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First published online 29 September 2004
doi: 10.1242/dev.01397

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Zebrafish and fly Nkx6 proteins have similar CNS expression patterns and regulate motoneuron formation

Sarah E. Cheesman¹, Michael J. Layden^2,3, Tonia Von Ohlen^1,3,*, Chris Q. Doe^1,2,3 and Judith S. Eisen^1,

¹ Institute of Neuroscience, 1254 University of Oregon, Eugene OR, 97403-1254, USA
² Institute of Molecular Biology 1229 University of Oregon, Eugene OR, 97403-1229, USA
³ Howard Hughes Medical Institute, 1254 University of Oregon, Eugene OR, 97403-1254, USA

View larger version (43K): [in a new window]   Fig. 1. Schematized morphogenesis of vertebrate and arthropod neurectoderm. Vertebrate and arthropod ectoderm viewed in cross-section (grey circles represent non-neural ectoderm). Vertebrate neurectoderm (colored boxes) is positioned dorsally (top of figure) on the body. Later neurectoderm undergoes morphogenesis (direction indicated by arrowhead) to form a neural tube, internal to the non-neural ectoderm. Dorsal CNS (D; red) is initially lateral, intermediate CNS (I; green) is initially intermediate and ventral CNS (V; blue) is initially medial. Arthropod neurectoderm (colored boxes) is positioned ventrally (bottom of figure) on the body. The nerve cord forms as neuroblasts bud off toward the interior of the embryo (direction of arrowhead). Later in development, the arthropod nerve cord contains layers of neurons. Early, members of the vnd/Nkx gene family are expressed in the ventral domain (blue) and members of the msh/Msx gene family are expressed in the dorsal (red) domain of both vertebrates and flies (see text for details). In flies, ind is expressed early in the intermediate domain. Although vertebrate ind homologs (Gsh) are expressed in a subset of intermediate domain cells, they are expressed early only in anterior CNS and much later in spinal cord (Deschet et al., 1998; Cheesman and Eisen, 2004; Valerius et al., 1995).

View larger version (44K): [in a new window]   Fig. 2. Homology between Nkx6 proteins. (A) Alignment of fly and zebrafish Nkx6 translated amino acid sequence reveals several regions of high identity: the almost invariant NK domain (dashed line) and homeodomain (solid line), and two other regions indicated by asterisks. Fly Nkx6 was trimmed of the N-terminus. (B) Maximum likelihood tree. Fly Nkx6 is orthologous to vertebrate Nkx6 proteins, distinct from Vnd/Nkx2.2 proteins. Numbers at the nodes represent bootstrap values. Dm, Drosophila melanogaster; Dr, Danio rerio.

View larger version (88K): [in a new window]   Fig. 3. nkx6.1 is expressed in zebrafish ventral spinal cord. (A) Expression is initiated in the embryonic shield at 50% epiboly, approximately 5 hpf (animal pole view, optical section of embryo without yolk). Asterisk denotes epiblast (outer) layer. (B) By bud stage, 10 hpf, nkx6.1 is broadly expressed in medial neurectoderm (dorsal view, rostral to left). (C) At 26 hpf, nkx6.1 is strongly expressed in ventral CNS from the midbrain through the tail (lateral view; rostral to left). Arrowhead denotes pancreas expression. (D) Higher magnification view of spinal cord shown in (C). Scale bar: 50 µm for A,B,D; 25 µm for C. sc, spinal cord; n, notochord; y, yolk.

View larger version (131K): [in a new window]   Fig. 4. Nkx6.1 is expressed in zebrafish motoneurons. Nkx6.1 (green) Islet1/2 (red) antibody double labels. (A) 14 hpf lateral view reveals Rohon-Beard neurons (RBs) in dorsal spinal cord express only Islet proteins and PMNs in ventral spinal cord are double labeled (yellow). (B) By 18 hpf Nkx6.1 is largely absent from PMNs; most RBs are out of focal plane. (C) Cross-section of 24 hpf embryo showing Nkx6.1-positive nuclei, which comprise about five cell rows in the ventral spinal cord including medial (^) and lateral (*) floorplate. (D) Cross-section of 48 hpf embryo double labeled with Nkx6.1 (green) and Neurolin (red) antibodies. Nkx6.1-positive nuclei are surrounded by Neurolin-labeled cell surfaces. Laterally-located slow muscle cells are also Neurolin-positive. (E) 20 hpf lateral view of individually labeled VeLD interneuron (yellow) expressing Nkx6.1 (green). Most individually labeled VeLDs in 20-22 hpf embryos expressed Nkx6.1 (n=11, 82%). (F) By 24 hpf VeLDs have downregulated Nkx6.1 (n=13, 85%). Scale bar: 50 µm for A,B,D,E,F; 75 µm for C.

View larger version (82K): [in a new window]   Fig. 5. Nkx6 is expressed in fly ventral CNS and motoneurons. (A) Ventral view of stage 9 embryo showing Nkx6 RNA in cephalic neurectoderm and CNS midline. (B) At stage 10, Nkx6 is ventrally restricted to rostral neuroblasts in each hemisegment. (C) By the end of gastrulation Nkx6 is expressed in neurons throughout the CNS. (D-F) Nkx6 is expressed in neuroblasts 1-1, 1-2, 2-2, 3-1, 3-2 and 4-2. Neuroblasts expressing Nkx6 mRNA (blue) were identified by their position relative to Eagle-positive neuroblasts (brown; NB2-4, arrowhead; NB3-3, arrow; NB6-4, asterisk; NB7-3, white arrowhead) and other neuroblasts. The same two segments of a stage 11 embryo are shown at different focal planes (D, superficial; E, middle; F, deep). White line, ventral midline. (G) At stage 14, Nkx6 (RNA in situ, purple) is confined to a population of neurons distinct from those expressing the dorsal motoneuron determinant Eve (antibody, brown). The prominent brown cells near the midline are RP2 motoneurons (*). (H) pMAD (antibody, brown) and Nkx6 (RNA in situ, purple) expression are coincident in many cells at stage 14. (I) By stage 16, cells positioned laterally are positive for pMAD and Nkx6 (bracket), but more medial cells are clearly only pMAD-positive. Rostral is up in all images. Scale bar: 50 µm for A-C; 16 µm for D-I.

View larger version (98K): [in a new window]   Fig. 6. Hh signaling induces zebrafish nkx6.1 expression. (A) 18 hpf wild-type expression of nkx6.1. (B) Dramatic dorsal expansion of nkx6.1 in an 18 hpf embryo ectopically expressing shh (shh OE, 87% of injected animals, n=283). (C) 12 hpf wild-type expression of nkx6.1. (D) 12 hpf syu mutant with reduced nkx6.1 expression. (E) 12 hpf syu mutant injected with ehh and twhh MOs. nkx6.1 expression is largely absent (28% of injected clutch from syu heterozygous parents, n=120). (F) Wild-type expression of nkx6.1 in spinal cord at 24 hpf. (G) 24 hpf syu mutant has wild-type levels of nkx6.1 expression in spinal cord. (H) Few nkx6.1-positive cells remain in a 24 hpf smu mutant spinal cord. (I) 24 hpf syu mutant injected with ehh and twhh MOs has no nkx6.1 expression in spinal cord (21% of injected clutch from syu heterozygous parents, n=128). C-D dorsal views; all others are lateral. Rostral to left in all images. Scale bar: 50 µm for A,B; 100 µm for C-E; 25 µm for F-I.

View larger version (75K): [in a new window]   Fig. 7. nkx6.1 is sufficient to induce zebrafish motoneurons and repress interneurons. (A) isl2-positive CaPs and VaPs at 18 hpf. Wild-type embryos have 1.3±0.7 isl2-positive cells per spinal hemisegment (n=5). (B) Embryos injected with nkx6.1 mRNA (Nkx6.1 OE) display supernumerary isl2-positive cells (2.3±0.5 per spinal hemisegment; n=7). These numbers are significantly different (P<0.001). Most RBs are not in focus in these images. Inset is a cross-section indicating the more dorsal position of supernumerary PMNs (arrowhead). (C) 24 hpf wild type labeled with zn1 and znp1 antibodies reveals 1 or 2 CaP/VaP cell bodies and axons as well as RBs. (D) Embryos injected with nkx6.1 RNA have 3-4 CaP/VaPs with normal axons, but some are more dorsally located (arrowheads). RBs are out of focus. (E) Wild-type 24 hpf GATA-2:GFP embryo has clusters of 3.0±1.7 SMNs in each rostral spinal cord hemisegment (8 hemisegments in 2 embryos). (F) nkx6.1 RNA-injected embryos have 4.0±2.8 SMNs per cluster (n=4; numbers are significantly different, P<0.009). All views are lateral, rostral to left. Scale bar: 50 µm for A,B; 33 µm for C-H.

View larger version (96K): [in a new window]   Fig. 8. Nkx6.1 is required for zebrafish secondary motoneuron formation. (A) Nkx6.1 protein in 24 hpf wild-type spinal cord. (B) nkx6.1 MO-injected embryo has no detectable protein in spinal cord at 24 hpf. (C) Wild-type and (D) MO-injected 12 hpf embryos have the same pattern of isl1-positive PMNs. RBs are out of focus on each side of PMNs. (E) At 48 hpf, ventral spinal cord is full of Neurolin-positive SMNs. (F) MO-injected embryo has few remaining SMNs (75% of injected animals, n=565). The brightly-stained cuboidal cells are floorplate. (G) GABA antibody labels KA and VeLD interneurons in ventral spinal cord at 24 hpf. (H) In MO-injected embryos there is a dramatic increase in GABA-positive VeLD interneurons (n=21). Rostral to left in all images. A,B,E-H lateral views. C,D dorsal views Scale bar: 50 µm for A-D; 33 µm for E-H.

View larger version (113K): [in a new window]   Fig. 9. Nkx6 function is conserved between fish and flies. (A) isl2-positive CaPs and VaPs in an18 hpf zebrafish embryo. isl2-positive RBs are out of focus. Wild-type embryos have 1.3±0.7 isl2-positive motoneurons per spinal hemisegment (n=5). (B) 18 hpf embryo ectopically expressing Nkx6 (fly gene, Nkx6 OE) RNA with supernumerary isl2-positive cells (2.3±0.3 isl2-positive cells per hemisegment), similar to the phenotype caused by ectopic expression of zebrafish nkx6.1 RNA (compare with Fig. 7B). The number of isl2-positive motoneurons is significantly different (P<0.0003). (C) PMNs in 24 hpf wild-type embryo labeled with zn1 and znp1; RBs and several axon tracts are also visible. (D) 24 hpf embryo ectopically expressing Nkx6 (fly gene) RNA generates ectopic PMNs (compare with Fig. 7D). (E) Stage 17 wild-type fly embryo stained with Fas II. Arrowheads indicate SNb nerves. (F) sca>Nkx6.1 (fish gene) fly embryos with visibly thicker SNb nerves (88%, n=42 hemisegments). (A-D) Lateral views of zebrafish spinal cord. (E,F) Dissected fly embryo musculature. Scale bar: 50 µm for A,B; 33 µm for C-F.

View larger version (49K): [in a new window]   Fig. 10. Nkx6 misexpression affects both motoneurons and interneurons in flies. (A) Wild-type fly embryo with normal numbers of aCC, pCC, and RP2 (arrow) neurons (n=48). (B) In sca>Nkx6 (fly gene) embryos RP2 is duplicated (arrow) in 75% of hemisegments (n=34). (C) Wild-type embryos have 5.0±0.14 U motoneurons (asterisks; n=48) and 10.0±0.73 EL interneurons (circle; n=46). (D) In sca>Nkx6 embryos both types of neurons are reduced (Us, 2.5±1.4, n=34; ELs, 5±3.4, n=34). (E) In wild types there are 3.8±0.48 RP1,3,4,5 motoneurons (circle; n=80) and 2.8±0.37 EW interneurons (arrowhead; n=25). (F) In sca>Nkx6 (fly gene overexpression, OE) RP1,3,4,5 are normal (circle; 3.6±0.65; n=73) and EWs are reduced (arrowhead; 1.0±1.2; n=30). In some cases, as shown here, an EW-like cell ectopically expresses pMAD, indicating an interneuron-to-motorneuron fate change; these cells were not scored as EWs. (G) In sca>nkx6.1 (fish gene) RP1,3,4,5 numbers are normal (circle, 3.5±0.66; n=55). (H) Wild-type embryos have one medial lateral motoneuron (ML) per hemisegment (asterisk; 80±0; n=80) and 4.7±0.54 HB9-, Islet-, pMAD-positive lateral cluster motoneurons (LC; circle; n=50). (I) sca>Nkx6 (fly gene) embryos have one ML motoneuron (asterisk; 94% of hemisegments; n=44) and 8.5±2.3 LC motoneurons (n=80). (J) sca>nkx6.1 (fish gene) also have one ML motoneuron (asterisk, 89.4% of hemisegments; n=57) and 8.0±1.89 LC motoneurons; (n=60). (K) Schematic depicting average wild-type number of assayed motoneurons and interneurons. (L) Schematic depicting average number of motoneurons and interneurons in sca>Nkx6 (fly gene) embryos. Scale bar: 15 µm. All images are ventral views; rostral is up, medial is left and lateral is right.