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First published online 6 October 2004
doi: 10.1242/dev.01418

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Specification of Drosophila motoneuron identity by the combinatorial action of POU and LIM-HD factors

Sarah J. Certel and Stefan Thor^*,

Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA

View larger version (66K): [in a new window]   Fig. 1. Colocalization of Isl, Lim3 and Dfr in the embryonic ventral nerve cord. (A) Schematic representation of Isl, Lim3 and Dfr expression in the TN, ISNb and ISNd motoneuron subclasses. (B) Staining for Tau-Myc (red), ß-galactosidase (blue) and Dfr (green) in a stage 16 embryo carrying the isl-tau-myc and Lim3A-lacZ transgenes. Isl, Lim3 and Dfr are co-expressed in the ISNb neurons, including the RP neurons (arrows). Only Isl and Lim3 are expressed in the TN neurons (arrowheads). (C) A stage 16 Lim3A-tau-myc transgenic embryo labeled for Tau-Myc (green) and Dfr (red) expression. Co-expression is seen in the RP neurons (arrow) and the lateral cluster of motoneurons that include MN14/30 and MN 28 (arrowheads). (D) An early stage 15 isl-tau-mycEGFP transgenic embryo labeled for EGFP (green) and Dfr (red) expression. Several lateral EGFP-expressing cells, presumably the ISNd neurons, do not express Dfr (arrows). Dfr and EGFP co-expression is observed in two serotonergic EW neurons at this stage (arrowheads). (E) An abdominal neuromere from a late stage 15 egP289 lacZ enhancer trap line. ß-Gal (green) expression is visible in the three EW neurons and the GW (ISNd) motorneuron. Dfr (red) is not expressed in the GW (ISNd) motoneuron (arrows). (F) Dfr expression (green) is unaffected in an isl37Aa/islDf isl-tau-myc transgenic embryo. The RP (ISNb) motoneurons (arrows) are identified by Myc (red) expression from the transgene. (G) Tau-myc expression (arrows) is not altered in dfrB129/dfrDf Lim3A-tau-myc transgenic embryos.

View larger version (189K): [in a new window]   Fig. 2. Transheterozygous combinations between isl, Lim3 and dfr show motoneuron innervation defects. In each panel, the dissected embryos are at stage 16 and stained with anti-Fas2 antibodies. The ventral nerve cord is towards the left, anterior is upwards. (A) In the wild-type embryo, the ISNb motoneurons innervate muscles 6, 7, 12 and 13 in a stereotypical fashion, extending processes into the muscle clefts. (B) In isl37Aa Lim3BD6/+; dfrB129/+ embryos, the ISNb motoneurons fail to project to and innervate their specific target muscles, including muscles 12 and 13 (arrow). (C) Muscle innervation is also affected in isl37Aa/+; dfrB129/+ embryos (arrow) and in some hemisegments, the ISNb motor axons target the TN fascicle (arrowhead). (D) Lim3BD6/+; dfrB129/+ embryos also show severe defects in motor axon targeting and muscle innervation (arrow).

View larger version (129K): [in a new window]   Fig. 4. Dfr is required for ISNb motoneuron specification. (A) In isl37Aa/islDf embryos, the ISNb motor axons fail to correctly target the ventral muscles. The arrow indicates an absence of muscle cleft innervation at muscles 12 and 13. (B) Schematic summary of wild-type and dfr phenotypes. In the wild-type embryo, the ISNb (green) and TN (blue) neurons project axons to their appropriate target areas. In dfr mutants, the ISNb motor axons do not target the ventral muscles correctly and/or project ectopically in the TN fascicle area. (C) Two hemisegments of a stage 16 Lim3B-Gal4; dfrB129 UAS-dsdfr/+ embryo labeled with anti-Fas2 antibodies. The arrow indicates a lack of innervation at muscles 12 and 13. (D) Removing Dfr function can also cause ISNb motor axons to target the TN fascicle (arrow) in ftzng-Gal.20/dfrB129 UAS-dsdfr embryos.

View larger version (49K): [in a new window]   Fig. 3. Dfr expression can be reduced in the ISNb motoneurons. (A) Wild-type Dfr expression (green) in the ventral nerve cord of a stage 16 dfrB129 UAS-dsdfr/+transgenic embryo. Dfr is expressed in the ISNb neurons, including the RP neurons (lower arrow). Slit expression (magenta) is used to mark the Dfr-expressing midline glia (white/colocalization). (B) Dfr expression (green) is severely reduced or eliminated in Lim3B-Gal4;dfrB129 UAS-dsdfr/+ embryos. The absence of Dfr labeling in the RP neurons is indicated by the arrows. Dfr expression remains in the Slit-expressing midline glia (arrowhead) as well as in lateral neuronal clusters. (C) Two neuromeres of a Lim3B-Gal4;UAS-taumycEGFP VNC labeled with GFP (green) and the Lim3 antibody (magenta). Lim3 expression is co-localized with the reporter GFP expression in the RP neurons (lower arrow) and the TN neurons (arrowhead). The process of the TMNp neuron is also visualized in this image (upper arrow).

View larger version (120K): [in a new window]   Fig. 5. Dfr misexpression in the TN neurons results in a redirection of TN motor axons to ISNb muscle targets. (A and B1) In the wild-type embryo, two TN motoneurons leave the VNC. One, TMN 25, innervates ventral muscle 25 (out of the plane of focus) and the other, TMNp, contacts the ventral process of the LBD neuron. The TN and ISNb fascicles do not ever meet and are on different focal planes (arrowhead). All embryos are stained with anti-Fas2 antibodies. (B) Schematic summary of wild-type and Dfr misexpression phenotypes in the TN neurons (blue). (B2) When Dfr is misexpressed in the TN neurons, the TMNp motor axons target the ISNb muscle field. (C,D) In Lim3B-Gal4;UAS-dfr embryo late stage 16 embryos, the TN axons have retargeted to either enter or innervate the ISNb muscle field (arrows).

View larger version (101K): [in a new window]   Fig. 6. isl and Lim3 genetically interact with beat Ic in TN axon targeting and fasciculation. (A) In situ hybridization of beat Ic in a wild-type stage 15 VNC. Expression is evident in lateral clusters (arrows) but not in the RP neurons (arrowhead). (B) In situ hybridization and antibody labeling in a Lim3B-Gal4;UAS-taumycEGFP late stage 15 embryo. Both GFP (brown) and beat Ic (blue) expression is observed in the TN neurons (arrow). The cluster of RP neurons (arrowhead) stain very darkly for GFP expression but do not express beat Ic (see A). (C) Embryos that lack beat Ic were generated using the overlapping chromosomal deficiencies, beat IcDf/beat IcDf1(Df(2L)TE35D-GW19/Df(2L)RM5). In these embryos, the TMNp motor axon did not completely fasciculate with the LBD projection, resulting in bifurcation (arrow) and aberrant ventral muscle exploration. (D) In isl mutants, isl37Aa/islDf embryos, the same failure of the TMNp motor axon to fasciculate with the LBD projection is observed (arrow). (E,F) TN targeting and fasciculation is also affected (arrows) in embryos in which one copy of isl, Lim3 and beat Ic have been removed, isl37Aa Lim3BD6 beat IcDf (C) and isl37Aa Lim3BD6/beat IcDf1 (E). (G) Schematic diagram of the beat Ic locus. Isl1 (CTAATG) and Lhx3 (AATTAATTA) consensus sites within this locus are graphically represented.

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