The products of the homeotic genes in Drosophila are transcription factors that are necessary to impose regional identity along the anterior-posterior axis of the developing embryo. However, the target genes under homeotic regulation that control this developmental process are largely unknown. We have utilized an immunopurification method to clone target genes of the Antennapedia protein (ANTP). We present here the characterization of centrosomin (cnn), one of the target genes isolated using this approach. The spatial and temporal expression of the cnn gene in the developing visceral mesoderm (VM) of the midgut and the central nervous system (CNS) of wild-type and homeotic mutant embryos is consistent with the idea that cnn is a homeotic target. In the VM, Antp and abdominal-A (abd-A) negatively regulate cnn, while Ultrabithorax (Ubx) shows positive regulation. In the CNS, cnn is regulated positively by Antp and negatively by Ubx and abd-A. Characterization of a cDNA encoding CNN predicts a novel structural protein with three leucine zipper motifs and several coiledcoil domains exhibiting limited homology to the rod portion of myosin. Immunocytochemical results demonstrate that the cnn encoded protein is localized to the centrosome and the accumulation pattern is coupled to the nuclear and centrosome duplication cycles of cleavage. In addition, evidence suggests that the expression of the cnn gene in the VM correlates with the morphogenetic function of Ubx in that tissue, i.e., the formation of the second midgut constriction. The centrosomal localization of CNN and the involvement of microtubules in midgut morphogenesis suggest that this protein may participate in mitotic spindle assembly and the mechanics of morphogenesis through an interaction with microtubules, either directly or indirectly.

The homeotic selector (HOM) genes in Drosophila encode transcription factors that are necessary for the regional specification of characteristic structures and organs. The biological importance of these genes is apparent in their high conservation across many species (Slack et al., 1993) and the gross abnormalities in the body plan that result from misexpression or absence of expression of these genes (Lewis, 1978; Karch et al., 1985; Sanchez-Herrero et al., 1985; Kaufman et al., 1990). HOM genes are thought to fulfil their function in the specification of segmental identity through the transcriptional activation or repression of a set of downstream genes (Andrew and Scott, 1992; Botas, 1993). The identification of homeotic target genes is now an active endeavor in the field of homeotic research. Currently, there are few characterized target genes and we need to know more about the various roles that target genes serve in morphogenetic events. It has been postulated that homeotic target genes encode a variety of different proteins that have both regulatory and/or structural functions in processes of morphogenesis (Botas, 1993). These proteins might be involved in the regulation of cellular activities such as cell migration, communication, division, adhesion, differentiation and neuronal target innervation. Indeed, identified homeotic target genes include molecules that are regulatory in nature such as distal-less, a transcription factor that controls limb development in the thoracic region (Vachon et al., 1992), and structural molecules such as connectin, that specify the innervation of a subpopulation of motoneurons with their muscles (Gould and White, 1992; Nose et al., 1992).

The VM represents an example of a tissue in which the homeotic genes regulate specific events of morphogenesis. The homeotic genes are expressed in non-overlapping domains of the VM surrounding the midgut and have specific roles in midgut morphogenesis (Tremml and Bienz, 1989; Reuter and Scott, 1990). Molecules related to mammalian growth factors have been implicated as candidate target genes of the homeotics in events of midgut morphogenesis. One of these genes is decapentaplegic (dpp), a member of the TGFβ family of growth factors (Padgett et al., 1987) and another is wingless, a member of the int family of growth factors (Immerglück et al., 1990). These molecules are implicated in signaling pathways between the VM and endoderm in aspects of midgut morphogenesis (Immerglück et al., 1990; Reuter et al., 1990; Panganiban et al., 1990). However, no homeotic target genes have been identified in the VM that participate in the mechanical events of midgut morphogenesis.

We have generated a library of candidate Antennapedia (Antp) binding sites by the immunopurification of chromatin bound in vivo by ANTP (Roche et al., unpublished data). Using a probe generated from this library, we have identified a gene, which we have named centrosomin (cnn), that is regulated by Antp and other homeotic genes in the CNS and VM. We describe here the initial molecular and genetic characterization of the cnn gene with a focus on the function of the cnn gene product in the process of midgut morphogenesis. Our data indicates that the cnn gene is located in a genomic region required for specific events of midgut morphogenesis and encodes a novel type of structural protein that localizes to the centrosome. As centrosomes represent microtubule organizing centers (MTOCs) (Mitchison and Kirschner, 1984) and microtubules have been implicated in midgut morphogenesis (Reuter and Scott, 1990), we suggest that cnn is involved in the assembly of mitotic spindles and the mechanics of midgut morphogenesis as a homeotic target gene through its direct or indirect interaction with microtubules.

Drosophila stocks

Drosophila stocks were raised at 25°C on standard medium supplemented with live yeast. The wild-type stock of Drosophila melanogaster used was Oregon-R. The homeotic mutant stocks used were Scr4red e/TM6B, which is protein null for Scr (Wakimoto and Kaufman, 1981), AntpA74red e/TM3, which is a transcription null for Antp (Abbott and Kaufman, 1986) and Df(3R)Ubx109/Dp(3,3)P5,Sb, which is deficient for the Ubx and abd-A transcription units (Lewis, 1978; Karch et al., 1985). Other mutant stocks utilized were dpps14/CyO, which removes dpp function in the embryonic midgut (Lindsley and Zimm, 1992). The transformant line P{w+mCGal4hsp70=GawB}24B was a kind gift of Andrea Brand and expresses the yeast transcriptional activator GAL4 (Ptashne et al., 1988) in the entire mesoderm (Brand and Perrimon, 1993). The transformant lines P{w+mCScrUAS=UAS::Scr}EE2, P{w+mCAntpUAS=UAS::Antp}W2 and P{w+mCUbxUAS=UAS::Ubx}M2A carry the Scr, Antp, and Ubx cDNAs respectively under the transcriptional control of GAL4 (Kalkbrenner et al., in preparation). The UBX protein expressed by the last construct is the Ia isoform. Deficiency and P-element stocks used for genetic analysis include Df(2R)vg-B and Df(2R)vg135 (Lasko and Pardue, 1988), P{F}1/CyO (Levis et al., 1985) and P{PZ}drk10626/CyO (Mlodzik and Hiromi, 1992), all on the second chromosome. The transformant line that expresses lacZ in the midgut carries a reporter construct called RD2 that places shv region sequences of the dpp gene upstream of the minimal hsp70 promotor to drive β-galactosidase expression in ps 4 and 7 of the midgut and is described in Hursh et al. (1993). This construct is on the third chromosome and was a kind gift from William Gelbart. The lacZdppshv containing construct P{w+mClacZdppshv=dppshv::lacZ}RD2 was crossed into the Df(2R)cnn background for analysis of the midgut in cnn mutant embryos.

Df(2R)cnn was created by X-ray induced deletion of P{F}1. Males of the genotype w; P{w+=F}1/CyO were treated with 4,000r of Xirradiation, and mated to w; Sp/SM5 females. P-element deletion events were recovered as white-eyed progeny that were either Sp or Cy but not both. Male ‘w’/Sp and both sexes of ‘w’/SM5 were crossed back to w; Sp/SM5 flies. A stock was established by inbreeding the Cy progeny. Cytological examination of the mutagenized chromosomes revealed that one of the recovered lines was associated with a small deletion that removes all of section 50A and may delete some of the material from the 50B1,2 doublet. Embryos from the balanced stock of this candidate cnn deficiency were stained with CNN-specific antibodies to determine if homozygotes lacked the gene product. We have called this deletion Df(2R)cnn.

Antibodies and staining procedures

The collection, fixation and staining of embryos was performed essentially according to the method of Mahaffey and Kaufman (1987). Double staining of embryos was performed using simultaneous incubations with polyclonal and monoclonal primary antibodies followed by secondary incubations with goat anti-mouse alkaline phosphatase (AP) and goat anti-rabbit horseradish peroxidase (HRP). The HRP reaction was developed first, followed by 2× 5 minute washes in PBT, 1× 5 minute wash in AP buffer, and then the AP reaction was developed. Following the AP reaction, the embryos were washed in PBT several times, then dehydrated in ethanol and either rehydrated and mounted in glycerol or cleared in methyl salicylate. Embryos that were double stained for CNN and either DNA or α-Tubulin were fixed with methanol according to the method of Matthews et al. (1993). Staining of ovaries was done according to the method of Matthews et al. (1990). The anti-DNA and anti-Tubulin monoclonal antibodies were kindly provided by K. Matthews. The anti-LAB antibody was generated as described by Diederich et al. (1989). The hybridoma lines 8C11.1 (anti-ANTP; J. Condie and D. Brower, unpublished results) and FP3.38 (anti-UBX; White and Wilcox, 1984) were kindly provided by D. Brower and M. Muskavitch respectively. The supernatant from the hybridoma line BP104 (anti-Neuroglian) was kindly provided by C. Goodman and is described by Hortsch et al. (1990). The F2 rat polyclonal antibody (anti-CUT) was kindly provided by Y. Jan and is described by Blochlinger et al. (1990). The β-galactosidase monoclonal antibody was purchased from Boehringer Mannheim and used at a dilution of 1:1000. The anti-DNA and antiTubulin antibodies were used at 1:200, the antibodies 8C11.1 and FP3.38 were used as purified IgG at 1-3 μg/ml, while the BP104 monoclonal was used as hybridoma supernatant at a 1:5 dilution. The F2 (anti-CUT) and R19 (anti-CNN) polyclonal antibodies were both used at a dilution of 1:500, while the LAB polyclonal antibody was used at 1:100. The secondary antibodies used for immunostaining were goat anti-mouse HRP, goat anti-rabbit HRP and goat anti-mouse AP (Jackson Labs). The secondary HRP antibodies were used at a dilution of 1:500 and the secondary AP antibody was used at 1:1000. Stained embryos were examined and photographed under Nomarski optics with a Zeiss axiophot microscope. Photographs were taken with Kodak Gold color print film with a blue filter in place for color correction and a tungsten light source.

Genomic and cDNA cloning

The preparation and characterization of the immunopurified library of candidate Antp target genes will be described elsewhere (Roche et al., unpublished data). cDNA library screening, cloning and phage DNA preparation were done according to standard protocols (Sambrook et al., 1989). The Drosophila embryonic cDNA library was purchased from Novagen. Gel purified genomic fragments were radiolabeled with [32P]dATP (Amersham) by a random priming kit (Boehringer Mannheim). Genomic and cDNA phage clones were mapped according to standard methods by restriction enzyme digestion followed by Southern blot hybridization (Sambrook et al., 1989). All restriction enzymes used were purchased from NEB.

DNA sequencing

Restriction fragments of the cnn cDNA were subcloned into both pBluescript KS+ and KS− plasmids (Stratagene). Single stranded DNA from recombinant pBluescript clones was obtained by infection with M13K07 helper phage (kindly provided by N. Pace) and isolated according to a protocol from Stratagene. Both strands of the cDNA were completely sequenced with a kit to generate nested deletions by Exo III-mung bean nuclease treatment (Stratagene) and synthesis of internal primers to extend the sequence range in both directions. The templates were sequenced with the T7 and T3 primers (Stratagene) and several internal primers from the cDNA sequence in dideoxy sequencing reactions (Sanger et al., 1977) with 35S-dATP (Amersham) and Sequenase version 2.0 (USB) according to the vendor’s instructions. Internal primers for extending sequence were generated on an Applied Biosystems DNA synthesizer and were used without further purification. Compressions were resolved with dITP according to instructions from USB. Sequencing reactions were resolved on 5% polyacrylamide wedge gels containing 7 M urea, fixed for 30 minutes in an acetic acid/methanol bath, dried for 1 hour and subjected to autoradiography with Amersham film. DNA sequence was analyzed with MacVector 2.1, DNA strider 1.2 and DNASIS software.

Production of CNN fusion protein and CNN specific antibodies

To produce the CNN protein in E. coli, a lacZ::cnn fusion construct was made with a pWR590-1 lacZ fusion cassette (Guo et al., 1984). The CNN portion of the fusion protein encodes amino acids 271-1034 and was derived from the cnn cDNA. The fusion protein was expressed in JM101 E. coli cells and isolated according to the method of Mahaffey and Kaufman (1987). The 158×103Mr fusion protein was isolated from a 10% SDS-PAGE gel, emulsified in Freund’s adjuvant and injected intramuscularly into rabbits. Prior to injection of fusion protein, the rabbits were bled to obtain preimmune serum. β-gal/CNN fusion protein was purified from gel slices according to the method of Mahaffey and Kaufman (1987). Approximately 2 mg each of purified β-gal/CNN fusion protein and βgalactosidase protein were individually coupled to Affigel-10 (Biorad) to make two affinity chromotography columns for each protein. Bleeds from rabbits were obtained after two subsequent boosts with fusion protein, the serum was allowed to clot and complement was subsequently heat inactivated. CNN specific antibodies were obtained by first passing the anti-serum over two columns of Affigel-10 coupled to βgalactosidase, followed by two rounds of purification over Affigel-10 coupled to β-gal/CNN fusion protein. The purified antibodies were neutralized with Tris buffer pH 7.5, BSA was added to 1 mg/ml and sodium azide was added to 0.025%. Antibodies were then aliquoted and stored at −70°C.

Western blot analysis

Small amounts of ovaries and embryos were homogenized in an Eppendorf tube with a small pestle in 1× Laemmli buffer. Protein samples were boiled for 20 minutes in Laemmli buffer, seperated on 10% SDSPAGE gels according to standard methods (Sambrook et al., 1989) and transferred electrophoretically using the mini-Protean II equipment (Biorad) to PVDF membranes (Biorad) according to the manufacturer’s instructions. Western analysis was performed with CNN-specific antibodies at a dilution of 1:5,000 and a goat anti-rabbit secondary antibody coupled to alkaline phosphatase at a dilution of 1:2,000 (Promega). Blots were blocked in 5% BSA for 1 hour, incubated for 1 hour with primary antibody, washed 3× for 10 minutes each and then incubated with secondary antibody for 1 hour. The blots were then washed again 3× for 10 minutes each and developed for alkaline phosphatase activity with NBT and X-phos as substrates (Boehringer) and then photographed with Kodak black and white film.

Whole-mount in situ hybridization to embryos

Whole-mount in situ hybridization to embryos was performed according to the method of Tautz and Pfeifle (1989). Double staining of embryos for protein and RNA was done with in situ hybridization first, followed by immunocytochemistry for the protein. Digoxigeninlabeled cRNA was prepared with kits (Promega, Boehringer Mannheim) and digoxigenin-dUTP or digoxigenin RNA labeling mixture (Boehringer Mannheim). Digoxigenin-labeled probes were detected with an alkaline phosphatase coupled anti-digoxigenin antibody with NBT and X-phos as substrates (Boehringer Mannheim).

Northern blot analysis

Total RNA was isolated from an overnight collection of embryos with Trizol reagent (BRL) and the manufacturer’s protocol. Poly(A)+ selected mRNA was obtained with a kit (Boehringer Mannheim) and quantitated by absorbance at 260 nm. 3 μg of poly(A)+ mRNA per well was seperated electrophoretically on a formaldehyde gel and transferred electrophoretically to Nytran (S&S) in 1× TAE buffer, probed and analyzed according to standard techniques (Sambrook et al., 1989).

Cytogenetic analysis

In situ hybridization to polytene chromosomes was performed according to standard methods (Roberts, 1986). Digoxigenin-labeled DNA was prepared by random priming and detected with a kit from Boehringer Mannheim. Chromosome squashes were analyzed under phase contrast optics on a Zeiss axiophot microscope.

A novel protein with three leucine zippers and limited homology to coiled-coil proteins is encoded by cnn

The immunopurified DNA fragment of a clone designated no. 18 was obtained from a library of immunopurified chromatin bound by ANTP in vivo (Roche et al., unpublished data) and used to isolate genomic clone jh18-A. A 6.8 kb SalI fragment derived from this genomic clone contains a transcription unit as indicated by whole-mount in situ hybridization to wild-type embryos. The expression pattern of this transcription unit suggested homeotic regulation, i.e., there were higher levels of expression in the thoracic region of the CNS compared to the abdominal region. The 6.8 kb SalI fragment hybridizes to two major species of polyadenylated RNA on a northern blot of sizes 4.8 and 5 kb (data not shown). To characterize the gene further this fragment was used as a probe to isolate a cDNA clone from an embryonic library. A single cDNA of 4,332 bp in size was isolated that recognizes the same species of mRNA on a northern blot as the 6.8 kb genomic fragment. Assuming a poly A tail addition of 400 bases, the size of this cDNA is in close agreement to the sizes of polyadenylated RNA species observed on a northern blot (4.8 and 5 kb) and thus appears to be near full length. Both strands of the entire 4,332 bp cDNA were sequenced by a combination of nested primers and exonuclease III treatment of both strands. Within the entire 4,332 bp, a single long open reading frame (ORF) of 3,102 bp was found. This ORF agrees well with Drosophila codon usage bias (Ashburner, 1989). There is a single translation start site at position no. 130, yet the sequence surrounding this codon does not agree well with the Drosophila consensus sequence (CAAAAUG) for translation start sites (Cavener, 1987). However, there are no other translation initiator codons and there are stop codons in all three reading frames upstream of this one. The cDNA sequence therefore contains 129 bp of 5′ non-translated sequence, 1,101 bp of 3′ non-translated sequence and a polyadenylation signal sequence AATAAA is found at its most 3′ end (data not shown). The long ORF encodes a potential polypeptide of 1,034 amino acids in length with a predicted Mr of 118×103. The amino acid sequence is shown in Fig. 1. The predicted polypeptide is acidic with a computed pI of 5.2, is hydrophilic, and rich in leucine (13%) and glutamate (13%). Hydropathy plot analysis reveals no significant hydrophobic regions that might encode signal leader peptide or transmembrane sequences. The most striking structural features of the protein are the three putative leucine zipper motifs (Landschulz et al., 1988). The first putative leucine zipper is located from amino acids 100 –126 and is bounded by several proline residues, the second is located at amino acids 525 –551 and the third is from amino acids 946 –972. All three zippers are within predicted coiled-coil regions (Lupas et al., 1991). Helical wheel plots of the three zipper regions show that all contain a continuous spine of hydrophobicity greater than six helical turns and the opposite faces are rich in charged amino acid residues for the formation of salt bridges (data not shown). No other protein motifs were identified, although the protein does contain a number of protein kinase recognition sites. These include 2 sites for cAMP-dependent kinase, 2 sites for Ca2+-dependent kinase II, 2 sites for GSK3 kinase, 2 sites for protein kinase C and one site for tyrosine kinase (Fig. 1A). The polypeptide sequence was compared with known protein sequences in several databases. The region of CNN extending from amino acids 85–970 shows limited homology with coiledcoil domains of myosin heavy chain from several species. This region of CNN contains long stretches of alpha helices interrupted by small regions that lack heptad periodicity (Fig. 1A), often containing proline residues which are known to disrupt alpha helices. There are no other significant homologies to known proteins in the databases.

Fig. 1.

The deduced amino acid sequence of CNN. (A) The predicted amino acid sequence of the cnn cDNA coding region. Putative leucine zippers are indicated by lines above and below and the leucine residues are outlined. Consensus phosphorylation sites for various protein kinases are in italics and lowercase letters. A glutamine enriched region is underlined and a highly positively charged region which is a potential nuclear localization signal is underlined by a shaded box. (B) Schematic representation of CNN. Approximate locations of the three putative leucine zippers are indicated by ovals (shaded) and the glutamine rich region is indicated by a shaded box. (C) Western blot with CNN antibodies. Western blot analysis of total cellular protein isolated from ovaries (lane 1), 0to1-hour embryos (lane 2) and 3to 15hour (lane 3) embryos with affinity purified CNN antibodies. Approximately 10–20 μg of total protein was loaded per lane, however samples do not represent equivalent loadings. Molecular mass markers shown (bars) are in kilodaltons. A single band representing CNN is detected between 116 and 200 kd in each lane (arrow).

Fig. 1.

The deduced amino acid sequence of CNN. (A) The predicted amino acid sequence of the cnn cDNA coding region. Putative leucine zippers are indicated by lines above and below and the leucine residues are outlined. Consensus phosphorylation sites for various protein kinases are in italics and lowercase letters. A glutamine enriched region is underlined and a highly positively charged region which is a potential nuclear localization signal is underlined by a shaded box. (B) Schematic representation of CNN. Approximate locations of the three putative leucine zippers are indicated by ovals (shaded) and the glutamine rich region is indicated by a shaded box. (C) Western blot with CNN antibodies. Western blot analysis of total cellular protein isolated from ovaries (lane 1), 0to1-hour embryos (lane 2) and 3to 15hour (lane 3) embryos with affinity purified CNN antibodies. Approximately 10–20 μg of total protein was loaded per lane, however samples do not represent equivalent loadings. Molecular mass markers shown (bars) are in kilodaltons. A single band representing CNN is detected between 116 and 200 kd in each lane (arrow).

Pattern of cnn transcripts in wild-type and homeotic mutant embryos

The detection of two transcripts by the cDNA in northern analysis indicates alternate splicing, though we have not characterized this further. In situ hybridizations to whole-mount embryos using the cDNA indicates that cnn is expressed in cells of the mesoderm and developing CNS and peripheral nervous system (PNS). The cnn mRNA is detected as early as stage 5 at cellular blastoderm (Fig. 2A). High levels of expression are detected in the gastrulating embryo along the ventral and cephalic furrows (Fig. 2B) and later in the mesoderm (Fig. 2C). At the time of germ band extension, at stage 10, cnn mRNA begins to accumulate in large cells presumed to be neuroblasts of the CNS (Fig. 2D) and by stage 11 is seen in cells of the PNS (Fig. 2E). By stage 12, cnn expression predominates in the CNS and PNS (Fig. 2F). At a later time, expression diminishes in the PNS, and more cells in the thoracic part of the nerve cord contain cnn mRNA relative to the abdominal region (Fig. 2G). From stage 11 to stage 16, cnn mRNA is observed in two domains of the VM surrounding the midgut. Double labeling experiments for cnn mRNA and SCR, ANTP or UBX in the VM indicate that the anterior domain (domain 1) of cells expressing cnn is located just anterior to cells expressing SCR and that the posterior domain (domain 2) overlaps with cells expressing ANTP or UBX (Figs 2H, 3A and data not shown).

Fig. 2.

Expression of cnn in wildtype embryos. Digoxigeninlabeled cRNA probes derived from the 4.3 kb cDNA and wholemount in situ hybridization were used to detect cnn mRNA (shown in black) in wild-type embryos. Digoxigenin-labeled product was detected by an antibody coupled to peroxidase with DAB and NiCl2 as the substrate. All embryos are oriented anterior to the left and ventral side down unless otherwise stated. (A) Cellular blastoderm embryo showing uniform expression except in the pole regions (arrowheads). (B) A ventral view of an early gastrula embryo. cnn mRNA is detected in cells surrounding the cephalic (arrows) and ventral (arrowhead) furrows. (C) cnn transcripts are visible in the mesoderm of an elongating germ band embryo (arrowheads). (D) In an extended germ band embryo, CNN expression is present in the developing neuroblast cell layer of the CNS (white arrowheads). (E,F) During germ band shortening, cnn mRNA is visible in cells of the developing PNS (open and solid arrows). (G) cnn expression persists in the developing CNS during germ band shortening and is expressed in more cells of the thoracic neuromeres (white arrows) than the abdominal neuromeres (black arrows). Expression can also be seen in the brain hemispheres (open arrow). (H) In the VM of a shortened germ band embryo, cnn transcripts are indicated between the black bars in two domains (arrowheads) labeled 1 and 2 that are separated by a gap.

Fig. 2.

Expression of cnn in wildtype embryos. Digoxigeninlabeled cRNA probes derived from the 4.3 kb cDNA and wholemount in situ hybridization were used to detect cnn mRNA (shown in black) in wild-type embryos. Digoxigenin-labeled product was detected by an antibody coupled to peroxidase with DAB and NiCl2 as the substrate. All embryos are oriented anterior to the left and ventral side down unless otherwise stated. (A) Cellular blastoderm embryo showing uniform expression except in the pole regions (arrowheads). (B) A ventral view of an early gastrula embryo. cnn mRNA is detected in cells surrounding the cephalic (arrows) and ventral (arrowhead) furrows. (C) cnn transcripts are visible in the mesoderm of an elongating germ band embryo (arrowheads). (D) In an extended germ band embryo, CNN expression is present in the developing neuroblast cell layer of the CNS (white arrowheads). (E,F) During germ band shortening, cnn mRNA is visible in cells of the developing PNS (open and solid arrows). (G) cnn expression persists in the developing CNS during germ band shortening and is expressed in more cells of the thoracic neuromeres (white arrows) than the abdominal neuromeres (black arrows). Expression can also be seen in the brain hemispheres (open arrow). (H) In the VM of a shortened germ band embryo, cnn transcripts are indicated between the black bars in two domains (arrowheads) labeled 1 and 2 that are separated by a gap.

The expression pattern in the VM suggests homeotic regulation. Therefore, we examined the transcript pattern of cnn in the VM of homozygous mutant embryos for homeotic proteins. In Antp mutant embryos, cnn transcripts are detected ectopically in cells that normally express Antp (Fig. 3B). Accumulation of cnn mRNA appears normal in Scr mutant embryos (data not shown). Embryos that lack both Ubx and abd-A, express cnn in a pattern clearly different than wild type. The cnn expression that normally overlaps with Ubx is absent, but cnn transcripts are now found in cells of the VM where abd-A is normally expressed (Fig. 3C).

Fig. 3.

Expression of cnn in the CNS and VM is regulated by homeotic proteins. cnn transcripts (in black) were detected by whole-mount in situ hybridization in shortened germ band embryos. Embryos in A,B and C are optical cross sections of whole mounts and are oriented anterior to the left. Embryos in D,E and F are oriented anterior to the left and ventral side down. The embryo in D has also been stained for UBX (shown in brown). (A) A wild-type embryo showing cnn transcripts in the VM in two discrete domains (indicated between the arrowheads and labeled 1 –2). (B) An Antp mutant embryo showing the extent of cnn expression in the VM between the white arrowheads. cnn transcripts are ectopically expressed in cells that normally express the ANTP (open arrow). (C) An embryo that is mutant for the Ubx and abd-A showing three domains of cnn expression in the VM (indicated between the arrowheads and labeled 1-3). cnn mRNA is absent from some cells that normally express the UBX (open arrow) and is detected in cells that normally express the ABD-A (arrow). (D) A wild-type embryo showing cnn expression in the CNS. cnn expression is present in more cells of the thoracic region (arrowheads) than of the abdominal region (open arrows).

Transcripts are also present in cells of the cerebral hemisheres (solid arrow). (E) cnn expression in the CNS of an Antp mutant embryo showing a decrease in transcripts of the thoracic region (arrowheads). (F) An embryo deficient for Ubx and abd-A has greater expression of cnn mRNA in cells of the abdominal region (arrows).

Fig. 3.

Expression of cnn in the CNS and VM is regulated by homeotic proteins. cnn transcripts (in black) were detected by whole-mount in situ hybridization in shortened germ band embryos. Embryos in A,B and C are optical cross sections of whole mounts and are oriented anterior to the left. Embryos in D,E and F are oriented anterior to the left and ventral side down. The embryo in D has also been stained for UBX (shown in brown). (A) A wild-type embryo showing cnn transcripts in the VM in two discrete domains (indicated between the arrowheads and labeled 1 –2). (B) An Antp mutant embryo showing the extent of cnn expression in the VM between the white arrowheads. cnn transcripts are ectopically expressed in cells that normally express the ANTP (open arrow). (C) An embryo that is mutant for the Ubx and abd-A showing three domains of cnn expression in the VM (indicated between the arrowheads and labeled 1-3). cnn mRNA is absent from some cells that normally express the UBX (open arrow) and is detected in cells that normally express the ABD-A (arrow). (D) A wild-type embryo showing cnn expression in the CNS. cnn expression is present in more cells of the thoracic region (arrowheads) than of the abdominal region (open arrows).

Transcripts are also present in cells of the cerebral hemisheres (solid arrow). (E) cnn expression in the CNS of an Antp mutant embryo showing a decrease in transcripts of the thoracic region (arrowheads). (F) An embryo deficient for Ubx and abd-A has greater expression of cnn mRNA in cells of the abdominal region (arrows).

The pattern of cnn expression in the CNS of a shortened germ band embryo also suggests homeotic regulation, so we analyzed the CNS for cnn expression in homeotic null mutants. Transcripts of cnn are normally visible in cells of the brain hemispheres and ventral nerve cord, with higher levels in the thoracic neuromeres compared to the abdominal neuromeres (Figs 2G, 3D). Expression of cnn appears reduced in the thoracic neuromeres and is more similar to the abdominal neuromeres in Antp mutant embryos (Fig. 3E). In contrast, embryos deficient for both UBX and ABD-A express higher than normal levels of cnn mRNA throughout the posterior ventral nerve cord (Fig. 3F).

The misexpression of homeotic genes in the VM alters the distribution of cnn transcripts

To gain more information about the homeotic regulation of cnn in the VM, we employed the two component GAL4 system (Greig and Akam, 1993; Brand and Perrimon, 1993) to create homeotic gain of function mutants. The transformant line P{GawB}24B drives GAL4 expression in the mesoderm (Brand and Perrimon, 1993). This line was crossed to the transformant lines P{UAS::Scr}EE2, P{UAS::Antp}W2 and P{UAS::Ubx}M2A to ectopically express SCR, ANTP or UBX in the entire VM of 100% of the progeny. The ectopic expression of these proteins in the VM results in abnormalities of gut development and embryonic lethality (Kalkbrenner et al., unpublished data). Ectopic expression of ANTP in the VM is associated with a decrease in cnn expression and some embryos exhibit a complete repression of cnn expression (Fig. 4A,B). The overexpression of UBX in the VM results in an activation of cnn expression. In these embryos, cnn mRNA is ectopically expressed in the VM from the anterior end of the midgut to domain 2 of normal cnn expression (Fig. 4D). As the ectopic expression of cnn in some of these cells could be due to loss of Antp, we examined these embryos for Antp expression. We found a complete absence of ANTP accumulation in the VM of these embryos (Fig. 4C), indicating that the downregulation of Antp by Ubx might be responsible for the activation of cnn in some of these cells. The overexpression of SCR in the VM had no discernable effects on cnn expression (data not shown).

Fig. 4.

Transcript accumulation of cnn can be deregulated in the VM through the misexpression of homeotic proteins. The two component Gal4 system was employed to ectopically drive ANTP and UBX in the VM. ANTP (in brown) was detected by a monoclonal antibody and a peroxidase coupled secondary antibody. cnn mRNA (in black) was detected by whole-mount in situ hybridization. All panels show optical cross sections of whole mount embryos with anterior to the left and they are at the shortened germ band stage. (A) An embryo in which ANTP is ectopically expressed in the entire mesoderm. Note the high levels of ANTP in the VM (arrowheads). (B) The same type of embryo shown in A stained for cnn mRNA showing a down-regulation of transcripts in cnn domain 1 of the VM (arrowhead) and a complete absence of mRNA in domain 2 (wide arrow). (C) An embryo in which UBX is ectopically expressed in the entire mesoderm and stained for ANTP. Although the CNS and epidermal patterns of Antp expression are normal, there is a complete absence of ANTP in the VM (arrowhead). (D) The same type of embryo as shown in C stained for cnn mRNA (indicated between the white arrowheads). Note the ectopic expression of cnn mRNA in cells anterior to cnn domain 1 and in cells found between cnn domains 1 and 2.

Fig. 4.

Transcript accumulation of cnn can be deregulated in the VM through the misexpression of homeotic proteins. The two component Gal4 system was employed to ectopically drive ANTP and UBX in the VM. ANTP (in brown) was detected by a monoclonal antibody and a peroxidase coupled secondary antibody. cnn mRNA (in black) was detected by whole-mount in situ hybridization. All panels show optical cross sections of whole mount embryos with anterior to the left and they are at the shortened germ band stage. (A) An embryo in which ANTP is ectopically expressed in the entire mesoderm. Note the high levels of ANTP in the VM (arrowheads). (B) The same type of embryo shown in A stained for cnn mRNA showing a down-regulation of transcripts in cnn domain 1 of the VM (arrowhead) and a complete absence of mRNA in domain 2 (wide arrow). (C) An embryo in which UBX is ectopically expressed in the entire mesoderm and stained for ANTP. Although the CNS and epidermal patterns of Antp expression are normal, there is a complete absence of ANTP in the VM (arrowhead). (D) The same type of embryo as shown in C stained for cnn mRNA (indicated between the white arrowheads). Note the ectopic expression of cnn mRNA in cells anterior to cnn domain 1 and in cells found between cnn domains 1 and 2.

Generation of a polyclonal antiserum to CNN and western blot analysis of the protein

The transcription of the cnn gene is dynamic throughout development and is regulated by the homeotic genes in the CNS and VM. In order to study the distribution of CNN in embryos, we produced polyclonal antibodies against a CNN-β-gal fusion protein. The fusion protein contains sequences from amino acids 271-1034 of CNN fused to β-galactosidase with a predicted Mr of 158×103. Affinity purified polyclonal antibodies to CNN specifically recognize the CNN fusion protein but do not recognize the β-galactosidase protein or any other bacterial proteins on a western blot (data not shown). These antibodies were used in a western blot analysis of ovarian, 0to 1-hour and 3to 15-hour embryonic total protein. The affinity purified CNN antibodies, but not preimmune IgG, specifically recognize a single band of approximately 140×103Mr in both ovaries and embryos (Fig. 1C).

CNN localizes to the centrosome and cytoplasm of early embryos in a staining pattern that changes with the cell cycle

RNA encoded by cnn is detectable in the egg before cellularization, thus we examined the distribution of CNN in early Drosophila embryos with CNN-specific antibodies. No detectable signal was observed with preimmune IgG in the immunohistochemical staining of embryos. Before the first nuclear cycle, CNN is found distributed throughout the cytoplasm, yet concentrated in a cytoplasmic island (Ashburner, 1989) surrounding an intensely stained dot, presumed to be the centrosome provided by the sperm (Fig. 5A). With subsequent nuclear cycles, CNN continues to accumulate in dots and cytoplasmic islands that surround the dividing nuclei (Fig. 5B). At higher magnification, CNN is detectable on the mitotic spindle of dividing nuclei and the dots appear to represent the poles of the spindle apparatus (Fig. 5C). At cellular blastoderm, CNN is visible in the cytoplasm and in two dots located at the periphery of each cell (Fig. 5D-F).

Fig. 5.

Immunohistochemical staining of CNN in early Drosphila embryos. (A) CNN (in brown) is detected throughout the cytoplasm of a fertilized egg. In addition, a single dot of staining is detected near the anterior end of the egg (arrowhead) surrounded by slightly more intense CNN staining. (B) At approximately the tenth nuclear cycle, CNN accumulates in dots (arrowheads) and cytoplasmic islands surrounding the dots. (C) At cellular blastoderm, CNN is evenly distributed in every cell. (D) A higher magnification of the embryo seen in B indicates that CNN is detectable on spindles (small arrowheads) and the dots appear to be located at the poles of the spindles (large arrowheads). (E) A high magnification surface view at cellular blastoderm between anaphase and telophase, CNN is not present in tight dots, but rather in large dispersed clusters with many extensions and smaller dots. (F) A high magnification optical cross section of the cellular blastoderm embryo seen in C. CNN is detectable in every cell in both the cytoplasm (arrow) and in two dots per cell at the periphery of the cell (arrowhead).

Fig. 5.

Immunohistochemical staining of CNN in early Drosphila embryos. (A) CNN (in brown) is detected throughout the cytoplasm of a fertilized egg. In addition, a single dot of staining is detected near the anterior end of the egg (arrowhead) surrounded by slightly more intense CNN staining. (B) At approximately the tenth nuclear cycle, CNN accumulates in dots (arrowheads) and cytoplasmic islands surrounding the dots. (C) At cellular blastoderm, CNN is evenly distributed in every cell. (D) A higher magnification of the embryo seen in B indicates that CNN is detectable on spindles (small arrowheads) and the dots appear to be located at the poles of the spindles (large arrowheads). (E) A high magnification surface view at cellular blastoderm between anaphase and telophase, CNN is not present in tight dots, but rather in large dispersed clusters with many extensions and smaller dots. (F) A high magnification optical cross section of the cellular blastoderm embryo seen in C. CNN is detectable in every cell in both the cytoplasm (arrow) and in two dots per cell at the periphery of the cell (arrowhead).

The localization of CNN in dots at the spindle poles suggests that the protein is localized to the centrosome. Therefore, we examined early embryos double stained for CNN and either DNA or tubulin. The results of this analysis are shown in Fig. 6. There is a lack of cytoplasmic staining for CNN in these embryos due to fixation with methanol rather than paraformaldehyde. These results and analysis of double stained embryos with confocal microscopy (data not shown) confirm that CNN is localized to the centrosome at all stages of the cell cycle. The centrosomal staining pattern of the protein appears to change with different phases of mitosis. At prophase, CNN is detected in the two centrosomes located at opposite ends of the nucleus (Fig. 6A). In metaphase, CNN is associated in a compact manner with the two centrosomes located at the poles of the mitotic spindle (Fig. 6B). During anaphase, CNN distribution changes from compact to dispersed with many projections and appears to increase with the duplication of the centrosome (Fig. 6C). Between anaphase and telophase, we also observe many small staining dots in addition to the larger dots. This was much more apparent with paraformaldehyde fixation than with methanol fixation (Fig. 5E). At telophase, CNN is associated with the newly duplicated centrosomes that appear at each pole (Fig. 6D).

Fig. 6.

CNN accumulates at the centrosome during the nuclear cycle of cleavage. Embryos at approximately nuclear cycle 10 are shown in the different phases of the cell cycle and stained for either α-tubulin (top row) or DNA (bottom row) in blue and CNN in brown. At prophase, CNN is present in the two centrosomes at the periphery of the nuclear membrane (arrowheads). During metaphase, CNN staining appears as compact dots localized to the centrosomes at the poles of the mitotic spindle (arrowheads). At anaphase, material staining for CNN at the centrosome appears to increase and become less compact compared to metaphase (arrowheads). In telophase, the CNN staining pattern becomes more compact again as the centrosomes duplicate and appear as two dots at each pole (arrowheads).

Fig. 6.

CNN accumulates at the centrosome during the nuclear cycle of cleavage. Embryos at approximately nuclear cycle 10 are shown in the different phases of the cell cycle and stained for either α-tubulin (top row) or DNA (bottom row) in blue and CNN in brown. At prophase, CNN is present in the two centrosomes at the periphery of the nuclear membrane (arrowheads). During metaphase, CNN staining appears as compact dots localized to the centrosomes at the poles of the mitotic spindle (arrowheads). At anaphase, material staining for CNN at the centrosome appears to increase and become less compact compared to metaphase (arrowheads). In telophase, the CNN staining pattern becomes more compact again as the centrosomes duplicate and appear as two dots at each pole (arrowheads).

Immunohistochemical localization of CNN at later stages of embryogenesis in wild-type and homeotic mutant embryos

The spatial and temporal distribution of CNN was analyzed in wild-type embryos with CNN-specific antibodies and is shown in Figs 7 and 8. Embryos were staged according to Campos-Ortega and Hartenstein (1985). The pattern of protein expression is essentially identical to the pattern of mRNA expression, thus only a brief description will be presented that is relevant to CNN protein compared to mRNA accumulation. Both cytoplasmic and centrosomal staining with CNN antibodies are detected in cells that express CNN. In the CNS, detection of CNN clearly demonstrates that there are more cells toward the ventral midline in the thoracic neuromeres that express CNN compared to the abdominal neuromeres (Fig. 8D). CNN can also be detected in cells of the developing gonads (Fig. 12B). The cnn mRNA and protein expression patterns are not identical in the VM. RNA is expressed in two discrete domains of the VM beginning at late germ band retraction (stage 12; Fig. 2H). These two domains overlap with the domains of dpp expression in the VM (data not shown). CNN is not detectable in cells of the VM at this stage of development. However, from stages 14-16, CNN is present in the VM of the gastric caecae and the second midgut constriction (Fig. 12B,C).

Fig. 7.

CNN expression in later stages of embryogenesis. The embryonic pattern of CNN accumulation (shown in brown) was analyzed with CNN specific antibodies. All embryos are oriented anterior to the left and ventral side down. (A) An early germ band extended embryo, CNN is expressed in the cytoplasm and centrosomes of virtually all cells.(B) An optical cross section of a germ band extended embryo, showing strong CNN expression in the mesoderm (arrows) and much lower levels in cells of the epidermis (small arrowheads). CNN is also present in the pole cells (large arrowhead) and mesoderm surrounding the invaginating hindgut (open arrow) and the neuroblast cell layer of the developing CNS (arrows). (C) An optical cross section of a germ band extended embryo showing strong CNN expression in cells of the neuroblast cell layer of the developing CNS (arrowheads). (D) A lateral view of an extended germ band embryo showing CNN accumulation in neuroblast cells of the developing PNS (arrowheads).

Fig. 7.

CNN expression in later stages of embryogenesis. The embryonic pattern of CNN accumulation (shown in brown) was analyzed with CNN specific antibodies. All embryos are oriented anterior to the left and ventral side down. (A) An early germ band extended embryo, CNN is expressed in the cytoplasm and centrosomes of virtually all cells.(B) An optical cross section of a germ band extended embryo, showing strong CNN expression in the mesoderm (arrows) and much lower levels in cells of the epidermis (small arrowheads). CNN is also present in the pole cells (large arrowhead) and mesoderm surrounding the invaginating hindgut (open arrow) and the neuroblast cell layer of the developing CNS (arrows). (C) An optical cross section of a germ band extended embryo showing strong CNN expression in cells of the neuroblast cell layer of the developing CNS (arrowheads). (D) A lateral view of an extended germ band embryo showing CNN accumulation in neuroblast cells of the developing PNS (arrowheads).

Fig. 8.

CNN expression in cells of the CNS and PNS of late stage embryos. CNN was detected with affinity purified CNN antibodies and is shown in brown. All embryos are oriented anterior to the left. (A) A lateral view of a stage 12 embryo showing segmental CNN expression in cells of the PNS (arrowheads). The thoracic segments are marked with bars.(B) A higher magnification of the embryo seen in A showing the thoracic region. CNN staining is both cytoplasmic and centrosomal (dots) in cells of the PNS (arrows) and in the anlagen of the anterior spiracle (arrowhead). (C) A ventral view of a stage 13 embryo showing the segmental accumulation of CNN in cells of the ventral nerve cord (arrows). The thoracic neuromeres are labeled with bars. (D) A higher magnification of the embryo shown in C reveals the cytoplasmic and centrosomal (dots) staining of CNN in more cells of the thoracic neuromeres (arrows) than the abdominal neuromeres (arrowheads).

Fig. 8.

CNN expression in cells of the CNS and PNS of late stage embryos. CNN was detected with affinity purified CNN antibodies and is shown in brown. All embryos are oriented anterior to the left. (A) A lateral view of a stage 12 embryo showing segmental CNN expression in cells of the PNS (arrowheads). The thoracic segments are marked with bars.(B) A higher magnification of the embryo seen in A showing the thoracic region. CNN staining is both cytoplasmic and centrosomal (dots) in cells of the PNS (arrows) and in the anlagen of the anterior spiracle (arrowhead). (C) A ventral view of a stage 13 embryo showing the segmental accumulation of CNN in cells of the ventral nerve cord (arrows). The thoracic neuromeres are labeled with bars. (D) A higher magnification of the embryo shown in C reveals the cytoplasmic and centrosomal (dots) staining of CNN in more cells of the thoracic neuromeres (arrows) than the abdominal neuromeres (arrowheads).

The homeotic genes Antp, Ubx, and abd-A are observed to regulate cnn mRNA expression in the later stages of CNS development (Fig. 3). To determine if CNN expression is also regulated by the homeotic genes in a similar manner, we analyzed the protein expression pattern in the CNS of homozygous AntpA74 and Df(3R)Ubx109 mutant embryos by immunohistochemistry (Fig. 9). We found that the homeotic regulation of protein and mRNA expression in the CNS are identical.

Fig. 9.

CNN expression in the ventral nerve cord of homeotic mutant embryos. CNN (in brown) was detected in stage 13 embryos with specific CNN antibodies. All embryos show ventral views of the ventral nerve cord with anterior to the left. The thoracic neuromeres are labeled. (A) A wild-type embryo showing that more cells express CNN in the thoracic neuromeres (arrows) than the abdominal ones (arrowheads). (B) A Ubx109 embryo, which is deficient for both Ubx and abd-A, has more cells in the abdominal neuromeres (arrowheads) that express CNN relative to the wild-type pattern. (C) An AntpA74 embryo, which is mutant for Antp, shows fewer cells in the thoracic region that stain for CNN (arrowheads).

Fig. 9.

CNN expression in the ventral nerve cord of homeotic mutant embryos. CNN (in brown) was detected in stage 13 embryos with specific CNN antibodies. All embryos show ventral views of the ventral nerve cord with anterior to the left. The thoracic neuromeres are labeled. (A) A wild-type embryo showing that more cells express CNN in the thoracic neuromeres (arrows) than the abdominal ones (arrowheads). (B) A Ubx109 embryo, which is deficient for both Ubx and abd-A, has more cells in the abdominal neuromeres (arrowheads) that express CNN relative to the wild-type pattern. (C) An AntpA74 embryo, which is mutant for Antp, shows fewer cells in the thoracic region that stain for CNN (arrowheads).

To determine if CNN distribution is conserved across Drosophila species, we examined embryos derived from D. virulis, D. funibris, D. hydei and D. pseudobscura by immunohistochemistry with CNN-specific antibodies. These species are over a million years diverged from each other. We observed the same patterns of staining for CNN in these species of Drosophila as in D. melanogaster (data not shown).

Genetic analysis of the cnn gene and generation of a deficiency that removes CNN

We initiated a genetic analysis of cnn by in situ hybridization to salivary polytene chromosomes with a digoxigenin-labeled probe derived from genomic clone jh18-A. The probe hybridizes to the dark band in the 50A3-6 region of the second chromosome (Fig. 10). To define further the region that contains cnn, we identified a deficiency, Df(2R)vg-B, that extends into this cytological subdivision (Linsley and Zimm, 1987). Immunohistochemical analysis for CNN in embryos homozygous for this deficiency reveals that Df(2R)vg-B does not remove cnn (data not shown). We also identified two P-element transformant lines (stocks P{F}1 and P{PZ}drk10626) that also map to this cytological region. The Pelement insertion in the P{F}1 line does not complement Df(2R)vg-B, yet maps cytologically to 50A, thus we conclude that this insertion is in the 50A1-5 region. This P-element insertion does not disrupt cnn as CNN distribution is normal in these embryos (data not shown). The P-element insertion in the P{PZ}drk10626 line maps cytologically to the 50A12-14 region and is inserted in the drk gene, which encodes a protein with SH2 and SH3 domains (Simon et al., 1993). This P-element insertion complements Df(2R)vg-B and also does not interrupt cnn, based on immunohistochemical analysis with CNN antibodies (data not shown). As we could not find any deficiencies or P-element insertions in the 50A subdivision that affect the accumulation of CNN, we created a deficiency that removes cnn function. The P-element insertion line P{F}1 maps in the vicinity of cnn and carries the white gene as a visible marker, thus we irradiated males from the P{F}1 line with X-rays to excise the Pelement and looked for progeny with white eyes. A single line was established that had white eyes and one-quarter of the embryos derived from this line failed to stain for CNN by immunohistochemical analysis (Fig. 11B). CNN is still detectable up to germ band extension in homozygous embryos carrying this deficiency, albeit at much lower levels compared to their heterozygous siblings (Fig. 11A). Analysis of salivary gland polytene chromosomes from this line indicates that this deficiency, Df(2R)cnn, removes almost the entire 50A interval including the dark band that the cnn probe hybridizes to (data not shown). This deficiency does not complement Df(2R)vg-B, P{F}1 or P{PZ}drk10626, but compliments Df(2R)vg135 and a breakpoint in the 50B region. Homozygous embryos carrying Df(2R)cnn develop up to the end of embryogenesis, yet fail to hatch. These embryos appear grossly normal, but analysis with molecular markers indicates the presence of defects in the nervous system and midgut.

Fig. 10.

A genetic map of the 49DF-50AB region and molecular map of the cnn locus. A schematic representation of the genes in the 49DF-50AB region on the second chromosome and approximate location of the cnn locus, based on cytological analysis and complementation analysis of Df(2R)cnn with Df(2R)vg-B and P-element insertions in the 50A region. Below is shown a molecular map of the cnn gene, showing the intron-exon structure, locations of coding sequences and the immunopurified ANTP target site.

Fig. 10.

A genetic map of the 49DF-50AB region and molecular map of the cnn locus. A schematic representation of the genes in the 49DF-50AB region on the second chromosome and approximate location of the cnn locus, based on cytological analysis and complementation analysis of Df(2R)cnn with Df(2R)vg-B and P-element insertions in the 50A region. Below is shown a molecular map of the cnn gene, showing the intron-exon structure, locations of coding sequences and the immunopurified ANTP target site.

Fig. 11.

Df(2R)cnn eliminates CNN accumulation and results in CNS and PNS abnormalities. (A) An extended germ band embryo is shown that is homozygous for Df(2R)cnn and is a protein null for CNN (arrow) next to similar staged embryo that expresses CNN (open arrow). In the cnn mutant embryo, some remaining maternally deposited protein can still be detected in cells of the developing CNS (arrowhead). (B) A germ band shortened embryo (stage 12) that is homozygous for Df(2R)cnn completely lacks CNN (arrow), while a similar staged embryo nearby expresses CNN (open arrow). (C) A wild-type embryo stained for neuroglian with the BP104 antibody shows the axon scaffold and neurons in the CNS (arrow) and peripheral nerves of the PNS (arrowheads). (D) A homozygous Df(2R)cnn embryo stained with the BP104 antibody has fewer stained neurons in the CNS and an apparently reduced axon scaffold (arrow). The peripheral nerves are also disorganized and do not fasciculate properly (arrowheads). (E) A wild-type embryo stained for the Cut protein with the F2 antibody. The antibody labels nuclei of cells in the CNS (arrow) and external sensory organs in the PNS (arrowheads). (F) A homozygous Df(2R)cnn embryo stained with the F2 antibody for Cut protein shows fewer cells staining in the CNS (arrow) and a disorganized array of es organs in the PNS (arrowheads).

Fig. 11.

Df(2R)cnn eliminates CNN accumulation and results in CNS and PNS abnormalities. (A) An extended germ band embryo is shown that is homozygous for Df(2R)cnn and is a protein null for CNN (arrow) next to similar staged embryo that expresses CNN (open arrow). In the cnn mutant embryo, some remaining maternally deposited protein can still be detected in cells of the developing CNS (arrowhead). (B) A germ band shortened embryo (stage 12) that is homozygous for Df(2R)cnn completely lacks CNN (arrow), while a similar staged embryo nearby expresses CNN (open arrow). (C) A wild-type embryo stained for neuroglian with the BP104 antibody shows the axon scaffold and neurons in the CNS (arrow) and peripheral nerves of the PNS (arrowheads). (D) A homozygous Df(2R)cnn embryo stained with the BP104 antibody has fewer stained neurons in the CNS and an apparently reduced axon scaffold (arrow). The peripheral nerves are also disorganized and do not fasciculate properly (arrowheads). (E) A wild-type embryo stained for the Cut protein with the F2 antibody. The antibody labels nuclei of cells in the CNS (arrow) and external sensory organs in the PNS (arrowheads). (F) A homozygous Df(2R)cnn embryo stained with the F2 antibody for Cut protein shows fewer cells staining in the CNS (arrow) and a disorganized array of es organs in the PNS (arrowheads).

CNS and PNS defects are observed in Df(2R)cnn homozygous embryos

The CNS and PNS are derived from subsets of ectodermal cells within neurogenic regions (Campos-Ortega and Hartenstein, 1985). Neurogenesis begins when neuroblasts delaminate from the ectoderm and segregate into the interior of the embryo. The neuroblasts then undergo a series of mitotic divisions to give rise to specific cell lineages and produce different types of neurons (Campos-Ortega, 1990). Major sites of cnn expression in the developing embryo include the CNS and PNS. Therefore, we examined the nervous system of homozygous Df(2R)cnn embryos for abnormalities with nervous system-specific molecular markers. The BP104 antibody recognizes the nervous system-specific form of neuroglian that localizes to the cell surface of neurons in the CNS and PNS (Bieber et al., 1989). The F2 antibody recognizes the nuclear CUT, a homeodomain-containing transcription factor that is expressed in cells of the PNS and CNS (Blochlinger et al., 1990). Homozygous Df(2R)cnn embryos, identified by their lack of CNN accumulation, were examined for nervous system defects with the BP104 and F2 antibodies. The normal distribution of neuroglian and CUT expression in the nervous system of stage 14 embryos are shown in Fig. 11. There is a dramatic reduction in the number of cells in the CNS of homozygous Df(2R)cnn embryos as detected with both the BP104 antibody (Fig. 11D) and the F2 antibody (Fig. 11F). There is also a reduction in the number of cells in the PNS (data not shown) as well as defects in peripheral nerve fasciculation and projection (Fig. 11D). The phenotypic defects observed with the BP104 antibody were specific for cnn mutant embryos as these defects were not observed in Df(2R)vgB/Df(2R)cnn, P{F}1/Df(2R)cnn, or P{PZ}drk10626/Df(2R)cnn embryos (data not shown).

Df(2R)cnn embryos have specific defects in midgut morphogenesis

The cnn gene is transcriptionally regulated by several homeotic genes in the VM and the protein is expressed in the domains of the gastric caecae and second midgut constriction at times in development when these structures are forming (Fig. 12B,C). For these reasons, we were interested in examining midgut morphogenesis in homozygous Df(2R)cnn embryos with the use of several markers expressed in the developing midgut. For this analysis we used LAB antibodies, which detect the nuclear labial protein in a band of endoderm cells of the second compartment of the midgut (Chouniard et al., 1991), the FP3.38 antibody, which recognizes the nuclear Ubx protein in the VM surrounding the second midgut constriction (White and Wilcox, 1984; Reuter et al., 1990), and the RD2 lacZdppshv reporter construct (see Materials and Methods) that is expressed in the domains of dpp expession in the VM surrounding the midgut (Hursh et al., 1993).

Fig. 12.

Mutant cnn embryos fail to form the second midgut constriction and extend the gastric caecae. All embryos are stage 16-17, oriented anterior to the left and represent optical cross sections of whole mounts. (A) cnn mRNA expression detected by whole-mount in situ hybridization. cnn mRNA expression (in blue) is seen in the visceral mesoderm of the second midgut constriction (arrows). (B) CNN accumulation (in brown) in the visceral mesoderm surrounding the midgut detected with CNN-specific antibodies. CNN is detected in the visceral mesoderm of the second midgut constriction (arrow) and gastric caecae (arrowhead). CNN is also expressed in the gonads (open arrow). (C) A Df(2R)cnn/SM5 embryo double stained for the CNN (cytoplasmic) and the LAB (nuclear). LAB is seen in nuclei of endodermal cells in a band surrounding the second compartment of the midgut (arrows). All three midgut constrictions are indicated. (D) A homozygous Df(2R)cnn embryo stained for the labial protein. The first and third midgut constrictions are labeled. The normal boundary of labial protein expression in the endoderm is maintained (arrows), yet the second midgut constriction does not form. (E-F) Immunohistochemical localization of β-galactosidase (in blue) in a dpp pattern of the visceral mesoderm directed by the RD2 construct (see Materials and methods) in a Df(2R)cnn/SM5 embryo (E) and a homozygous Df(2R)cnn embryo (F). The midgut constrictions are labeled in both embryos. (E) CNN is shown in brown. The dpp midgut enhancer construct normally expresses in the evaginating gastric caecae (arrowheads) and in a band that surrounds the second compartment of the midgut (arrows). All three midgut constrictions are present. (F) In the cnn mutant embryo, the second midgut constriction fails to form, but the boundaries of dpp expression in the second compartment of the midgut are normal (arrows). Although dpp expression appears normal in the gastric caecae, they fail to evaginate (arrowheads).

Fig. 12.

Mutant cnn embryos fail to form the second midgut constriction and extend the gastric caecae. All embryos are stage 16-17, oriented anterior to the left and represent optical cross sections of whole mounts. (A) cnn mRNA expression detected by whole-mount in situ hybridization. cnn mRNA expression (in blue) is seen in the visceral mesoderm of the second midgut constriction (arrows). (B) CNN accumulation (in brown) in the visceral mesoderm surrounding the midgut detected with CNN-specific antibodies. CNN is detected in the visceral mesoderm of the second midgut constriction (arrow) and gastric caecae (arrowhead). CNN is also expressed in the gonads (open arrow). (C) A Df(2R)cnn/SM5 embryo double stained for the CNN (cytoplasmic) and the LAB (nuclear). LAB is seen in nuclei of endodermal cells in a band surrounding the second compartment of the midgut (arrows). All three midgut constrictions are indicated. (D) A homozygous Df(2R)cnn embryo stained for the labial protein. The first and third midgut constrictions are labeled. The normal boundary of labial protein expression in the endoderm is maintained (arrows), yet the second midgut constriction does not form. (E-F) Immunohistochemical localization of β-galactosidase (in blue) in a dpp pattern of the visceral mesoderm directed by the RD2 construct (see Materials and methods) in a Df(2R)cnn/SM5 embryo (E) and a homozygous Df(2R)cnn embryo (F). The midgut constrictions are labeled in both embryos. (E) CNN is shown in brown. The dpp midgut enhancer construct normally expresses in the evaginating gastric caecae (arrowheads) and in a band that surrounds the second compartment of the midgut (arrows). All three midgut constrictions are present. (F) In the cnn mutant embryo, the second midgut constriction fails to form, but the boundaries of dpp expression in the second compartment of the midgut are normal (arrows). Although dpp expression appears normal in the gastric caecae, they fail to evaginate (arrowheads).

In embryos homozygous mutant for cnn, the second midgut constriction fails to form, yet the expression patterns of the Ubx, lab and dpp genes in the midgut are normal (Fig. 12D,E and data not shown). The finger-like projections of the gastric caecae also fail to extend (Fig. 12F). The wild-type patterns of lab and dpp expression in the VM surrounding the midgut of stage 16 embryos is shown in Fig. 12 (C,E). As the expression patterns of cnn and dpp overlap with each other in the VM and the second midgut constriction and gastric caecae do not form in dpps14 mutant embryos (Panganiban et al., 1990; Hursh et al., 1993), we decided to examine the accumulation of CNN in the VM of these mutant embryos. We found that CNN is still detectable in the VM surrounding both the gastric caecae and the second midgut constriction of these mutant embryos, despite the defects that occur in these structures (data not shown).

We report the initial characterization of cnn, a candidate homeotic target gene identified using a library of immunopurified chromatin bound by the ANTP protein (Roche et al., unpublished data). We conclude that cnn is a target of several of the homeotic genes and that it may participate directly in processes of morphogenesis for the following reasons. (1) The cnn gene was isolated based on a close association with ANTP in vivo and cnn is ‘regulated’ by homeotic genes in two tissues. However proof as to whether the homeotic regulation is direct or indirect will require further analysis. (2) CNN represents a unique structural protein with features that suggest it oligomerizes with itself or other proteins and is localized to the centrosome, a known center for the organization and orientation of microtubules. The observation that both the homeotic gene products and microtubules are necessary for morphogenetic events supports the logical assumption that homeotic target genes might control microtubule function in morphogenesis. (3) The homeotic gene Ubx is necessary for the formation of the second midgut constriction and has been shown to positively regulate cnn in the VM. A deficiency that removes CNN protein accumulation also results in a failure to form the second midgut constriction. (4) The regional differences in cnn expression in both the CNS and VM that suggest homeotic regulation have been conserved in evolution across several Drosophila species. Our analysis of cnn in this report represents a first step towards defining the developmental roles of this gene as a homeotic target.

A centrosomal protein with three leucine zippers and limited homology to the coiled-coil domains of myosin is encoded by cnn

Sequence analysis indicates that CNN is rich in coiled-coil regions and contains three predicted leucine zippers, one near the amino terminus, one in the central region of the protein and one near the carboxyl terminus. The leucine zipper motif is considered to be a subset of the more general coiled-coil structure (O’Shea et al., 1991) and both structures are associated with protein-protein interactions (Landschulz et al., 1988; Lupas et al., 1991). The leucine zipper motif was originally identified in many transcription factors (Landschulz et al. 1988), but has since been found in many other classes of proteins including ion channels (McCormack et al., 1991), glucose transporter glycoproteins (Thorens et al., 1988), P transposase (Simonelig and Anxolabehere, 1991) and heat shock factors (Rabindran et al., 1991; Sarge et al., 1991). Although these proteins have different functions in the cell, the leucine zipper motifs within these proteins serve the common function of protein dimerization or oligomerization. The presence of coiled-coil regions and several leucine zippers suggests that CNN oligomerizes with itself or with other proteins to carry out its function. Another important structural feature of CNN worth noting is the presence of several protein kinase recognition sites, many of these being located near the leucine zipper motifs. As protein kinases are known to regulate the functions of many proteins through phosphorylation, the presence of such sites in CNN implies that phosphorylation may be an important mechanism in the regulation of CNN activity. This idea is supported by the observation that many centrosomal proteins are phosphorylated at the transition from interphase to mitosis (Kuriyama and Nislow, 1992).

The centrosomal localization and predicted structure of CNN have implications regarding its function. Centrosomes are microtubule organizing centers (MTOCs) in eucaryotic cells that control microtubule nucleation and spindle assembly and orientation (Mitchison and Kirschner, 1984; Kalt and Schliwa, 1993). Microtubules are essential for many cellular processes including mitosis, meiosis, changes in cell shape, axon elongation and migration of cells. These same cellular processes are thought to be regulated by the homeotic genes in controlling morphogenesis. The sites of cnn expression in the embryo correlate with regions that are either mitotically active or undergoing changes in cell shape, processes that involve microtubules. However, not all regions in the embryo that use microtubules express cnn, eg., the first and third midgut constrictions and many neurons in the CNS. Thus, cnn is compatible as a homeotic target gene with specific functions in morphogenesis through a direct or indirect interaction with microtubules at the centrosome.

A centrosome is composed of a pair of centrioles surrounded by pericentrolar material and many proteins have been identified that localize to the centrosome (Kalt and Schliwa, 1993). One of these proteins, named Pericentrin, has a strikingly similar protein localization pattern and secondary structure to CNN (Doxsey et al., 1994). Specifically, the centrosomal staining pattern for Pericentrin shows dynamic changes with the cell cycle and the protein is rich in coiled-coil regions. Pericentrin seems to be required for proper microtubule organization, and Doxsey et al. (1994) suggest that it might provide a scaffold for the binding of other centrosomal proteins. Although there is no significant identity at the primary amino acid sequence level between these two proteins, their similar secondary structure and localization suggest similar functions.

Homeotic regulation of cnn

The patterns of RNA and protein accumulation in wild-type and mutant animals support the idea that cnn expression is modulated by several of the homeotic genes. Analysis of homeotic mutant embryos implies that cnn expression in certain cells of the thoracic CNS is dependent on Antp expression whereas the BX-C gene products are negative regulators of cnn in the abdominal CNS. In the VM of the midgut, the regulation of cnn by the homeotics appears more complicated. In contrast to its positive role in the CNS the Antp locus acts as a negative regulator of cnn in the mesoderm. The cnn gene is normally expressed in two domains of the VM, an anterior and a posterior domain. It is in this latter posterior region that cnn expression is affected by Antp and Ubx. ANTP and UBX are normally accumulated in two contiguous domains in the VM and the posterior domain of cnn overlaps that of Antp at its posterior end and that of Ubx at its anterior end. ANTP expression in the VM consists of two cell populations with the posterior population initially expressing lower levels of protein relative to the anterior population (Tremml and Bienz, 1989; J. Heuer, unpublished observations) and it is in this posterior ANTP low region that CNN is accumulated. Thus, the negative regulation of cnn by Antp is apparently dependent on high levels of ANTP accumulation. This idea is supported by the observation that the ectopic expression of high levels of ANTP in the VM with the two component GAL4 system produces embryos which do not detectably express the cnn gene in the VM.

The cnn gene is under positive regulation by UBX in the VM of the midgut. The observation that cnn is not expressed in all cells that express Ubx in the VM suggests that a co-factor protein may be involved in this regulation. A loss of Ubx function results in a loss of cnn expression in the domain where UBX is normally found. In addition, the ectopic expression of Ubx with the GAL4 system in the VM can activate cnn expression anteriorly. The failure of Ubx to activate cnn posteriorly in the presence of abd-A and the fact that removal of abd-A in Ubx109 embryos leads to an activation of cnn in these cells suggests that abd-A is a negative regulator of cnn in the VM.

Df(2R)cnn removes the cnn gene and results in nervous system defects

Cytogenetic analysis of the Df(2R)cnn deficiency indicates that it removes the dark polytene band within the 50A region where cnn probes hybridize, as well as almost the entire remaining 50A region. Complementation tests with breakpoints in the 49E and 50B region support the cytological analysis of this deficiency, placing it between 49F and 50B. The hybridization of cnn to the dark polytene band located at 50A3-6, and the exclusion of cnn from Df(2R)vg-B, places cnn somewhere within 50A3-6. Although Df(2R)cnn removes almost the entire 50A interval and does include several genes, we conclude that phenotypes observed in homozygous deficiency embryos are actually due to a lack of cnn function. The phenotypes observed correlate with a loss CNN accumulation and are only seen in Df(2R)cnn/Df(2R)cnn and are not observed in Df(2R)vg-B/Df(2R)cnn, P{F}1/P{F}1, or P{PZ}drk10626/ P{PZ}drk10626 embryos, all of which express CNN and inactivate the other know genes within Df(2R)cnn. Moreover, tissues affected by the cnn deficiency are normally prominent sites of cnn expression. Specifically, CNN is accumulated in a segmentally modulated fashion in the CNS and severe CNS and PNS defects were observed in deficiency animals (but not the other previously noted genotypes). Immunological staining using nervous system specific antibodies suggests that these defects are associated with a gross reduction in cell numbers in the nevous system. An observation consistent with our hypothesis that CNN is required for proper centrosomal function in spindle assembly. That the CNS and PNS are the only tissues obviously mitotically defective is likely due to the fact that the neuroblasts are the only cells that remain mitotically active after embryonic mitosis 16. It would appear that the maternally deposited CNN remains at high enough levels to support cleavage up to blastoderm and the three post-blastoderm divisions. Antibody staining of homozygous deficiency embryos is consistent with this conclusion since maternally supplied protein is detectable (albeit at lower than normal levels) until stages 7-8 but not thereafter.

The presence of CNN at the centrosome during the rapid cycles of nuclear cleavage also suggests that the protein may be necessary for spindle function. If true, a loss of CNN during cleavage would result in an arrest of mitosis following fertilization. However, as noted above, CNN is maternally provided to the egg and the deficiency we have created that removes cnn is embryonic lethal, making an assessment of the preblastoderm function of cnn difficult at present. An analysis of the function of CNN during the early cycles of nuclear cleavage will have to await the creation of temperature sensitive mutant alleles of cnn and/or the creation of germ line mutant clones. However, cells that are still mitotically active following germ band extension (i.e. neuroblasts) are affected by lack of CNN accumulation and the apparent hypoplasia of the CNS in cnn animals supports the idea that CNN may be important for spindle function in mitosis.

A potential function for CNN in midgut morphogenesis

Drosophila midgut development involves cell shape changes in the VM that produce four finger-like projections called the gastric caecae that extend forward from the anterior end of the midgut and three constrictions that subdivide the midgut into four compartments (Reuter and Scott, 1990). The Scr gene is necessary for the formation of some anterior minor constrictions and indirectly for the formation of the gastric caecae. The Antp gene is required for the formation of the first midgut constriction, Ubx for the middle constriction and abd-A for the middle and third constrictions. The mechanisms by which the homeotic genes control midgut morphogenesis is just beginning to be elucidated and clearly involves the homeotic regulation of target genes such as dpp. Ubx has been reported to directly regulate dpp in the VM (Capovilla et al., 1994) and dpp is necessary for the formation of the second midgut constriction (Panganiban et al., 1990). The dpp gene product is believed to transmit a signal to the underlying endoderm to express the homeotic gene labial (lab), which imposes a copper cell phenotype on these cells (Hoppier and Bienz, 1994). The formation of the gastric caecae is also dependent on the expression of the dpp gene in the anterior region of the midgut (Panganiban et al., 1990). Although dpp represents a homeotic target gene that is involved in the regulation of midgut constriction formation, there are no identified homeotic target genes that might be directly involved in the mechanics of this process.

We have demonstrated that cnn is regulated by the homeotic genes in the VM and is a candidate for direct regulation. The expression of cnn in the VM coincides with regions that will form the gastric caecae and second midgut constriction, structures that are also under the control of the dpp gene. Interestingly, CNN is not detectable in these cells until stages 14-15, despite the expression of cnn mRNA at earlier stages, suggesting that translational control may represent a mechanism for the regulation of cnn activity in these cells. This is particularly significant because CNN becomes detectable in these regions only at a time when mechanical events of morphogenesis are occurring, i.e., the extension of the gastric caecae and the formation of the second midgut constriction. Moreover, in homozygous Df(2R)cnn embryos, in which cnn is deleted, the second midgut constriction fails to form and the gastric caecae do not elongate, even though the dpp, Ubx, and lab genes are all expressed in their normal domains.

How might the cnn gene product be involved in these events of midgut morphogenesis? The VM cells, in which the homeotic genes are expressed, have been shown to impose the shape changes on the underlying endoderm and yolk in midgut constriction formation. Moreover, these cell shape changes are associated with the presence of dense bundles of microtubules found in the VM cells close to the inner limits of the midgut constrictions (Reuter and Scott, 1990). It has been suggested that the microtubules either exert a force that drives the constriction or they stabilize the constrictive movements (Reuter and Scott, 1990). As cnn encodes a predicted structural protein that localizes to centrosomes, it is conceivable that CNN could participate in the organization and/or function of microtubules in the cell shape changes associated with gastric caecae elongation and second midgut constriction formation. It should be noted that although the CNS and PNS defects associated with CNN deficiency can be attributed to a blockage in mitosis, the gut constriction defect cannot. Thus the absence of CNN is apparently associated with two aspects of MTOC and microtubule function. The observation that cnn and dpp are involved only in the formation of the second midgut constriction yet all three constrictions form in a similar manner, raises an intriguing question concerning the significance of the observed homeotic regulation and involvement of molecules such as DPP and CNN in midgut morphogenesis. This suggests redundancy in the evolution of other molecules that function in a similar manner to DPP and CNN for the formation of the other constrictions. Another interesting observation concerns the midgut phenotype of dpps14 mutant embryos. These embryos do not express dpp in the VM nor form the second midgut constriction, yet do express CNN. This suggests that CNN is necessary but not sufficient for constriction formation and the activity of CNN, i.e., phosphorylation state or interaction with other proteins, may be regulated through the dpp signaling pathway.

The authors wish to thank Kathy Matthews for the anti-DNA and anti-Tubulin antibodies, and are grateful for her advice, analysis of data and sharing of thoughts on this project. We also thank Yuh Nung Jan for the F2 antibody, Mark Seeger and Corey Goodman for the BP104 antibody, Marc Muskavitch for the FP3.38 antibody, Danny Brower for the 8C11.1 antibody, Marie Mazzula for preparation of the LAB antibody and Dave Miller for his help and advice on the preparation of the R19 anti-CNN antibody. We would also like to thank William Gelbart for the dppshv reporter construct line and the Bloomington Stock Center for supplying us with mutant fly stocks. We are grateful to Barb Wakimoto, Tim Megraw and Bill Saxton for sharing their thoughts on this project, Bryan Rogers for critically reviewing the manuscript and Dee Verostko for her helpful administrative assistance. J. G. H. was an associate for and T. C. K. is an investigator of the Howard Hughes Medical Institute.

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Accession number for cnn sequence reported in this paper is GenBank U35621.