Development 121, 429-438 (1995) 429 Printed in Great Britain © The Company of Biologists Limited 1995 Proneural genes influence gliogenesis in Drosophila Angela Giangrande Institut de Génétique et de Biologie Molécularie et Cellulaire, CNRS UPR6520-INSERM U.184-ULP, BP163 67404 Illkirch Cedex, CU de Strasbourg, France SUMMARY Fly glial cells in the wing peripheral nervous system of pathway. Despite these similarities, however, the Drosophila melanogaster originate from underlying epithe- mechanism of action of the achaete-scute complex seems to lial cells. Two findings indicate that gliogenesis is closely be different in the two processes. Neural precursors express associated with neurogenesis. First, it only occurs in products of the complex, therefore the role of these genes regions that also give rise to sensory organs. Second, in on neurogenesis is direct. However, markers specific to glial mutants that induce the development of ectopic sensory cells do not colocalize with products of the achaete-scute organs glial cells develop at new positions. These findings complex, showing that the complex affects gliogenesis indi- prompted a genetic analysis to establish whether glial and rectly. These observations lead to the hypothesis that glio- sensory organ differentiation depend on the same genes. genesis is induced by the presence of sensory organ cells, Loss of function mutations of the achaete-scute complex either the precursor or its progeny. lead to a significant reduction of sensory bristles and glial cells. Genes within the complex affect gliogenesis with different strength and display some functional redundancy. Key words: proneural genes, gliogenesis, PNS, Drosophila, achaete- Thus, neurogenesis and gliogenesis share the same genetic scute complex INTRODUCTION because the proneural clusters and the SOP cells fail to form (Ghysen and O’Kane, 1989; Romani et al., 1989). Accord- Little is known about the genes inducing the first steps of gli- ingly, ASC ectopic expression due to a heat shock promoter or ogenesis. In the Drosophila wing, glial cells differentiate from to gain of function mutations such as Hairy-wing, (Hw), induce epithelial cells, from regions that also give rise to sensory ectopic sensory organs, due to the formation of additional SOP neurons (Giangrande, 1994). Moreover, mutations that induce cells (Campuzano et al., 1986; Balcells et al., 1988; Rodriguez ectopic sensory organs, also induce gliogenesis at these ectopic et al., 1990; Blair et al., 1992). Other genes are needed for positions, indicating a strong association between gliogenesis sensory organ differentiation. hairy (h) and extramacrochaete and neurogenesis (Giangrande, 1994). Since several genes (emc) repress sensory organ formation at ectopic positions involved in sensory organ differentiation are known (for through inhibitory interactions with the ASC (Moscoso del reviews, see Ghysen and Dambly-Chaudière, 1993; Jan and Prado and Garcia-Bellido, 1984a,b; Skeath and Carroll, 1991; Jan, 1994), it was important to ask whether they also act on Van Doren et al., 1991, 1992; Cubas and Modolell, 1992). gliogenesis. Using different glial markers in ASC mutant backgrounds I The so called proneural genes are necessary for the defini- show here that proneural genes as well as h are required for tion of the proneural cluster, a group of epithelial cells from proper gliogenesis in the wing PNS. I also show that the which one is singled out to become the sensory organ precursor products of the ASC are not expressed in the early steps of gli- cell (SOP) (for reviews, see Ghysen et al., 1993; Jan and Jan, ogenesis. These results strongly suggest that the mechanisms 1994). To this class of genes belong those of the achaete-scute of action of ASC are different in gliogenesis and neurogenesis. complex (ASC): achaete (ac), scute (sc) and asense (ase) (Garcia-Bellido, 1978; for a review, see Campuzano and Modolell, 1992). ac and sc are initially expressed in the MATERIALS AND METHODS proneural cluster and then in the SOP cell (Cubas et al., 1991; Skeath and Carroll, 1991, 1992). Their expression fades after Stocks the SOP cell has been singled out, when ase starts being The wild-type stocks used were Oregon R and Sevelen. The mutant expressed (Brand et al., 1993; Dominguez and Campuzano, stocks were the following: h1, ase (sc2 w s), Hw49c (In(1) Hw49c w 1993; Jarman et al., 1993). These genes code for bHLH type s/FM7), ac sc (In(1) ac3 sc10-1 f36a/FM7), sc (In(1) sc8L-sc4R v f/In (1) transcription factors that form heterodimers and activate gene dl49), ac (In(1)y3PL-sc8R), Hw1 (y Hw1m2g2/y2Y67g). The sc and ac expression (Murre et al., 1989a,b; Cabrera and Alonso, 1991). stocks were gifts from J. Modolell, the Hw1 stock was a gift from C. ASC loss of function mutations lead to lack of sensory organs, Dambly-Chaudière. As glial markers, the following enhancer trap 430 A. Giangrande lines were used: AE2 and rA87 (gifts from V. Auld and C. Goodman; Auld and Goodman, 1992; Giangrande et al., 1993); 2206 (gift from M. Schubiger; Giangrande et al., 1993; Schubiger et al., 1994). The lines A409.1F3/TM3Sb and B72.1M3 (gifts from W. Gehring), which carry a P element on the third chromosome, were identified as being glial specific in a screen in adult flies (Giangrande, unpublished results). All lines label subsets of glial cells, the only exception being rA87, which most likely transiently labels all or nearly all glial nuclei (Giangrande, unpublished results). Mutant heterozygous females were crossed with males of the enhancer trap line. In the F1 generation, males of the appropriate genotype were selected on the basis of the y+ phenotype. For ase and ac, homozygous females were used in the cross and therefore all males were of the appropriate genotype. For Hw1, a stock homozygous mutant and heterozygous for the glial insert was obtained, in order to analyse homozygous females, which display a stronger phenotype than males. Glial markers were always analysed in heterozygotes, to avoid possible recessive phenotypes due to the insert. Control flies were obtained by crossing the enhancer trap line with a wild-type stock. The line ase-β-gal (Jarman et al., 1993) was a gift from A. Jarman. Immunohistochemistry X-gal labelling in adults was performed as described by Giangrande et al. (1993). Antibody labelling was as described by Giangrande (1994). In double labelling experiments with anti-β-gal and a mouse monoclonal antibody as primaries, polyclonal rabbit-anti-β-gal from Cappel was used at 1:4000, detected with Cy3 conjugated goat-anti- rabbit 1:600 (Jackson). The monoclonal antibody 22C10 (a gift from S. Benzer) was used at 1:100 and detected with FITC-conjugated goat-anti-mouse 1:400 (Jackson). Rabbit-anti-asense (a gift from A. 10-1 Jarman) was used at 1:8000; mouse-anti-achaete supernatant was Fig. 1. Glial labelling in wild-type and sc adults. The 2206 prepared from the H990ΣSF1 line (a gift from J. Skeath and S. enhancer trap line was used as a glial marker. (A) 2206/+ and (B) 10-1 Carroll) and used at 1:50. These two antibodies were revealed with sc /Y; 2206/+ wings, labelled with X-gal. (A) Wild-type array of Cy3-conjugated goat-anti-rabbit 1:300 and FITC goat-anti-mouse glial nuclei (arrowheads) and sensory organs. cb and mb indicate the 1:300 (both from Jackson). chemo- and mechanosensory bristles on the anterior margin, Confocal images were obtained using a Zeiss axiophot and a Leica respectively; acv, the anterior cross vein sensillum; L3, the sensilla DMRE. present on L3; c and a, the costa and the alula; TSM, the position of the twin sensillum on the margin. L1-L3 shows the junction between veins L1 and L3. (B) Mutant wing shows dramatic defects at sensory organs and glial cells. Chemosensory bristles on the anterior margin RESULTS and on the costa are absent (see asterisks). Other types of bristles are also partially missing and all sensilla on acv and L3 are absent. No ASC loss of function mutations affect gliogenesis glial nuclei can be detected. To facilitate X-gal penetration, wings were cut at several positions. Bar 100 µm. Glial cell organization in ac sc mutants Adult wings carry two major nerves, L1 and L3, travelling bristles of the anterior margin as well as all the large campan- along the L1 and the L3 veins respectively (Murray et al., 1984; iform sensilla are absent (Fig. 1B). I have crossed the 2206 Hartenstein and Posakony, 1989). These purely sensory nerves glial-specific enhancer trap line (Giangrande et al., 1993; contain axons from the chemo- and mechanosensory bristle Schubiger et al., 1994) with sc10-1 flies and looked at the glial neurons located on the anterior margin and from the neurons labelling in adult mutant wings. Since 2206, as well as most of the large campaniform sensilla located on the L3 vein and glial specific lines, mark subsets of glia, two other lines have on the margin (Figs 1A, 2A). Wing sensory organs differenti- ate from precursor cells that divide two or more times to give rise to the neuron(s) and to the sensory organ accessory cells Fig. 2. Glial labelling in wild-type and sc10-1 wings at 25-38 hours (Hartenstein and Posakony, 1989). Glial cells present along the AP. 2206 and rA87 enhancer trap lines were used as glial markers. two nerves (Figs 1A, 2B) differentiate from wing epithelial Anti-HRP detects neurons and nerves (A,C,E); anti-β-gal detects cells, in the same regions in which sensory organs develop, and glial nuclei (B,D,F). G is a double exposure of E and F. then migrate for short distances along the underlying nerves (A,B) rA87/+ wing: arrows indicate glial nuclei along the L1(L1) and L3 (L3) nerves.