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First published online 29 September 2004
doi: 10.1242/dev.01394

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Drosophila homeodomain protein Nkx6 coordinates motoneuron subtype identity and axonogenesis

Heather T. Broihier^1,*,,, Alexander Kuzin^2,, Yi Zhu¹, Ward Odenwald² and James B. Skeath¹

¹ Department of Genetics, Washington University School of Medicine, 4566 Scott Avenue, St Louis, MO 63110, USA
² Neural Cell-Fate Determinants Section, NINDS, NIH, 36 Convent Drive MSC 4130, Bethesda, MD 20892-4130, USA

View larger version (81K): [in a new window]   Fig. 1. Nkx6 allele generation and expression analysis. (A) Diagram of the Nkx6 locus, with the location of the P[JG] insertion and the extent of the D25 deletion indicated. (B) Stage 9, (C) stage 11, and (D) stage 14 wild-type embryos stained for Nkx6. (B) Nkx6 is expressed in midline precursors at stage 9. (C) At stage 11, Nkx6 is expressed in compact clusters of neural precursor cells and neurons flanking the midline. (D) Nkx6 is expressed in 30 to 40 postmitotic neurons at stage 14. Note, Nkx6 expression levels vary widely between neurons. (E-J) Wild-type embryos labeled with indicated antibodies. (E) Nkx6 and Dpn are co-expressed in medial neuroblasts in stage 11 embryos. Arrowhead points to a cluster of NBs including NBs 1-2, 3-1, 3-2, and 4-2. Arrow points to NB5-2. (F) Confocal projection of stage 14 CNS with Nkx6 and Eve expressed in complementary subsets of postmitotic neurons. (G) Nkx6 and a lim3-taumyc reporter are co-expressed in the medial 1 and 3-5 RP MNs (arrowheads) at stage 14. (H-J) Confocal projection of stage 15 embryo with Nkx6 and Hb9 expressed in largely overlapping populations of postmitotic neurons. Three segments of dissected nerve cords are shown in B-J with anterior oriented up.

View larger version (97K): [in a new window]   Fig. 4. Nkx6 is necessary for axon growth. Three hemisegments of Stage 16 dissected (A) wild-type (B) Nkx6D25/Nkx6D25 (C) Nkx6P[JG]/Nkx6D25, and (D) UAS-Nkx6/+; Nkx6Gal4/Nkx6D25 embryos labeled with -Fas2 to mark motor axon projections. Schematics summarizing the observed phenotype are shown below each panel. (A) In wild type, ISNb (arrowheads) and ISNd innervate the ventral muscle field. (B) In Nkx6D25 homozygous embryos, ISNb/d do not innervate their muscle targets. Arrowheads indicate ventral muscle field. (C) In Nkx6P[JG]/Nkx6D25 mutants the ventral muscle field is not innervated, though presumptive ISNb axons extend along ISN (arrowheads). (D) In Nkx6 rescue embryos ISNb axons usually contact their appropriate target muscles. (E) Penetrance of mutant phenotypes for different Nkx6 allelic combinations. See text for details. (F) Three hemisegments of wild-type and (G) UAS-GAPGFP/+; hb9GAL4 Nkx6D25/Nkx6D25 mutants labeled with -GFP (green) to mark Hb9-positive axons and -MHC (red) to mark muscles. Hb9-positive axons extend along ISNb to contact ventral muscles in wild-type (F), but not mutant (G) embryos. (H) Three segments of wild-type and (I) UAS-GAPGFP/+; hb9GAL4 Nkx6D25/Nkx6D25 mutant nerve cords labeled with -GFP to mark Hb9-positive axons. In wild type (H), numerous Hb9-positive longitudinal fascicles are visible, while in I very few Hb9-positive axons are present. Anterior is up in all panels.

View larger version (94K): [in a new window]   Fig. 5. Nkx6 overexpression drives ISNb overgrowth. (A) wild-type and (B-D) elavGAL4:2xUAS-Nkx6 embryos labeled with -Fas2 to mark motor axons. Schematics outlining motor axon phenotypes are below each panel. Single hemisegments are shown in (A-C), in (D) adjacent hemisegments are shown. (A) In wild type, ISNb innervates ventral muscles 7, 6, 13, and 12 in a stereotyped fashion. (B-D) In Nkx6 overexpression embryos ISNb appears to exhibit exuberant growth. In (B) the terminal arbor between muscles 13 and 12 is expanded. (C) ISNb contains supernumerary branches. (D) ISNb in adjacent hemisegments have crossed the segment boundary and fused. Anterior is up in all panels.

View larger version (80K): [in a new window]   Fig. 2. Nkx6 and hb9 antagonize vnd. (A) Stage 9, (B) stage 11, (C) stage 14 wild-type embryos stained for Nkx6 and Vnd. (A) At stage 9 Nkx6 is expressed in midline precursor cells and Vnd is expressed in flanking ventral neuroectoderm. (B) At stage 11 Nkx6 is expressed in clusters of GMCs and neurons situated within stripes of Vnd-positive GMCs and neurons. (C) At stage 14 Nkx6 and Vnd are expressed in mutually exclusive populations of postmitotic neurons. (D) In stage 15 wild-type embryos approximately 30 neurons express Nkx6 while (E) elavGAL4:UAS-vnd embryos are devoid of Nkx6-positive neurons. (F) In stage 15 wild-type embryos approximately ten neurons express Vnd while (G) elavGAL4:UAS-Nkx6 mutants and (H) elavGAL4:UAS-hb9 mutants exhibit fewer Vnd-positive neurons. (I) Stage 15 hb9KK30 Nkx6D25 double mutant embryo displays ectopic Vnd-positive neurons. Three segments of dissected nerve cords with anterior up are shown in all panels.

View larger version (54K): [in a new window]   Fig. 3. Nkx6 and hb9 collaborate to regulate neuronal fate. (A) Stage 15 wild-type and (B) stage 15 elavGAL4:UAS-eve embryos stained for Nkx6. Ectopic eve expression largely abolishes Nkx6 expression. Stage 15 (C) wild-type, (D) elavGAL4:UAS-Nkx6 (E) hb9KK30, and (F) hb9KK30 Nkx6D25 embryos labeled with -Eve. (D) elavGAL4:UAS-Nkx6 embryos exhibit a specific reduction of Eve expression in the U MNs and EL interneurons, but not in RP2 or a/pCC. (E) hb9 mutant displays two ectopic Eve-positive neurons per hemisegment (arrowheads). (F) hb9 Nkx6 mutant displays roughly six ectopic Eve neurons per hemisegment. (G-J) Confocal projections of stage 15 (G,I) wild-type and (H, J) hb9 Nkx6 mutant embryos stained for Lim3 (G,H) or Islet (I,J). The 1 and 3-5 RP MNs express Lim3 (G) and Islet (I) while in hb9 Nkx6 double mutants, Lim3 (H) and Islet (J) are no longer expressed in RP MNs (arrowheads). Three segments of dissected nerve cords with anterior up are shown in all panels.

View larger version (96K): [in a new window]   Fig. 6. Nkx6 promotes axon growth and guidance of RP1,3,4,5 MNs. Confocal projections of four micron series from dissected stage 16 (A) wild-type and (B-D) Nkx6D25 nerve cords carrying a lim3-tmyc transgene. -Myc marks axon projections of RP1,3,4,5. (A-D) An asterisk indicates the RP MN cell bodies and arrowheads mark the extent of the RP axon projection. (A) In wild type, RP motor axons leave the CNS. Nkx6D25 mutant embryos RP motor axons display various defects. (B) RP motor axons frequently truncate and exhibit aberrant morphology. (C) RP motor axons in the bottom left and both right hemisegments are markedly thinner than in wild-type embryos. (D) RP motor axons in the top left hemisegment diverge from their normal course and turn toward the midline. (E,F) Confocal projections of four micron series from dissected stage 16 nerve cords from (E) wild-type and (F) Nkx6D25 mutant embryos labeled with Fas3. (E) In wild type, the Nkx6-positive RP MNs express Fas3 (arrowheads) as do Nkx6-negative lateral neurons (arrows). (F) In Nkx6 mutant embryos, RP MNs lose Fas3 expression (arrowheads), while expression in more lateral Nkx6-negative neurons is unaffected (arrows). Anterior is up in all panels.

View larger version (17K): [in a new window]   Fig. 7. Transcription factor hierarchy in Drosophila MN specification. The factors activating Nkx6 and hb9 in ventrally and laterally projecting MNs are largely unknown. In ventrally and laterally projecting MNs, Nkx6 and hb9 act in parallel to activate the Lim-HD proteins Lim3 and Islet. Nkx6 and hb9 also repress the dorsally projecting MN determinant Eve. Since Eve also represses Lim3 and Islet, it is possible that eve repression is responsible for loss of Islet and Lim3 expression in Nkx6 hb9 double mutant embryos.