DAP-2, the Drosophila homolog of transcription factor AP-2

Mechanisms of Development 76 (1998) 191–195

Gene expression pattern

DAP-2, the Drosophila homolog of transcription factor AP-2 Ignacio Monge, Pamela J. Mitchell* Institute of Pharmacology, University of Zu¨rich, Winterthurerstr. 190, 8057 Zu¨rich, Switzerland Received 17 June 1998; revised version received 2 July 1998; accepted 6 July 1998

Abstract Transcription factor AP-2 is essential for craniofacial, nervous system, and limb development in the mouse. We report here the cloning and expression pattern analysis of DAP-2, the Drosophila homolog of AP-2 family genes. DAP-2 is expressed in discrete regions of procephalic neuroectoderm, the brain, ventral nerve cord, and maxillary segment during Drosophila embryogenesis, and in the brain, optic lobes, ventral nerve cord, antenno-maxillary complex, and antennal and leg imaginal disks in third instar larvae. Protein sequence conservation and parallels between the embryonic expression patterns of DAP-2 and mammalian AP-2 family genes indicate that transcription factor AP-2 has been structurally and functionally conserved during metazoan evolution.  1998 Elsevier Science Ireland Ltd. All rights reserved Keywords: Transcription factor AP-2; DAP-2; DNA binding domain; Drosophila embryogenesis; Procephalic neuroectoderm; Maxillary segment; Protocerebrum; Ventral nerve cord; Antennomaxillary complex; Imaginal disk; Antenna; Optic lobe; Sense organ development

1. Molecular cloning of DAP-2 A cDNA fragment homologous to vertebrate AP-2 genes was amplified from Drosophila melanogaster embryonic cDNA by polymerase chain reaction (PCR) and used to probe Drosophila embryonic cDNA libraries (see Section 3). Multiple cDNAs representing a single gene, hereafter designated DAP-2, were isolated. The longest cDNA (2.0 kb, not shown) encodes a 461-amino-acid protein with slightly more amino acid identities to murine AP-2 than to other murine AP-2 family members (Fig. 1). In vitro transcription–translation of the DAP-2 cDNA yielded a 50 kDa protein which bound specifically to a bandshift probe representing a recently defined AP-2 binding site in the mouse Hoxa-2 gene (R. Krishnamurthy, I. Monge, P.J. Mitchell, unpublished data; M. Maconochie, S. Nonchev, R. Krishnamurthy, P. Meier, P.J. Mitchell, R. Krumlauf, unpublished data; Nonchev et al., 1996).

* Corresponding author. Tel.: +41 1 6355949; fax: +41 1 6355708; e-mail: mitchell @ pharma.unizh.ch

0925-4773/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0925-4773 (98 )0 0125-7

2. DAP-2 expression in Drosophila embryos and third instar larvae The developmental expression pattern of DAP-2 was analyzed by in situ hybridization of DIG-labeled-DAP-2-antisense RNA probe to wild-type Drosophila embryos (Figs. 2–4) and dissected larval tissues (Fig. 5). Zygotic transcripts were first detected at stage 9 in a restricted area of procephalic neuroectoderm (PNE) (brain anlage) (Fig. 2). Expression in the PNE expanded between stages 10 and 12 to include several discrete subregions (Campos-Ortega and Hartenstein, 1997). During stages 11–13, DAP-2 transcripts were also detected in presumed neuroblasts and daughter cells underlying PNE sites. At stage 14 and thereafter, DAP-2-expressing cells were located in the brain, mainly in the anteriormost neuromere, the protocerebrum (Fig. 2E,F). When viewed together with procephalic fate map data (Schmidt-Ott and Technau, 1994; Younossi-Hartenstein et al., 1996, 1997), the spatiotemporal pattern of DAP-2 expression in these dorsal head regions strongly suggests a lineage relationship between DAP-2-expressing cells in the PNE and in the brain thereafter. Aspects of this

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Fig. 1. DAP-2 and murine transcription factor AP-2 family proteins compared. Percent amino acid identities are indicated for the most highly conserved portion and regions adjacent to it (see Section 3 for DAP-2 cDNA cloning and PCR primers d51, d52 and d32). Amino acid positions and domains for transcriptional activation (transcr. act.), DNA binding and dimerization functions of AP-2 (Williams and Tjian, 1991) are indicated. The filled circle denotes a sequence (PPYFPPPY/F) present in the transcriptional activation domain of AP-2 that is perfectly conserved in the four proteins. Fig. 2. DAP-2 transcripts in procephalic region and embryonic brain. Dorsal views, anterior to left. (A–D) Expression in restricted areas of procephalic neuroectoderm (stages 9–13) and underlying presumed neuroblasts and daughter cells (stages 11–13). (E,F) Expression in the embryonic brain, mainly in the protocerebrum (pro). mx, maxillary segment (out of focal plane).

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pattern are reminiscent of proneural gene expression (e.g. achaete–scute complex (AS-C) genes expressed in neuroectoderm sites and neuroblasts selected from these sites) (Campos-Ortega, 1993). DAP-2 was expressed in the ventral nerve cord (VNC) beginning at stage 12 in two clusters of cells in every hemineuromere (Fig. 3). The relatively late onset of VNC expression indicates that DAP-2 is not involved in selection of VNC neuroblasts from ventral neuroectoderm (VNE), but rather functions in a subset of differentiating neurons or glia.

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Expression was detected in subregions of the maxillary segment beginning at stage 10 (not shown) and persisted as this head segment moved anteriorly to become part of the larval antenno-maxillary complex (Fig. 4). The maxillary segment generates larval mouth hooks, maxillary cirri and two chemoreceptive and mechanoreceptive sensory organs of the antennomaxillary complex: the ventral organ (VO) and maxillary sense organ (MxSO) (terminal organ) (ChuWang and Axtell, 1972a,b). MxSO and VO anlage roughly map to dorsomedial and ventromedial parts of the maxillary

Fig. 3. DAP-2 transcripts in the embryonic ventral nerve cord are restricted to two pairs of cell clusters per neuromere. Closed arrowhead indicates a lateral cluster; open arrowhead indicates a mediolateral cluster. (A,B) Ventral views, anterior up; (A) late stage 12, lateral clusters and incipient mediolateral clusters, (B) clusters contain 2–4 cells each. (C,D) Lateral views, anterior to left. mx, maxillary segment; pro, protocerebrum (mx and pro out of focal plane); s, nonspecific salivary gland staining. Fig. 4. DAP-2 maxillary segment expression. Anterior is left; lateral views in A–C, dorsal in D. Insets in B–D are labeled for orientation. (A–D) Expression in dorsal (d), posterior–dorsal (pd), posterior–ventral (pv), and lateral (lat) subregions of the maxillary lobe. to, terminal organ (or maxillary sense organ); pro, protocerebrum.

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lobe at stage 12 (Ju¨rgens et al., 1986), and may be flanked by or partly coincident with DAP-2-expressing cells. DAP-2 expression in the maxillary segment overlaps with expression of proboscipedia (pb) and Deformed (Dfd) (group 2 and 4 Hox genes) which regulate development of adult labial and maxillary palps, and larval mouth hooks, ventral organs and maxillary cirri, respectively (O’Hara et al., 1993; Aplin and Kaufman, 1997). In third instar larvae, DAP-2 was expressed in discrete parts of the optic lobes, brain, VNC, and antennal and leg imaginal disks (Fig. 5). The homeodomain gene Distalless (Dll) is also expressed, like DAP-2, in the maxillary segment and antennal and leg disks (O’Hara et al., 1993); and similarities exist between the expression patterns of AP-2 and Dlx (murine Dll-related) family members in the branchial arches, limbs, and forebrain during mouse embryogenesis (Mitchell et al., 1991; Bulfone et al., 1993; Chazaud et al., 1996; Anderson et al., 1997; Qiu et al., 1997). In summary, the DAP-2 developmental expression pattern described here strongly parallels the embryonic expression patterns of murine AP-2 family members in the CNS, maxillary (proximal) portion of the mandibular arch, and frontonasal and distal limb regions (Mitchell et al., 1991; Meier et al., 1995; Moser et al., 1995, 1997 Chazaud et al., 1996; Oulad-Abdelghani et al., 1996; Schorle et al., 1996). Thus, expression in discrete CNS regions and tissues that develop extensive sensory innervation is a conserved feature of Drosophila and mammalian transcription factor AP2 family genes.

served portions of the DNA binding domain of vertebrate AP-2 proteins. The PCR primers were as follows: d51: 5′-gctctaga(AG)GTIT(AT)(CT)TG(CT)(AT)C (AGT)GT(CT)C-3′; d52: 5′-cgggatcc(ACG)CCIGA(AG)TG(CT)(CT)T (ACT)AA(CT)GC-3′; d32: 5′-ccactagtG(AG)A(CT)IGCTTC(ACT)(CT)C (CT)TC(ACT)AC-3′. PCR conditions are available upon request. A 192 bp second-round PCR product digested with BamHI–SpeI was cloned into pBluescript (Stratagene); multiple clones were sequenced. The cloned fragment was used to screen Drosophila embryo cDNA libraries (stages 9–12 and 17hatching). Six overlapping cDNA clones were obtained and sequenced. The deduced protein sequence of DAP2 was compared to murine AP-2 family proteins using FASTA (GCG Wisconsin Sequence Analysis Package). DAP-2 is only slightly more similar to AP-2 than to AP-2ß and AP-2.2 (overall amino acid identities of 46%, 44%, and 43%, respectively), and the murine proteins are more similar to each other than to DAP-2. DAP-2 likely represents the homolog of the ancestral gene from which the mammalian AP-2 family arose subsequent to divergence of chordates and arthropods. The GenBank accession number for the longest DAP-2 cDNA is AJ006868. 3.2. In situ hybridization

3. Experimental procedures 3.1. Isolation and sequence analysis of Drosophila DAP-2 cDNA A DAP-2 cDNA fragment was synthesized from Drosophila embryo RNA by random-primed reverse transcription and PCR using degenerate PCR primers specific for con-

Antisense and sense riboprobes representing the longest DAP-2 cDNA (2.0 kb) were prepared (DIG-RNA Labeling Kit, Roche Diagnostics) and in situ hybridized to Oregon-R embryos (Tautz and Pfeifle, 1989) using AP-coupled anti-DIG-Fab fragment antibody and NBT and BCIP substrates (Roche Diagnostics). Staining of salivary gland and trachea was nonspecific as it was also seen in sense-probe controls.

Fig. 5. DAP-2 expression in third instar larval tissues. (A) Ventral view of CNS: brain (br), optic lobes (op) and ventral nerve cord (vnc). (B) Eye-antennal disk. (C) Leg disk. (D) Partly everted leg disk.

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Acknowledgements We thank Markus Noll and Joy Alcedo for kindly providing Drosophila cDNA libraries and staged embryo cDNA; Heinrich Reichert, Barry Dickson, Ernst Hafen, Markus Noll, Markus Affolter, Joy Alcedo, Doris Brentrup, Thomas Gutjahr, and Lei Xue for stimulating discussions and advice; and Eric Kubli for fly-culture space. This work was funded by the Swiss National Science Foundation (31-47228.96), Sandoz-Stiftung, and the Roche Research Foundation.

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