Yan B , Neilson KM , Moody SA .

NS23158 NINDS NIH HHS , R01 NS023158 NINDS NIH HHS , R01 NS023158-20 NINDS NIH HHS , R01 NS023158-20S1 NINDS NIH HHS , R01 NS023158-20S2 NINDS NIH HHS , R01 NS023158-21 NINDS NIH HHS , R01 NS023158-22 NINDS NIH HHS

	Figure 2. Whole mount in situ hybridization assays of embryos injected with FoxD4L1/D5 mRNA. A: Examples of FoxD4L1/D5 causing an increase in downstream gene expression. In neural plate stage embryos, the expression domains of FoxB1 and XSpr2 are broader on the FoxD4L1/D5-injected side (right; indicated by βGal-expressing (red) cells). Lines indicate width of neural plate on injected (right) and control (left) sides of embryos. Dorsal views with anterior to the bottom. The FoxB1 expression domain also extends more anterior (bracket) compared to anterior limit on control side (arrow). At gastrula stages, the endogenous domains of Fgf8b and Oct-1 surround the yolk plug; the anterior extent of the domain is indicated by an arrow on the control side. The cells expressing FoxD4L1/D5 (red nuclei) in the neural plate (bracket) also express increased levels of Fgf8b and Oct-1. Dorsal views with anterior to the bottom. Inset in Oct-1 embryo: higher magnification showing FoxD4L1/D5-cells with red nuclei and blue cytoplasm. At gastrula stages, VegT/Brat and BJ051739 are ectopically induced in the animal cap ectoderm. Animal pole views. B: Examples of FoxD4L1/D5 causing a decrease in downstream gene expression. In neural plate stage embryos, MGC52564, MGC52940, Neptune, Dlx6, and Blimp1 are expressed in the border zone that separates the anterior neural plate (np) from the epidermis (epi). For each gene, this region shows decreased expression at the site of FoxD4L1/D5-expressing cells (red nuclei, arrows). MGC52564, MGC52940, Dlx6, Blimp1 are anterior views with dorsal to the top; control side is on the left. Neptune is a side view with anterior to top and dorsal to right. In a gastrula embryo, the normal high level of FoxI1 expression in the animal cap ectoderm (epi) is greatly reduced in the FoxD4L1/D5-expressing clone (*), which is outlined for clarity.
	Figure 3. RT-PCR assays illustrate the temporal expression patterns of uncharacterized up-regulated (A) and down-regulated (B) genes in normal, unmanipulated embryos. RT-, minus RT step; numbers across the top indicate the developmental stages (st). H4 is an internal control.
	Figure 4. Whole mount in situ hybridization assays illustrating the expression patterns of 7 uncharacterized genes that are up-regulated by FoxD4L1/D5. Gastrula stages are side views oriented with animal cap (An) to the top and vegetal pole (Veg) to the bottom. Transcripts for all 7 genes are detected in the animal cap ectoderm. At neural plate stages, embryos are oriented in an anterior frontal view, with dorsal to the top. Transcripts for all 7 genes are detected in the neural plate (np) and in the ectoderm bordering the neural plate (arrow) that will give rise to the cranial placodes and neural crest. At neural tube stages, MGC132176 is expressed throughout the neural tube (nt), with intense staining in a band in the midbrain-hindbrain (hb) region, in adjacent neural crest (nc), and the dorsal rim of the cement gland (cg). Left embryo is a dorsal view with anterior at the bottom and right embryo is a ventral view of the head. At larval stages (st328), MGC132176 is detected throughout the brain and retina (r), in the lens (L), otocyst (oto), cement gland, branchial arch (BA), and nephric mesoderm (ne). This and all larval stages are side views with anterior to the left. Transverse sections of st38 embryo confirm the labeled tissues identified in whole mount embryos, and identify the profundal ganglion (Pg). At neural tube stages, LOC496224 is expressed throughout the neural tube, retina, and somites (so). Side view with anterior to the left. At larval stages, LOC496224 is additionally expressed in the lens, otocyst, and branchial arches. BC097640 is expressed throughout the neural tube, with strong expression in the pineal primordium (pin), cement gland, several cranial placodes (dlp, dorso-lateral placode; olf, olfactory placode; Vp, trigeminal placode), and scattered cells on the ventral midline (bracket) caudal to the cement gland. Left embryo is an anterior frontal view with dorsal to the top; right embryo is a ventral view with anterior to the top. At larval stages, BC097640 is additionally expressed in the lens, otocyst, branchial arches, head mesoderm (hm), heart (ht), ventral blood islands (bl), somites, and nephric mesoderm. At neural tube stages, BJ051730 is weakly expressed throughout the neural tube, with strong expression in the midbrain-hindbrain region and adjacent neural crest. Dorsal anterior-view from the top. At larval stages, BJ051730 is additionally expressed in the lens, otocyst, branchial arches, somites, and nephric mesoderm. At neural tube stages, BJ043686 is expressed in the neural tube and the ectoderm bordering it (bz). Side view with anterior to the left. At larval stages, BJ043686 is additionally expressed at low levels in the lens, otocyst, somites, branchial arch, head, and nephric mesoderm. At neural tube stages, CB983184 is expressed in the neural tube and the ectoderm bordering it. At larval stages, CB983184 is additionally expressed in the olfactory pit, lens, retina, otocyst, somites, branchial arch mesoderm, and heart. At neural tube stages, MGC68716 is expressed in the neural tube, the ectoderm bordering it, and pineal primordium. At larval stages, MGC68716 is additionally expressed in the otocyst, somites, branchial arch mesoderm, and heart. fb, forebrain; mb, midbrain.
	Figure 5. Whole mount in situ hybridization assays illustrating the expression patterns of 5 genes that are down-regulated by FoxD4L1/D5. Abbreviations are the same as in Figure 4. A: Down-regulated, validated genes of unknown function. Gastrula stages are side views oriented with animal pole to the top and vegetal pole to the bottom. Transcripts for all 4 genes are detected in the animal cap ectoderm. At neural plate stages, embryos are oriented in an anterior frontal view, with dorsal to the top. MGC81002 is weakly detected in the neural plate, the ectoderm bordering it, and epidermis (epi). At neural tube stages, it is detected diffusely in the neural tube, retina and epidermis. Side view. At larval stages, MGC81002 is additionally detected in the otocyst and branchial arch mesoderm. Side view and transverse sections. At neural plate stages, LOC443682 is strongly expressed in the epidermis, the ectoderm bordering the neural plate, and the anterior neural ridge (anr). At neural tube stages, LOC443682 is detected throughout the epidermis, with enhanced expression bordering the retina, hatching gland (hg), and cement gland. Left embryo is a dorso-anterior view with anterior to the bottom; top right embryo is a dorsal view of the head with anterior to the left, and bottom right embryo is a ventral view of the head with anterior to the top. At larval stages, LOC443682 is detected throughout the epidermis, most strongly on the dorsal side, and in the nephric mesoderm. Side view and transverse sections. At neural plate stages, MGC53193 is detected in the epidermis, border of the neural plate, and anterior neural ridge. At neural tube stages, MGC53193 is strongly expressed in the head epidermis and tissue surrounding the cloaca (cl). Left embryo is a frontal view with dorsal to the top and right embryo is a ventral view with posterior to the left. At larval stages, MGC53193 is weakly detected in the epidermis of whole mount embryos (side view), but transverse sections reveal additional weak expression in brain (b), lens, otocyst, and anterior pharynx (ph). At neural plate stages, MGC52940 is strongly expressed in the ectoderm bordering the neural plate and cement gland, and weakly in the epidermis. At neural tube stages, MGC52940 expression is pronounced in a dorsal midline stripe in the epidermis, surrounding the retinas, in a patch over the forebrain (between the retinas), and in the cement gland. Anterior-frontal view with dorsal to the top. At larval stages, MGC52940 is highly expressed in the olfactory placode and in cells dotted throughout the epidermis; it is weakly expressed in the cement gland and anterior pharynx. Side view and transverse sections. B: Expression pattern of Xenopus Elf-1. Elf-1 is weakly expressed in the animal cap ectoderm at gastrulation, throughout the neural plate and border zone, and then is barely detectable in the anterior neural tube (*). At larval stages it is weakly detected in the brain, lens, and branchial arch mesoderm (side views and transverse sections).
	Supporting Figure S1: Expression pattern of BG161338 (eif5b) , which was up-regulated in the microarray assay, but not validated by RT-PCR or ISH assays. A: RT-PCR analysis of unmanipulated embryos shows that BG161338 is highly expressed at all developmental stages investigated. B: ISH analysis shows that at gastrula stages, BG161338 is expressed in the animal cap ectoderm (an) and the marginal zone (bracket) that will give rise to mesoderm; side view with animal to the top. Veg, vegetal pole. At neural plate stages, it is strongly expressed in the neural plate (np) and the ectoderm bordering the neural plate (arrow); anterior view with dorsal to the top. At neural tube stages, it is strongly expressed throughout the neural tube (nt), in the migrating neural crest (nc), and in the lens (L); anterior view with dorsal to the top. At larval stages, it is additionally expressed in the otocyst (oto), branchial arch mesoderm (BA), somites (so), nephric mesoderm (ne), and ventral blood islands (bl); side view with anterior to the left.
	eif5b (eukaryotic translation initiation factor 5B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, lateral view, dorsal up.
	eif5b (eukaryotic translation initiation factor 5B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, anterior view, dorsal up.
	eif5b (eukaryotic translation initiation factor 5B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, anterior view, dorsal up.
	eif5b (eukaryotic translation initiation factor 5B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left.

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