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Figure 6. Xenopus Tmem79 is required in naive ectoderm for anterior and neural development. (A) Tmem79 is expressed throughout Xenopus embryogenesis from 1 cell (fertilized egg) to stage (st) 30, spanning blastula, gastrula, neurula and tailbud/organogenesis stages, as assayed by RT-PCR. (B) Tmem79 is expressed in dissected animal, vegetal, dorsal, and ventral regions (as schemed in drawing) of early gastrula embryos (stage 10.5). xnr3 is a control for dorsal. (C) Tmem79 expression during Xenopus embryogenesis by whole-mount in situ hybridization. An apparent lack of hybridization signals in vegetal pole and derived tissues is likely technical due to yolk. Top row (c1 to c6) Tmem79 is expressed in the animal pole at the four-cell stage and in naive ectoderm at blastula stages: (c1) Dorsal-animal view; (c2 to c4) Lateral-animal views; (c5 and c6) Lateral and bisected views. Second row (c7 to c12) Tmem79 is expressed broadly in naive ectoderm at early gastrula stages: (c7 and c8) dorsal-vegetal and lateral views with the arrow pointing to the dorsal; (c9) bisected view with an enlarged view (c10) showing expression in naive ectoderm; (c11 and c12) lateral and bisected views. Anterior to top in c11. Third row (c13 to c18) Tmem79 shows dynamic expression from late gastrula to neurula stages: (c13 and c14) lateral and bisected views showing broad expression including in neural plate. Anterior to top in c13 and left in c14. (c15 to c17) Dorsal views. Anterior to top. Tmem79 expression is downregulated in neural plate while remains strong in epidermis, as seen also in a cross-section (c18) along the dashed line in c17. (c18) an enlarged cross-section view with dorsal on top. Bottom row (c19 to c23) Tmem79 is expressed during organogenesis: (c19 and c20) Lateral and anterior views showing expression in the cement gland. (c21 and c22) A lateral view with an enlarged view (anterior to right) and (c23) a ventral view (anterior to top) showing expression in primitive kidney (black arrows) and otic vesicle (white arrows). (D and E) Dorsal-animal (naive ectoderm) injection of Tmem79MO causes anterior deficiency, which is rescued/over-rescued by co-injection of mouse Tmem79 mRNA or Usp8MO or β-cateninMO. CoMO, Control Morpholino. Anterior to right (E). Also see Supplementary file 1A. (F) Tmem79MO reduces neural plate formation and reciprocally expands epidermal differentiation, reflected by Sox2 and cytokeratin expression domains at Stage 16, respectively. These changes are rescued by Tmem79 mRNA or Usp8MO. Anterior to right. Also see Supplementary file 1C. (G) Neural induction by Noggin in animal pole explants is prevented by Tmem79MO, whose effect is rescued by Tmem79 mRNA or Usp8MO or β-cateninMO. Pan-neural (n-cam), anterior (bf1, otx2, and six3) and mid/hind (en2 and krox20) brain markers and a neuronal differentiation marker (n-tub) are assayed, as is an epidermal marker (keratin). |
