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Munster, a novel Paired-class homeobox gene specifically expressed in the Drosophila larval eye
Mechanisms of Development 88 (1999) 107±110
Gene expression pattern
www.elsevier.com/locate/modo
Munster, a novel Paired-class homeobox gene speci®cally expressed in the Drosophila larval eye Anne Goriely 1, Bertrand Mollereau, Catherine Cof®nier, Claude Desplan* Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA Received 16 June 1999; received in revised form 9 July 1999; accepted 9 July 1999
Abstract Munster (Mu) is a homeobox-containing gene of the Paired-class which is speci®cally expressed in the developing Bolwig organs, the Drosophila larval eyes. This expression is ®rst detected during early germ band retraction stage (stage 12 from 7 h 20 at 258C) and persists until the end of embryogenesis. Mu homeodomain is most similar to that of Aristaless and D-Goosecoid. Strikingly, the Munster gene maps within 6 kb of D-goosecoid, in the same genomic region as aristaless, suggesting that these genes are part of a homeobox gene cluster. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Munster; Larval eye; Paired-class; Drosophila
1. Sequence analysis and cloning The Munster (Mu) genomic clone was originally isolated during a low stringency screen for Drosophila homeoboxcontaining genes (Dessain and McGinnis, 1993). The original EMBL3 phage (called phage 1, Fig. 1A) contains another partial homeobox (HB) sequence which belongs to D-goosecoid (D-gsc) (Goriely et al., 1996; Hahn and Jackle, 1996). Mapping of this region reveals that Mu and D-gsc HB are only separated by 6 kb of genomic sequence (Fig. 1A). Moreover, both D-gsc and Mu map on the left arm of the second chromosome at position 21C5-6 (data not shown), very close to another HB-containing gene, aristaless (al) (Campbell et al., 1993; Schneitz and Noll, 1990; Schneitz et al., 1993). Mapping performed with P1 clones suggests that D-gsc and Mu are about 150 kb away from al (data not shown). Interestingly, sequence comparison reveals a close homology between the Mu, D-Gsc and Al homeodomains (HD) (Fig. 1B). All three belong to the Paired-class. However, although both Al and Mu bear a glutamine at position 50 of their HD (Q50), D-Gsc possesses a lysine (K50). Residue 50 has been shown to confer DNA binding speci®city to the HD (Treisman et al., 1989; Hanes and Brent, 1989). This suggests that al, * Corresponding author. Tel.: 11-212-327-7965; fax: 11-212-3278370. E-mail address: [email protected] (C. Desplan) 1 Present address: Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK.
Mu and D-gsc might be part of a cluster of related homeobox genes which could be involved in the formation of sense organs or appendages in the Drosophila embryo (Schneitz and Noll, 1990). Repo is another HD-containing molecule which is closely related to Mu (Fig. 1B) (Xiong et al., 1994). Interestingly, the third helix of the HD of Repo, Mu and DGsc has been conserved during evolution. There is a perfect conservation at the DNA level between Mu and repo over 44 bp (indicated as `1' in Fig. 1B), and only one base pair difference between Mu/repo and D-gsc. This difference accounts for one residue change which is responsible for the Q50 (encoded by the CAG codon) to K50 (encoded by the AAG codon) transition between these molecules. Al HD exhibits a more divergent DNA sequence over this stretch with 11 differences, despite the excellent conservation at the amino acid level. This suggests that D-Gsc, Mu and Repo could derive from a common ancestor. It should be noted, however, that repo maps to a different chromosomal location (90F3±4) than that of D-gsc, Mu and al. The high degree of homology between Mu, D-gsc and repo DNA sequences is likely to explain the cross-hybridisation observed when a probe containing the Mu homeobox sequence is used for in situ hybridisation (see later). A single 1.2 kb clone, called Munster 1 (Mu1) cDNA, was isolated from an embryonic pNB40 cDNA library (kindly provided by Nick Brown) during a high stringency screen using a 300 bp fragment located directly 3 0 of the Munster homeobox. Sequencing reveals that this cDNA contains the 3 0 end of the Munster gene but lacks the homeobox
0925-4773/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00168-9
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A. Goriely et al. / Mechanisms of Development 88 (1999) 107±110
Fig. 1. (A) Map of the Munster genomic region. As described previously (Schneitz and Noll, 1990; Dessain and McGinnis, 1993), two adjacent homeobox (HB) sequences (large arrows) are found in the 21C±D region on the left arm of chromosome 2. Mu and D-gsc HB are separated by approximately 6 kb of genomic DNA and are transcribed in opposite orientations. This map is consistent with the genomic sequence of this region which is contained in the P1 phage DS07610 (Berkeley Drosophila Genome project: Genbank accession number AC004573). Munster 73 is a 1.8 kb EcoR1 genomic fragment which contains the Mu HB sequence, Gh01528 is the EST sequence which corresponds to the Munster gene and Mu1 is the partial cDNA we isolated from the pNB40 library. Two small introns (indicated by small open triangles in Munster 73) of 120 bp (I1) and 60 bp (I2) interrupt the Munster cDNA. The sequences of the 1.2-kb partial Mu 1 cDNA as well as the full sequence of the EST Gh01528 have been deposited into the EMBL/Genbank database under the accession numbers AJ238667 and AJ238668, respectively. Phage 1 and phage A were isolated from an EMBL3 Drosophila genomic library using a 950 bp EcoR1±EcoRV fragment from Munster 73 and the 5 0 end of the D-gsc cDNA as probes, respectively. E, EcoR1 sites; B, BamH1 sites. (B) Homeodomain (HD) sequences of Munster and related Paired-class proteins. Amino acid at position 50 has been indicated in bold: Mu HD bears a glutamine (Q50), unlike D-Gsc (lysine, K50) and Paired (serine, S50). `1' indicates the 44 bp conservation observed in the third helix of Mu, Repo and D-Gsc DNA sequences. Repo (Xiong et al., 1994), Aristaless (Al) (Schneitz et al., 1993), r-Cart1 (Zhao et al., 1993), Alx3 (Rudnick et al., 1994), D-Rx (Liu et al., 1994), D-Gsc (Goriely et al., 1996; Hahn and Jackle, 1996), Paired (Prd) (Frigerio et al., 1986). (C) Octapeptide/GEH domain. Comparison of the Octapeptide/GEH region of Mu and other GEH domains (Galliot et al., 1999 and references therein).
sequence (Fig. 1A). However, submission of this partial cDNA to the Berkeley Drosophila Genome Project Database (BDGP) (http://fruit¯y.berkeley.edu/EST) reveals homology with an EST (Expressed Sequence Tag), called Gh01528. Sequencing and analysis of the full-length EST shows that it encompasses the whole open reading frame of the Mu gene (Fig. 1A). Comparison between the genomic sequence and that of the EST establishes the exon/intron boundaries (open triangles in Munster 73 on Fig. 1A). A short region which shows homology to the Octapeptide/ GEH domain (Goosecoid Engrailed homology domain) is found in the C-terminal part of the Mu protein (Fig. 1C). This region has been shown to be required for active transcriptional repression by HD-containing molecules (Smith and Jaynes, 1996; Mailhos et al., 1998; Galliot et al., 1999).
2. Pattern of expression In situ hybridisation performed with a genomic probe containing the Mu homeobox (950 bp EcoR1±EcoRV genomic fragment, corresponding to the 5 0 end of the Munster 73 clone), reveals an extensive and dynamic pattern of expression, including a head stripe in early cellularized embryos, expression in the peripheral nervous system at germ band elongation stage and, later in development, in the Bolwig organs and the central nervous system (not shown). However, when the partial cDNA (Mu1 cDNA) that does not contain the homeobox is used as a probe, the pattern of expression is limited to the Bolwig organs (Fig. 2). This suggests that the extensive early pattern of expression is a consequence of cross-hybridisation with other homeobox-
A. Goriely et al. / Mechanisms of Development 88 (1999) 107±110
109
Fig. 2. Expression pattern of Munster. (A) Expression of Mu is ®rst detected in stage 12 embryos at the germ band retraction stage. (B,D) Mu expression in stage 13 embryos. (C,E) stage 17 embryo. (F) KruÈppel staining in stage 17 embryo (Schmucker et al., 1992). Compare the staining of Kr in the BO with that of Mu on Fig. 2E. All embryos are shown with anterior to the left. (G) Section of an adult eye. Mu is expressed in the medulla (m), lamina (l) and in all photoreceptors of the retina (r). The Mu in situ hybridisation shown on this ®gure were performed with a 900 bp EcoR1 fragment corresponding to the 5 0 end of the partial Mu1 cDNA.
containing genes, such as D-gsc for the head stripe and late central nervous system and repo for the peripheral nervous system staining. Mu expression is con®ned to two small clusters of epidermal cells on each side of the embryonic head and is ®rst detectable during early stage 12 at the germ band retraction stage (7 h 20±9 h 20 at 258C) (Fig. 2A). At stage 13 (Fig. 2B,D), the Mu-expressing cluster adopts a rosettelike shape and contains a total of 12 cells, seven of which are super®cial, while the ®ve others are located deeper. After head invagination (stage 17), the expressing clusters appear more compact and elongated (Fig. 2C,E). These cell clusters clearly correspond to the Bolwig organs (BO), the primitive larval eyes (Fig. 2E,F). In Drosophila, the BO is typically composed of 12 light-sensitive neurones whose axons fasciculate and extend as a nerve towards the brain
(Green et al., 1993). Mu expression is unusual in that it is limited to the BO, unlike other genes such as KruÈppel (Hoch et al., 1990; Schmucker et al., 1992) (Fig. 2F), disconnected (Lee et al., 1991) or orthodenticle (Vandendries et al., 1996) which are also involved in other developmental processes. Its expression at stage 12 makes Mu the earliest gene so far described to be expressed in BO. Given the universal conservation of the Pax-6 genetic cascade in eye morphogenesis (Halder et al., 1995; Sheng et al., 1997; Desplan, 1997), it would be of interest to determine whether Mu is a target of Pax-6 during embryonic development. However, the role of Pax-6 in BO development has yet to be documented and no vertebrate homologues of Mu have been reported so far. Mu expression is not detected in the eye imaginal disc (not shown). However, Mu is found in the adult eye, where it
110
A. Goriely et al. / Mechanisms of Development 88 (1999) 107±110
is expressed in most of the cells of the retina as well as in the lamina and the medulla, parts of the optic lobes (Fig. 2G). Acknowledgements The authors wish to thank Bill McGinnis for the generous gift of phage 1 form which this study originated, the Berkeley Drosophila Genome project Database (BDGP) for providing the Gh01528 EST, Dietmar Schmucker for helpful discussions on BO formation and the past and present members of the Desplan lab for their encouragement. This work was supported by the Howard Hughes Medical Institute. A.G. was the recipient of an EMBO fellowship and B.M. was supported by the ARC (Association pour la Recherche contre le cancer), the Philippe Foundation and by the Human Frontier Science Program Organization (HFSPO). References Campbell, G., Weaver, T., Tomlinson, A., 1993. Axis speci®cation in the developing Drosophila appendage: the role of wingless, decapentaplegic, and the homeobox gene aristaless. Cell 74, 1113±1123. Desplan, C., 1997. Eye development: governed by a dictator or a junta? Cell 91 (7), 861±864. Dessain, S., McGinnis, W., 1993. Drosophila homeobox genes. Adv. Dev. Biochem. 2, 1±55. Frigerio, G., Burri, M., Bopp, D., Baumgartner, S., Noll, M., 1986. Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network. Cell 47, 735±746. Galliot, B., de Vargas, C., Miller, D., 1999. Evolution of homeobox genes: Q50 Paired-like genes founded the Paired-class. Dev. Genes Evol. 209, 186±197. Goriely, A., Stella, M., Cof®nier, A., Mailhos, C., Dessain, S., Kessler, D., Desplan, C., 1996. A functional homologue of Goosecoid in Drosophila. Development 122, 1641±1650. Green, P., Hartenstein, A.Y., Hartenstein, V.C.S., 1993. The embryonic development of the Drosophila visual system. Cell Tissue Res. 273, 583±598. Hahn, M., Jackle, H., 1996. Drosophila, Goosecoid participates in neural development but not in axis formation. EMBO J. 15, 3077±3084. Halder, G., Callaerts, P., Gehring, W.J., 1995. Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267, 1788±1792.
Hanes, S.D., Brent, R., 1989. DNA speci®city of the bicoid activator protein is determined by homeodomain recognition helix residue 9. Cell 57, 1275±1283. Hoch, M., Schroder, C., Seifert, E., Jackle, A.H., 1990. Cis-acting control elements for KruÈppel expression in the Drosophila embryo. EMBO J. 9, 2587±2595. Lee, J.L., Freeman, M., Steller, H., 1991. Expression of the disconnected gene during development of Drosophila melanogaster. EMBO J. 10, 817±826. Liu, I.S., Chen, J.D., Ploder, L., Vidgen, D., van der Kooy, D., Kalnins, V.I., McInnes, R.R., 1994. Developmental expression of a novel murine homeobox gene (Chx10): evidence for roles in determination of the neuroretina and inner nuclear layer. Neuron 13, 377±393. Mailhos, C., AndreÂ, S., Mollereau, B., Goriely, A., Hemmati-Brivanlou, A., Desplan, C., 1998. Drosophila Goosecoid requires a conserved heptapeptide for repression of Paired-class homeoprotein activators. Development 125, 937±947. Rudnick, A., Ling, T.Y., Odagiri, H., Rutter, W.J., German, M., 1994. Pancreatic beta cells express a diverse set of homeobox genes. Proc. Natl. Acad. Sci. USA 91, 12203±12207. Schmucker, D., Taubert, H., Jackle, H., 1992. Formation of the Drosophila larval photoreceptor organ and its neuronal differentiation require continuous KruÈppel gene activity. Neuron 9, 1025±1039. Schneitz, K., Noll, M., 1990. Isolation of prd-type homeobox gene cluster involved in Drosophila morphogenesis. Experientia 46, A82. Schneitz, K., Spielmann, P., Noll, M., 1993. Molecular genetics of aristaless, a prd-type homeo box gene involved in the morphogenesis of proximal and distal pattern elements in a subset of appendages in Drosophila. Genes Dev. 7, 114±129. Sheng, G., Thouvenot, E., Schmucker, D., Wilson, D., Desplan, C., 1997. Direct regulation of rhodopsin 1 by Pax-6/eyeless in Drosophila: evidence for a conserved function in photoreceptors. Genes Dev. 11, 1122±1131. Smith, S., Jaynes, J., 1996. A conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2-, and msh-class homeoproteins, mediates active transcriptional repression in vivo. Development 122, 3141±3150. Treisman, J., Gonczy, P., Vashishtha, M., Harris, E., Desplan, C., 1989. A single amino acid can determine the DNA binding speci®city of homeodomain proteins. Cell 59, 553±562. Vandendries, E., Johnson, D., Reinke, R., 1996. Orthodenticle is required for photoreceptor cell development in the Drosophila eye. Dev. Biol. 173, 243±255. Xiong, W., Okano, H., Patel, N.H., Blendy, J.A., Montell, C., 1994. repo encodes a glial-speci®c homeo domain protein required in the Drosophila nervous system. Genes Dev. 8, 981±994. Zhao, G.Q., Zhou, X., Eberspaecher, H., Solursh, M., De Crombrugghe, B., 1993. Cartilage homeoprotein 1, a homeoprotein selectively expressed in chondrocytes. Proc. Natl. Acad. Sci. USA 90, 8633±8637.
Gene expression pattern
www.elsevier.com/locate/modo
Munster, a novel Paired-class homeobox gene speci®cally expressed in the Drosophila larval eye Anne Goriely 1, Bertrand Mollereau, Catherine Cof®nier, Claude Desplan* Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA Received 16 June 1999; received in revised form 9 July 1999; accepted 9 July 1999
Abstract Munster (Mu) is a homeobox-containing gene of the Paired-class which is speci®cally expressed in the developing Bolwig organs, the Drosophila larval eyes. This expression is ®rst detected during early germ band retraction stage (stage 12 from 7 h 20 at 258C) and persists until the end of embryogenesis. Mu homeodomain is most similar to that of Aristaless and D-Goosecoid. Strikingly, the Munster gene maps within 6 kb of D-goosecoid, in the same genomic region as aristaless, suggesting that these genes are part of a homeobox gene cluster. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Munster; Larval eye; Paired-class; Drosophila
1. Sequence analysis and cloning The Munster (Mu) genomic clone was originally isolated during a low stringency screen for Drosophila homeoboxcontaining genes (Dessain and McGinnis, 1993). The original EMBL3 phage (called phage 1, Fig. 1A) contains another partial homeobox (HB) sequence which belongs to D-goosecoid (D-gsc) (Goriely et al., 1996; Hahn and Jackle, 1996). Mapping of this region reveals that Mu and D-gsc HB are only separated by 6 kb of genomic sequence (Fig. 1A). Moreover, both D-gsc and Mu map on the left arm of the second chromosome at position 21C5-6 (data not shown), very close to another HB-containing gene, aristaless (al) (Campbell et al., 1993; Schneitz and Noll, 1990; Schneitz et al., 1993). Mapping performed with P1 clones suggests that D-gsc and Mu are about 150 kb away from al (data not shown). Interestingly, sequence comparison reveals a close homology between the Mu, D-Gsc and Al homeodomains (HD) (Fig. 1B). All three belong to the Paired-class. However, although both Al and Mu bear a glutamine at position 50 of their HD (Q50), D-Gsc possesses a lysine (K50). Residue 50 has been shown to confer DNA binding speci®city to the HD (Treisman et al., 1989; Hanes and Brent, 1989). This suggests that al, * Corresponding author. Tel.: 11-212-327-7965; fax: 11-212-3278370. E-mail address: [email protected] (C. Desplan) 1 Present address: Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK.
Mu and D-gsc might be part of a cluster of related homeobox genes which could be involved in the formation of sense organs or appendages in the Drosophila embryo (Schneitz and Noll, 1990). Repo is another HD-containing molecule which is closely related to Mu (Fig. 1B) (Xiong et al., 1994). Interestingly, the third helix of the HD of Repo, Mu and DGsc has been conserved during evolution. There is a perfect conservation at the DNA level between Mu and repo over 44 bp (indicated as `1' in Fig. 1B), and only one base pair difference between Mu/repo and D-gsc. This difference accounts for one residue change which is responsible for the Q50 (encoded by the CAG codon) to K50 (encoded by the AAG codon) transition between these molecules. Al HD exhibits a more divergent DNA sequence over this stretch with 11 differences, despite the excellent conservation at the amino acid level. This suggests that D-Gsc, Mu and Repo could derive from a common ancestor. It should be noted, however, that repo maps to a different chromosomal location (90F3±4) than that of D-gsc, Mu and al. The high degree of homology between Mu, D-gsc and repo DNA sequences is likely to explain the cross-hybridisation observed when a probe containing the Mu homeobox sequence is used for in situ hybridisation (see later). A single 1.2 kb clone, called Munster 1 (Mu1) cDNA, was isolated from an embryonic pNB40 cDNA library (kindly provided by Nick Brown) during a high stringency screen using a 300 bp fragment located directly 3 0 of the Munster homeobox. Sequencing reveals that this cDNA contains the 3 0 end of the Munster gene but lacks the homeobox
0925-4773/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00168-9
108
A. Goriely et al. / Mechanisms of Development 88 (1999) 107±110
Fig. 1. (A) Map of the Munster genomic region. As described previously (Schneitz and Noll, 1990; Dessain and McGinnis, 1993), two adjacent homeobox (HB) sequences (large arrows) are found in the 21C±D region on the left arm of chromosome 2. Mu and D-gsc HB are separated by approximately 6 kb of genomic DNA and are transcribed in opposite orientations. This map is consistent with the genomic sequence of this region which is contained in the P1 phage DS07610 (Berkeley Drosophila Genome project: Genbank accession number AC004573). Munster 73 is a 1.8 kb EcoR1 genomic fragment which contains the Mu HB sequence, Gh01528 is the EST sequence which corresponds to the Munster gene and Mu1 is the partial cDNA we isolated from the pNB40 library. Two small introns (indicated by small open triangles in Munster 73) of 120 bp (I1) and 60 bp (I2) interrupt the Munster cDNA. The sequences of the 1.2-kb partial Mu 1 cDNA as well as the full sequence of the EST Gh01528 have been deposited into the EMBL/Genbank database under the accession numbers AJ238667 and AJ238668, respectively. Phage 1 and phage A were isolated from an EMBL3 Drosophila genomic library using a 950 bp EcoR1±EcoRV fragment from Munster 73 and the 5 0 end of the D-gsc cDNA as probes, respectively. E, EcoR1 sites; B, BamH1 sites. (B) Homeodomain (HD) sequences of Munster and related Paired-class proteins. Amino acid at position 50 has been indicated in bold: Mu HD bears a glutamine (Q50), unlike D-Gsc (lysine, K50) and Paired (serine, S50). `1' indicates the 44 bp conservation observed in the third helix of Mu, Repo and D-Gsc DNA sequences. Repo (Xiong et al., 1994), Aristaless (Al) (Schneitz et al., 1993), r-Cart1 (Zhao et al., 1993), Alx3 (Rudnick et al., 1994), D-Rx (Liu et al., 1994), D-Gsc (Goriely et al., 1996; Hahn and Jackle, 1996), Paired (Prd) (Frigerio et al., 1986). (C) Octapeptide/GEH domain. Comparison of the Octapeptide/GEH region of Mu and other GEH domains (Galliot et al., 1999 and references therein).
sequence (Fig. 1A). However, submission of this partial cDNA to the Berkeley Drosophila Genome Project Database (BDGP) (http://fruit¯y.berkeley.edu/EST) reveals homology with an EST (Expressed Sequence Tag), called Gh01528. Sequencing and analysis of the full-length EST shows that it encompasses the whole open reading frame of the Mu gene (Fig. 1A). Comparison between the genomic sequence and that of the EST establishes the exon/intron boundaries (open triangles in Munster 73 on Fig. 1A). A short region which shows homology to the Octapeptide/ GEH domain (Goosecoid Engrailed homology domain) is found in the C-terminal part of the Mu protein (Fig. 1C). This region has been shown to be required for active transcriptional repression by HD-containing molecules (Smith and Jaynes, 1996; Mailhos et al., 1998; Galliot et al., 1999).
2. Pattern of expression In situ hybridisation performed with a genomic probe containing the Mu homeobox (950 bp EcoR1±EcoRV genomic fragment, corresponding to the 5 0 end of the Munster 73 clone), reveals an extensive and dynamic pattern of expression, including a head stripe in early cellularized embryos, expression in the peripheral nervous system at germ band elongation stage and, later in development, in the Bolwig organs and the central nervous system (not shown). However, when the partial cDNA (Mu1 cDNA) that does not contain the homeobox is used as a probe, the pattern of expression is limited to the Bolwig organs (Fig. 2). This suggests that the extensive early pattern of expression is a consequence of cross-hybridisation with other homeobox-
A. Goriely et al. / Mechanisms of Development 88 (1999) 107±110
109
Fig. 2. Expression pattern of Munster. (A) Expression of Mu is ®rst detected in stage 12 embryos at the germ band retraction stage. (B,D) Mu expression in stage 13 embryos. (C,E) stage 17 embryo. (F) KruÈppel staining in stage 17 embryo (Schmucker et al., 1992). Compare the staining of Kr in the BO with that of Mu on Fig. 2E. All embryos are shown with anterior to the left. (G) Section of an adult eye. Mu is expressed in the medulla (m), lamina (l) and in all photoreceptors of the retina (r). The Mu in situ hybridisation shown on this ®gure were performed with a 900 bp EcoR1 fragment corresponding to the 5 0 end of the partial Mu1 cDNA.
containing genes, such as D-gsc for the head stripe and late central nervous system and repo for the peripheral nervous system staining. Mu expression is con®ned to two small clusters of epidermal cells on each side of the embryonic head and is ®rst detectable during early stage 12 at the germ band retraction stage (7 h 20±9 h 20 at 258C) (Fig. 2A). At stage 13 (Fig. 2B,D), the Mu-expressing cluster adopts a rosettelike shape and contains a total of 12 cells, seven of which are super®cial, while the ®ve others are located deeper. After head invagination (stage 17), the expressing clusters appear more compact and elongated (Fig. 2C,E). These cell clusters clearly correspond to the Bolwig organs (BO), the primitive larval eyes (Fig. 2E,F). In Drosophila, the BO is typically composed of 12 light-sensitive neurones whose axons fasciculate and extend as a nerve towards the brain
(Green et al., 1993). Mu expression is unusual in that it is limited to the BO, unlike other genes such as KruÈppel (Hoch et al., 1990; Schmucker et al., 1992) (Fig. 2F), disconnected (Lee et al., 1991) or orthodenticle (Vandendries et al., 1996) which are also involved in other developmental processes. Its expression at stage 12 makes Mu the earliest gene so far described to be expressed in BO. Given the universal conservation of the Pax-6 genetic cascade in eye morphogenesis (Halder et al., 1995; Sheng et al., 1997; Desplan, 1997), it would be of interest to determine whether Mu is a target of Pax-6 during embryonic development. However, the role of Pax-6 in BO development has yet to be documented and no vertebrate homologues of Mu have been reported so far. Mu expression is not detected in the eye imaginal disc (not shown). However, Mu is found in the adult eye, where it
110
A. Goriely et al. / Mechanisms of Development 88 (1999) 107±110
is expressed in most of the cells of the retina as well as in the lamina and the medulla, parts of the optic lobes (Fig. 2G). Acknowledgements The authors wish to thank Bill McGinnis for the generous gift of phage 1 form which this study originated, the Berkeley Drosophila Genome project Database (BDGP) for providing the Gh01528 EST, Dietmar Schmucker for helpful discussions on BO formation and the past and present members of the Desplan lab for their encouragement. This work was supported by the Howard Hughes Medical Institute. A.G. was the recipient of an EMBO fellowship and B.M. was supported by the ARC (Association pour la Recherche contre le cancer), the Philippe Foundation and by the Human Frontier Science Program Organization (HFSPO). References Campbell, G., Weaver, T., Tomlinson, A., 1993. Axis speci®cation in the developing Drosophila appendage: the role of wingless, decapentaplegic, and the homeobox gene aristaless. Cell 74, 1113±1123. Desplan, C., 1997. Eye development: governed by a dictator or a junta? Cell 91 (7), 861±864. Dessain, S., McGinnis, W., 1993. Drosophila homeobox genes. Adv. Dev. Biochem. 2, 1±55. Frigerio, G., Burri, M., Bopp, D., Baumgartner, S., Noll, M., 1986. Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network. Cell 47, 735±746. Galliot, B., de Vargas, C., Miller, D., 1999. Evolution of homeobox genes: Q50 Paired-like genes founded the Paired-class. Dev. Genes Evol. 209, 186±197. Goriely, A., Stella, M., Cof®nier, A., Mailhos, C., Dessain, S., Kessler, D., Desplan, C., 1996. A functional homologue of Goosecoid in Drosophila. Development 122, 1641±1650. Green, P., Hartenstein, A.Y., Hartenstein, V.C.S., 1993. The embryonic development of the Drosophila visual system. Cell Tissue Res. 273, 583±598. Hahn, M., Jackle, H., 1996. Drosophila, Goosecoid participates in neural development but not in axis formation. EMBO J. 15, 3077±3084. Halder, G., Callaerts, P., Gehring, W.J., 1995. Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267, 1788±1792.
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