Dichmann DS , Harland RM .

GM42341 NIGMS NIH HHS , R01 GM042341-25 NIGMS NIH HHS , R01 GM042341-26 NIGMS NIH HHS , R01 GM042341 NIGMS NIH HHS

	Fig. 1. Expression of Nkx6 genes in X. laevis. (A–E) Whole mount in situ hybridization showing Nkx6.1 expression. (A) Nkx6.1 is not expressed during gastrulation. (B) Expression begins at stage 13 in two lateral stripes (red arrowhead) and intensifies at stage 14 (C). At stage 17 (D) Nkx6.1 expression marks where Hb9-positive MNs will form (compare to (M). (E) At stage 34, Nkx6.1 is expressed in the ventral spinal cord and hindbrain as well as the foregut (red arrowhead). (F–J) Whole mount in situ hybridization showing Nkx6.2 expression. (F) Nkx6.2 is expressed during gastrulation in the dorsal ectoderm. Red line indicates the dorsal blastopore lip. (G) At stage 13, Nkx6.2 expression is detected in a broad part of the medial neural plate. At stage 14 (H) and stage 17 (I) Nkx6.2 expression extends through the medial developing hindbrain and spinal neural plate. (J) At stage 34, Nkx6.2 expression is detected in the ventral spinal cord and hindbrain as well as the foregut (red arrowhead). Nkx6.1 and Nkx6.2 expression is confined to the medial part of the neural plate when compared to the pan-neural marker Sox2 expression (compare (C, H) to (K); and (D, I) to, (L)). (N,O) Neither Nkx6.1 (N; red arrowhead) nor Nkx6.2 (O; red arrowhead) expression overlaps with Dbx1 expression (N,O; black arrows) at stage 16.
	Fig. 2. Effect of Nkx6 mRNA injection on neural development. Gene expression patterns analyzed by whole mount in situ hybridization. Top row: uninjected control embryos. Middle row: embryos injected with Nkx6.1 mRNA. Bottom row: embryos injected with Nkx6.2 mRNA. Injected side is to the right and red stain indicate beta-galactosidase lineage tracer. All embryos are analyzed at stage 16, except embryos stained for Pax3 (V–X), which were analyzed at stage 17. (A–C) Pan-neural marker Nrp1 is expanded in Nkx6 injected embryos. (D–F) Epidermal marker cytokeratin-81 (CK81) is reduced in Nkx6 injected embryos. (G–I) Primary neurogenesis is inhibited by Nkx6 mRNA injected as visualized by loss of N-tubulin (N-tub) staining. (J–L) Dbx1 expression is inhibited in the spinal neural plate when embryos are injected with Nkx6 mRNA. (M–O) Spinal neural plate expression of Pax2 is repressed by Nkx6 mRNA, whereas the MHB is intact. (P–R) Spinal, but not rhombomeric, expression of Irx3 is repressed by Nkx6 misexpression. (S–U) Pax6 expression in the neural plate is repressed by ectopic Nkx6 misexpression. (V–X) Pax3 expression and Msx1 (Y–æ) expression is unchanged in response to Nkx6 injection.
	Fig. 3. Effect of Nkx6 mRNA injection on Nkx6 gene expression and MN formation. Nkx6 mRNA injected embryos analyzed by whole mount in situ hybridization at stage 17. Injected side is to the right and red stain indicates the beta-galactosidase lineage tracer. (A,C,E) Uninjected control embryos, (B,G) embryos injected with Nkx6.2 mRNA, (D,F) embryos injected with Nkx6.1 mRNA. (A,B) Nkx6.1 expression is not repressed in response to Nkx6.2 mRNA injection. (C,D) Nkx6.2 is not repressed in embryos injected with Nkx6.1 mRNA. (E–G) Embryos injected with Nkx6 mRNA do not form ectopic Hb9-positive MNs.
	Fig. 5. Knockdown of Nkx6.1 has minor effects on neural plate patterning. Gene expression pattern in the neural plate of Nkx6.1MO injected X. laevis embryos. Top row shows uninjected control embryos, bottom row shows embryos injected with 37.5 ng Nkx6.1MO in one half of the embryos. The injected side is to the right. (A,B) N-tubulin expression pattern marking primary neurons is normal at stage 16. (C,D) Hb9-positive MNs form normally in Nkx6.1 knockdowns (stage 18). (E,F) Lhx3-positive cells are reduced in Nkx6.1MO injected embryos at stage 16 (black arrowhead). (G,H) Pax2-expressing interneurons are reduced at stage 15. Red brackets indicate medial and lateral Pax2-positive stripes in the spinal neural plate. Only one stripe is present on the injected side (H and insert). (I,J) At stage 18 the spinal Pax2+ interneurons form one stripe that is reduced in response to Nkx6.1 knockdown (J, red arrowhead). Interneuron progenitor markers such as Pax6 (K,L), Dbx1 (M,N), and Nkx2.2 (O,P) are not affected by Nkx6.1 knockdown. Nkx6.1 is not required for its own (Q,R) or for Nkx6.2 expression (S,T).
	Fig.4. Morpholino mediated knockdown of Nkx6 genes in X. laevis and X. tropicalis. MO design and morphology of MO injected tadpoles. (A) Alignment of Nkx6.1 sequences in X. laevis (Xl; top) and X. tropicalis (Xt; bottom) used for MO design. Vertical lines indicate identical residues and translation start codon is in bold type. Sequence targeted by Xl-Nkx6.1MO is shown in blue and is identical to the corresponding X. tropicalis sequence in 24/25 positions. (B) Uninjected stage 40 X. laevis tadpole. (C) Tadpole injected with 80 ng Xl-6.1MO at the 1-cell stage is morphologically normal but paralyzed. (D) Uninjected stage 40 X. tropicalis tadpole. (E) X. tropicalis tadpole injected with 8 ng Xl-6.1MO in both blastomeres at the 2-cell stage is morphologically normal but paralyzed, phenocopying the X. laevis injection in (C).
	Fig.6. Nkx6.1 knockdown results in paralysis and axon misguidance. (A) Narrative frames from a time lapse series showing uninjected controls (top panel) and Nkx6.1MO injected embryos (bottom). Uninjected embryos initiate escape reflex when poked with a pipette tip (frames 12 and 17; pipette tip painted black). Poking of Nkx6.1MO injected embryos does not trigger escape reflex (bottom). Nkx6.1MO injected embryos were injected in both blastomeres at the 2-cell stage. (B,C,F,H) Orthograde DiI-labeling of stage 39/40 embryos injected unilaterally with Nkx6.1MO. (B) Control side showing labeled nerve fibers emanating from the neural tube. (C) Nkx6.1MO injected side show labeled nerves. (D,E,G,I) Immunostaining for N-CAM using antibody 6F11 showing axonal extensions. (D) Uninjected side of stage 39/40 tadpole showing nerve fibers protruding from the spinal cord and hindbrain. (E) Nkx6.1MO injected side of same tadpole does have greatly reduced and disorganized axons and cranial nerves are severely affected (blue arrowhead). (F, H) High magnification of boxes in (B) and (D), respectively. White arrow heads in (F) indicate DiI labeled axons. (H,I) High magnification of boxes show in (C) and (E), respectively. Asterisks in (H) indicate fluorescence from the uninjected side out of the plane of focus. (J) Effect of different MO1 and MO2 doses, individually and in combination, on tadpole mobility. Embryos were injected in all blastomeres at the one or two cell stage. Total MO amount injected is listed under bar. Number in parenthesis indicates number of embryos analyzed. MO1 + 2 injections were performed with equal amounts of each. The two last bars show effect on MO1 when injected into X. tropicalis. Embryos were scored at stages 32–39.
	nkx6-1 (NK6 homeobox 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 14, anterio-dorsal view.
	nkx6-1 (NK6 homeobox 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 17, anterio-dorsal view.
	nkx6-1 (NK6 homeobox 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
	nkx6-2 (NK6 homeobox 2) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 13, anterio-dorsal view.
	nkx6-2 (NK6 homeobox 2) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 17, anterio-dorsal view.
	nkx6-2 (NK6 homeobox 2) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 34, lateral view, anterior left., dorsal up.
	nkx6-2 (NK6 homeobox 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, anterio-dorsal view.
	Ncam1 Ab2 (6f11) in a Xenopus laevis embryo, assayed via immunohistochemistry, NF stage 39, lateral view, anterior left, dorsal up, showing nerve fibers protruding from the spinal cord and hindbrain.

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