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Gastrulation begins at NF stage 10 with the appearance of the blastopore lip in the vegetal dorsal region of the embryo (Fig. 2E). The invaginating dorsal blastopore lip becomes obvious as a darker indentation, caused by apical constriction of bottle cells and condensation of pigment (Moosmann et al., 2013). As gastrulation proceeds, the involuting marginal tissue at the blastopore lip extends laterally towards the ventral sides of the embryo, reaching almost halfway around the blastopore circumference at NF stage 10.5 (Fig. 2E). Between NF stages 11 and 12 (Fig. 2E,F) the vegetal cells are increasingly internalized, and the blastopore diameter progressively decreases, owing in large part to mediolateral intercalation of the underlying mesodermal cells (Keller and Sutherland, 2020). The relative dimensions of yolk plug to blastopore diameter, in combination with the extent of pigmentation along the blastopore lip, are key landmarks for stage determination during gastrulation (Fig. 2E,F). Based on molecular analysis, it is now clear that neural induction begins as early as NF stage 10.5 with sox2 expression in the dorsal ectoderm (Gawantka et al., 1998). The neural plate is identifiable by NF stage 12 (Fig. 2F) with a posterior constriction of the midline dorsal ectoderm, and by NF stage 12.5 (blastopore closed completely) (Fig. 2F) the notochord forms from mediolateral convergence of dorsal axial mesoderm under the neural ectoderm, a process identifiable by chrd.1, nog and shh expression (Roelink et al., 1994; Sasai, 1994; Fletcher and Harland, 2008 ).
This process was essentially the same as that described previously (Zahn et al., 2017). Animals used in this project were wild-type J-strain X. laevis (NXR_0.0024), accessed at the National Xenopus Resource (NXR; RRID:SCR_013731) at the Marine Biology Laboratory (MBL) at Woods Hole, MA, USA. Animals were handled following the ARRIVE and NIH Guidelines for Use and Care of Laboratory Animals that were approved by the Institutional Animal Care and Use Committee of the MBL. Representative embryos from several different clutches were used. The developmental stages were prior to overt sexual identification. Vitelline membranes were removed from embryos at NF stages 13-15, because if not removed, the membrane flattens the neural plate, making it more difficult to tell NF stages 13-15 apart (Sive et al., 2007a). Likewise, the membranes were removed from stage NF 22-35 tailbud embryos before photographing to remove the curve in the body axis. Swimming tadpoles were anesthetized using approved methods. Specimens were photographed using a Zeiss steREO Discovery.V12 microscope with an AxioCam MRc camera. Larger tadpoles and froglets were imaged with an Apple iPhone XR camera.
The Zahn drawings presented here and those from the XenHead project (Zahn et al., 2017) are available as individual digital files (jpg format) and as a compiled printable sheet (pdf format) on Xenbase in the Anatomy & Development module ( ). Additionally, the Zahn drawings are used to illustrate the XAO pages ( =display) for relevant NF stages (Fig. S2).
All the Zahn drawings (those presented here and the earlier XenHead set) are available on Xenbase ( ) through an attributable, non-commercial creative commons license (CC BY-NC 4.0). To reproduce any of these images please use the following attribution: Illustration 2021 Natalya Zahn, CC BY-NC 4.0, and cite Xenbase ( , RRID:SCR_003280) and this article. This license permits reuse and manipulation of up to six illustrations, for any noncommercial use, with the attribution above. To use more illustrations, or for any commercial use, contact the illustrator, Natalya Zahn (email:natalya@natalya.com) for permission (fees may apply). Further information is available on the Xenbase page, and full details of the license can be read here: -nc/4.0/?ref=chooser-v1. Note that the full series of original Nieuwkoop and Faber drawings and the entire set of histology plates from Hausen and Riebesell (1991) are also available on Xenbase in the Anatomy and Development module.
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