced: Xenopus
Genes referenced: acta2 dlx3 egf egr2 fgf4 gata2 gata3 hoxa7 hoxb9 hoxc9-like kit ncam1 otx2 pax2 snai2 tbxt

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Fig. 1. Conserved boundaries of expression of GATA factors in the nonneural ectoderm of frog and fish gastrulae. Double whole-mount in situ hybridizations of Xenopus early neurula embryos stained for GATA-2 (a,c,e,g) or GATA-3 (b,d,f,h) with the anterior neural marker, Otx2 (a and bJ, the hindbrain marker Krox20 (c and d), the spinal cord marker HoxB9 (e and f) and neural crest marker, slug (g and h). Anterior is to the left and posterior to the right except for a and b which are anterior views, (a) Embryo stained for Otx2 (turquoise) and GATA-2 (purple). At the most anterior end GATA-2 is expressed in the cement gland and abuts Otx2 whereas more posteriorly a gap can be seen between GATA-2 and Otx2 (asterisk). (b) Double staining for GATA-3 (purple) and Otx2 (turquoise). At the most anterior end GATA-3 expressed in the cement gland overlaps the expression of Otx2. More posteriorly, a gap exists between GATA-3 and Otx2 expression (asterisk). (e) Dorsal view of an embryo stained for GATA-2 (purple) and Krox 20 (turquoise) marking rhombomeres 3 and 5 of the hindbrain (r3 and (5). A gap can be seen between GATA-2 and Krox20 expression in r5 (arrow). (d) Dorsal view of an embryo stained for GATA-3 (purple) and Krox20 (turquoise). A gap is seen between GATA-3 and Krox20 staining In r5 (arrow). (e and f) Lateral view of embryos stained for GATA-2 and 3 (purple) and HoxB9 (B9, turquoise). Note that GATA-2 is expressed more posteriorly than GATA-3. (g and h) Dorsal views of embryos stained for GATA-2 (purple, g) or GATA-3 (purple, h) and the neural crest marker slug (turquoise, g and h). Note the gap between GATA and slug expression (asterisk, g and h). (i and j) Double whole-mount in Situ hybridization of Xbra (turquoise, i and j) with GATA-2 (dark blue, i) and GATA-3 (dark blue, J) In Xenopus mid-gastrula embryos (the notocordal Xbra signal is not yet visible, stage 11). Note that GATA-2 abuts Xbra expression, (i), but a gap is seen between GATA-3 and Xbra expression, (arrow, j). yp, yolk plug; d, dorsal; v, ventral; anterior is to the left. (k) A 70% epiboly (gastrula) zebrafish embryo, stained for gta2 (blue) and ntl (red). gta2 expression abuts ntl at all gastrula stages. (I) An intact 70-80% (gastrula) zebra fish embryo stained for gta3 (blue) and ntl (red). Note the gap between gta3 and ntl (arrow), This gap is also seen at eartier gastrula stages (data not shown). At late gastrula, ntl is expressed in the notochord (noto). (m) 17 mm saggital section through the region marked by the arrow in l showing the gap (arrow) between gta3 (blue) and ntl (red) expression. a, anterior; p, postenor; v, ventral. (n) A flat mount of a zebrafish embryo stained for gta3 (blue) and dlx3 (expressed strongly in pIa codal tissue at this stage) and pax2 (a marker of the midbrain/hindbrain border) both stained in red. Note the gap between dlx3 and pax2 staining representing neural crest. a, anterior; p, posterior; v, ventral. (0) Diagrammatic representation ofaXenopus/zebrafish gastrula embryo showing the boundaries of expression of GATA-2 and 3 between neural and nonneural ectoderm and between nonneural ectoderm and mesoderm which are conserved between the two species.

Fig. 3. FGF represses the expression of GATA-2 and 3 (gata2 and 3) in the non-neural ectoderm of frog and fish embryos and restricts expression to regions outside the germ ring. Xenopus (a-d) and zebrafish (e-j) embryos were either untreated (a,c,e.g and j) or injected with 2pg of eFGF RNA at the single cell stage for Xenopus (b and d) or with 220 pg eFGF RNA (note that this RNA was not made with the Megascript kit and was capped less successfully, thus more was needed to produce an effect) at the 1-4 cell stage for zebra fish (f and h) or with 40pg XFD RNA at the 1- 4 cell stage (i). Embryos were examined by whole-mount in situ hybridization staining for GA TA- 2 (a and b), GA TA-3 (c and d), gta2 (e and f) and gta3 (g and h). Note that there is no gata2 staining in f): the background is caused by yolk which has been lost from the embryo in e). (i)Zebrafish gastrula embryo injected with XFD RNA and stained for gta2 (blue) and ntl (red), Note that gata2 expression reaches down to the involuting edge on the ventral side where ntl has been eliminated.