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. 2002 Nov 1;22(21):9143-9.
doi: 10.1523/JNEUROSCI.22-21-09143.2002.

Drosophila HB9 is expressed in a subset of motoneurons and interneurons, where it regulates gene expression and axon pathfinding

Joanne P Odden  1 , Scott Holbrook, Chris Q Doe
Affiliations

Drosophila HB9 is expressed in a subset of motoneurons and interneurons, where it regulates gene expression and axon pathfinding

Joanne P Odden et al. J Neurosci. .

Abstract

Motoneurons are an essential component of all metazoan nervous systems, but it is unknown whether there is an evolutionarily conserved mechanism for generating motoneurons during neurogenesis. In the vertebrate CNS, HB9/MNR2 transcription factors are specifically expressed in all somatic motoneurons and are necessary to distinguish motoneurons from interneurons, in part by repressing interneuron-specific gene expression. Here, we identify and characterize the single Drosophila ortholog of the HB9/MNR2 gene family. Drosophila HB9 is detected in a subset of motoneurons with ventral muscle targets and in a small group of interneurons, including the well characterized serotonergic interneurons. RNA interference knockdown of HB9 levels leads to defects in motoneuron ventral muscle target recognition, ectopic expression of a marker for dorsally projecting motoneurons (Even-skipped), and defects in serotonergic interneuronal projections. Conversely, ectopic HB9 expression causes an expansion of ventral motoneuron projections and repression of Even-skipped. Thus, Drosophila HB9 is required in a subset of motoneurons and interneurons for establishing proper axon projections but does not have a general role in distinguishing motoneuron and interneuron cell types.

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Figures

Fig. 1.
Fig. 1.
Identification and expression of theDrosophila HB9 gene. A–C, HB9 protein is first detected in the gut (asterisk), followed by the brain (arrowhead) and ventral CNS. Left, Anterior. A, B, Lateral views of stage 10 (A) and stage 13 (B) embryos; C, ventral view of a stage 15 embryo.VNC, Ventral nerve cord. D, HB9 protein is detected only in bilateral clusters of ∼50 cells in the third instar larval brain; anterior is up. E, HB9 (red) is coexpressed with the mitotic marker phosphohistone H3 (PPH3; green) in one or two cells per hemisegment; ventral view is of a late stage 10 embryo; anterior is left; the ventral midline is thebottom of each panel. F, Comparison of percentage amino acid identity between HB9/MNR2 proteins in the homeodomain (HD, gray).d, Drosophila; m, mouse;c, chick; AmphiMnx, amphioxus HB9.
Fig. 2.
Fig. 2.
HB9 protein is detected in a subset of motoneurons and interneurons. Localization of HB9 (red) compared with Eve (green) or Islet-tau:myc (green, anti-Tau) is shown. Two segments from a late stage 16 CNS are shown as projections of the superficial layer (A, B), intermediate layer (C, D), or internal layer (E, F) of the CNS. Anterior isup; the ventral midline is indicated by the white line. A, C, E, HB9 is not detected in any Eve+ motoneurons (U1–5,aCC, RP2) or interneurons (ELs, pCC). B, HB9 is detected in six interneurons (VHI neurons) and a single Islet+ neuron (arrowhead, VHN) but not in the Islet+ VUM motoneuron.D, HB9 is detected in the Islet+serotonergic interneurons (EW) and a lateral group of motoneurons (HL) that overlaps partially with a lateral group of Islet+ motoneurons but not in the Islet+ transverse nerve motoneurons (arrowhead, TN). F, HB9 is detected in the Islet+ RP1/3/4/5 motoneurons. Us, U1-5.
Fig. 3.
Fig. 3.
HB9 suppresses expression of the dorsal motoneuron determinant Eve. Wild-type,HB9RNAi, or HB9misexpression (ptc,en-Gal4/UAS-HB9 genotype) stage 16 embryos stained for HB9 (A–C) or Eve (D–I) are shown. Anterior isleft; the ventral midline is indicated by theblack line. A–C, Most HB9 protein is lost in HB9RNAi embryos, whereas all neurons, mesoderm, and epidermal cells express low levels of HB9 protein in HB9 misexpression embryos.D–F, Eve+ interneurons (ELs) are not affected significantly by loss of HB9 but are increased in number by HB9 misexpression. Eve+ U1–5 motoneurons have an extra Eve+ cell nearby (arrowhead) when HB9 is reduced but are almost completely missing after HB9 misexpression. G–I, Eve+ aCC/RP2 motoneurons and pCC interneuron have an extra Eve+ cell nearby (arrow) when HB9 is reduced but are almost completely missing after HB9 misexpression. J, Quantification of the Eve phenotype in wild-type,HB9RNAi, or two HB9misexpression genotypes in stage 16 embryos; Eve medial cells areaCC, pCC, RP2, and the U1–5 motoneurons.
Fig. 4.
Fig. 4.
HB9 promotes motoneuron ventral projections and inhibits motoneuron dorsal projections. Wild-type,HB9RNAi, or HB9misexpression (sca-Gal4/UAS-HB9 genotype) early stage 17 embryos stained for FasII (A–I) or β-gal (eagle-kinesin:lacZ genotype) (J–M) are shown. Anterior isleft; the ventral midline is indicated by theblack line; images are a montage of multiple focal planes (A–C, J–K), single focal planes (G–I), or camera lucida tracings (D–F: red, SNb; blue, ISN; brown, muscles 12, 13, 6, and 7 fromtop to bottom) (L–M:red, EW1–EW3 interneurons; blue, GW motoneuron). A–C, Dorsal, lateral, and ventral musculature are indicated with brackets. See Results for details.
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