Yan B , Neilson KM , Ranganathan R , Maynard T , Streit A , Moody SA .

R01 DE022065 NIDCR NIH HHS, R01 NS023158 NINDS NIH HHS , R03 DE018723 NIDCR NIH HHS

	Figure 2. Examples of changes in gene expression of putative Six1 targets after knock-down of endogenous Six1 levels by injection of antisense morpholino oligonucleotides (Six1 MOs), increased levels of wild-type Six1 by mRNA injection (Six1 WT), activation of Six1 targets by mRNA injection of an activating construct (Six1-VP16), or repression of Six1 targets by mRNA injection of a repressive construct (EnR-Six1) (see Brugmann et al., 2004 for validation of these constructs). Six1 MOs: Expression of Cdca7l in the segmenting somites (arrows on control [ctrl] side) is lost on the injected (inj) side (side views, anterior to left). Expression of HoxA3 in the neural crest (nc) is greatly reduced on the injected side, whereas hindbrain (hb) expression is maintained (side views, anterior to left). Expression of Ism1 is lost in the midbrain (arrow) (frontal view, dorsal to the top). Expression of IMAGE 3399268 is expanded in the neural plate (line depicts width from midline to lateral border) on the injected side (dorsal view, anterior to the bottom). Expression patterns of Cnfn1-a in the hatching gland (arrows) and of Snail1 in the migrating neural crest are reduced and disrupted on the injected sides (frontal views, dorsal to the top). Six1 WT: Expression of BG022063 is broader in the neural plate (line) and neural crest (bracket) on the injected side (frontal view, dorsal to the top). Expression of Nfya is broader in the neural tube (line) on the injected side (dorsal view, anterior to the top). Expression of IMAGE 3399268 in the segmented somites (arrows, ctrl side) is lost on the injected side (side views, anterior to left). Expression of IMAGE 4057931 in the neural crest (bracket), otocyst (oto) and trigeminal ganglion (arrow) is greatly diminished on the injected side (side views, anterior to left). Six1-VP16: Expression of Frizzled10b, Pbx1, and Zic2 in the neural plate is expanded (line) on the injected side (frontal views, dorsal to the top). EnR-Six1: Expression of Frizzled10b in the neural plate is reduced (line) on the injected side (frontal view, dorsal to the top). Expression of IMAGE 4057931 is reduced in the PPR (arrows on the control side; frontal view, dorsal to the top). Expression of Zic2 and Otx1 in the neural tube is reduced (line) on the injected sides (arrows; frontal views, dorsal to the top). In MO-injected embryos, injected sides were confirmed by the use of lysamine-labeled MOs (not shown). In mRNA-injected embryos, injected sides were confirmed by the presence of nuclear β-galactosidase lineage labeling (pink dots visible in most images).
	Figure 3. Semiquantitative RT-PCR assays illustrate the temporal expression patterns of previously uncharacterized up-regulated (A) and down-regulated (B) genes in normal, unmanipulated Xenopus embryos. RT−, minus RT step; numbers across the bottom indicate the developmental stages. H4 is an internal control. Im, IMAGE
	Figure 4. Whole-mount in situ hybridization assays at early developmental stages illustrating the common expression patterns of 14 up-regulated genes: Nrp1, Arrb2, BG022063, LOC100037047, BQ399495, Hrpt1, Rbm42-6, LOC414689, Uncx4.1, Xxylt1, Nfya, Tbc1d31, Cdca7l, Papd4-a. For all genes, expression is enhanced in the animal cells (an) of cleavage and blastula embryos (side views). At gastrula stages, expression is throughout the ectoderm, occasionally enhanced in the animal cells (e.g., Arrb2) or the dorsal cells (e.g., Nrp1) (side views). At early neural plate stages, all genes are expressed in the epidermis (epi), often more intensely on the dorsal (d) side, including neural plate (np) and border zone (bz) (all except Nrp1 are side views with anterior [a] to the left; Nrp1 is an anterior view with dorsal to the top). At late neural plate and neural tube stages, expression is strongest in the neural crest (nc), pre-placodal region (ppr), neural tube (nt) and occasionally cement gland (cem) (all are anterior views). For Nrp1, embryos were sectioned in the horizontal plane to demonstrate that the ppr stripe in the whole embryo is due to expression in the deep (d) layer of the ectoderm, not the superficial (s) layer of the ectoderm or the pharyngeal endoderm (phe); ph, lumen of pharynx.
	Figure 5. Whole-mount in situ hybridization assays at tail bud to larval stages illustrating more divergent expression patterns of 14 up-regulated genes: Nrp1, Arrb2, BG022063, LOC100037047, BQ399495, Hrpt1, Rbm42-6, LOC414689, Uncx4.1, Xxylt1, Nfya, Tbc1d31, Cdca7l, Papd4-a. Side views at tail bud (left column) and larval (middle column) stages, with anterior to the left and dorsal to the top, and transverse sections at larval stages (right side) with dorsal to the top. ba, branchial arches; cem, cement gland; ep, epibranchial placodes; epi, epidermis; fb, forebrain; h, heart; hb, hindbrain; IX/Xg, glossopharyngeal/vagal ganglion; l, lens; mb, midbrain; ne, nephric mesoderm; olf, olfactory placode; oto, otocyst; p, pineal; r, retina; spcd, spinal cord; so, somites; Vg, trigeminal ganglion; VIIg, facial ganglion.
	Figure 6. Whole-mount in situ hybridization assays illustrating the expression patterns of previously characterized up-regulated (Trhd-a, Pbx1b, Ism1, Arnt, HoxA3) or down-regulated (Foxi1-Xema) genes at stages previously unpublished. Abbreviations are as in Figures 4 and 5.
	Figure 7. Four examples of cleavage stage gene expression in embryos that were bisected before the in situ hybridization protocol to facilitate probe access to all of the cells. In each case the probe was detected in the animal pole blastomeres (a) and in marginal zone animal blastomeres (arrows), but not in vegetal blastomeres (v).
	Figure 8. Examples of embryos bisected after the in situ hybridization protocol to reveal expression patterns in internal cells. BQ399495: At blastula (left image, sagittal section), expression is primarily in the animal cap (AC) cells, and barely detected in the equatorial (eq) zone. At neural plate stages (right image, transverse section), expression is observed in the neural plate (np), somites (so), and epidermis (epi). Hprt1: At blastula (left image, sagittal section), expression is in both animal cap and equatorial cells. At gastrula stages (right image, sagittal section, anterior to the left), expression is observed in the surface ectoderm (ecto) and involuting mesoderm (meso); bl, dorsal blastoporal lip. Xxylt1: At blastula (left image, sagittal section), expression is in both animal cap and equatorial cells. At neural plate stages (right image, sagittal section, anterior to the left), expression is observed in the neural plate and underlying dorsal mesoderm (meso). IMAGE 3399268: At blastula (left image, sagittal section), expression is in both animal cap and equatorial cells. At gastrula stages (right image, sagittal section, anterior to the left), expression is observed in the animal ectoderm, but barely detected in the involuting mesoderm. Cnfn1-a: At gastrula stages (sagittal section, anterior to the left), expression is observed in the animal ectoderm, but not in the involuting mesoderm. IMAGE 7010138: At blastula (left image, sagittal section), expression is primarily in the animal cap cells, and barely detected in the equatorial zone. At gastrula stages (middle image, sagittal section, anterior to the left), there is strong expression in both the ectoderm and involuting mesoderm. At neural plate stages (right image, sagittal section, anterior to the left), weak expression is observed in both the neural plate and underlying dorsal mesoderm.
	Figure 9. Examples of Xenopus embryos hybridized with sense RNA probes to evaluate the extent of nonspecific staining at the early stages when expression patterns are diffuse (cf. Figs. 4, 5, 6, 10).
	Figure 10. Whole-mount in situ hybridization assays at early stages illustrating common expression patterns of 10 down-regulated genes: IMAGE 4174145, IMAGE 3399268, IMAGE 4057931, IMAGE 3400511, Socs3, MGC114680, IMAGE 7010138, Cnfn1-a, Ralgds, Dnaja4.2. For all genes, expression is enhanced in the animal cells (an) of cleavage and blastula embryos, and is throughout the ectoderm at gastrula stages (side views). At early neural plate stages, all genes are expressed in the epidermis (epi), often more intensely on the dorsal (d) side, including neural plate (np) and border zone (bz) (all except IMAGE 3400511 are side views with anterior [a] to the left; IMAGE 3400511 is an anterior view). At later neural plate and neural tube stages, expression is strongest in the neural crest (nc), pre-placodal region (ppr), neural tube (nt) and epidermis (epi) (anterior views). Cnfn1-a is uniquely expressed in the hatching gland (hg).
	Figure 11. Whole-mount in situ hybridization assays at tail bud to larval stages illustrating more divergent expression patterns of 10 down-regulated genes: IMAGE 4174145, IMAGE 3399268, IMAGE 4057931, IMAGE 3400511, Socs3, MGC 114680, IMAGE 7010138, Cnfn1-a, Ralgds, Dnaja4.2. Side views at tail bud (left column) and larval (middle column) stages, with anterior to the left and dorsal to the top, and transverse sections at larval stages (right side) with dorsal to the top. Abbreviations are as in Figure 10. For Cnfn1-a: hg, hatching gland; clo, cloaca.
	socs3 (suppressor of cytokine signaling 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up. Key: Branchial arch (ba), Otocyst (oto), Somites (so), Nephric mesoderm (ne).

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