1 Howard Hughes Medical Institute and Department of Biochemistry, Stanford University, Stanford, CA 94305-5307, USA
2 Department of Anatomy, University of Connecticut Health Center, Farmington, CT 06030, USA
* Present address: Mitchell College, New London, CT 06320, USA
Present address: 9 Cricket Lane, Simsbury, CT 06070, USA
Author for correspondence (e-mail: krasnow{at}cmgm.stanford.edu)
Accepted September 10, 2001
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SUMMARY |
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Key words: Drosophila, ribbon, BTB/POZ domain, Pipsqueak domain, Trachea, Branching morphogenesis, FGF, Epithelial migration
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INTRODUCTION |
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Because cells in an epithelium are attached, morphogenetic events require coordination of the behavior of cells located at different positions in the epithelium. These events also require coordination of the behaviors of different parts of each constituent cell. For example, the apical and basal surfaces of epithelial cells usually move together during morphogenetic events, but occasionally they show strikingly different behaviors. During epithelial invaginations, the apical surface constricts, while the basal surface expands (Fristrom, 1988). The coordination of events at the apical and basal sides of an epithelium is of special interest because, unlike isolated cells like fibroblasts, the environment on each side of an epithelial cell is very different. The composition of the extracellular matrix is distinct, and the signaling molecules and receptors that trigger the morphogenetic events are typically localized on one side (Kim, 1997).
The Drosophila tracheal (respiratory) system is a branched network of epithelial tubes that provides a tractable genetic system for analyzing epithelial morphogenesis (Manning and Krasnow, 1993; Samakovlis et al., 1996). Tracheal tubes are a simple epithelial monolayer, with the apical surface lining the lumen and the basal surface forming the tube exterior. Development of the tracheal system begins by invagination of 20 ectodermal placodes, each of which forms an epithelial sac of 80 cells. Over an 8 hour period in mid-embryogenesis, six primary branches bud from each sac, followed by about two dozen secondary branches. Subsequently, hundreds of fine terminal branches sprout during the several days of larval life. All of these budding events take place without loss of epithelial integrity and in the absence of cell division or death (Samakovlis et al., 1996): the extensively branched network forms exclusively by epithelial migration and morphogenesis.
Dozens of genes have been identified that are required for tracheal branching (Manning and Krasnow, 1993; Metzger and Krasnow, 1999; Affolter and Shilo, 2000). These have begun to separate the morphogenetic process into genetically distinct steps. Several genes are required for the initial specification of tracheal cell fate and invagination to form tracheal sacs. Another set of genes is required to assign distinct fates to each branch primordium before budding begins. Yet, other sets of genes are required for sprouting of primary, secondary and terminal branches.
Three primary branch genes have been characterized branchless (bnl), breathless (btl) and stumps (sms). These encode components of a fibroblast growth factor (FGF) signaling pathway that directs budding and outgrowth of the primary branches. bnl encodes a FGF expressed by small clusters of cells surrounding each sac, at each position where a primary branch buds (Sutherland et al., 1996). Bnl serves as a chemoattractant, activating the Btl FGF receptor, a receptor tyrosine kinase expressed on the tracheal epithelium (Klambt et al., 1992). This directs budding and outgrowth of primary branches to Bnl signaling centers. sms encodes a putative adapter molecule expressed in tracheal cells and appears to function downstream of Btl (Michelson et al., 1998; Vincent et al., 1998; Imam et al., 1999). The FGF pathway also plays important roles controlling subsequent sprouting events. It induces expression of some secondary (e.g. pointed) and terminal branching genes (e.g. blistered/DSRF) in cells located at the ends of outgrowing primary branches, which go on to form secondary and ultimately terminal branches (Sutherland et al., 1996).
We have characterized a fourth primary branch gene, ribbon (rib). Although the gross phenotype of rib mutants is similar to that of bnl, btl and sms, cellular analysis shows that there is not a complete block to primary branch outgrowth. Basal cytoplasmic extensions still form and grow towards Bnl signaling centers, but there is a severe impediment in the migration and morphogenesis of the tracheal cell bodies and apical surface. rib has previously been implicated in several epithelial morphogenetic events (Nusslein-Volhard et al., 1984; Jack and Myette, 1997; Blake et al., 1998; Blake et al., 1999). We show that rib encodes a putative transcription factor expressed in the trachea and other morphogenetically active epithelia, and we propose that it functions as a regulator of epithelial migration and morphogenesis.
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MATERIALS AND METHODS |
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Df(2R)P34 and Df(2R)PC29 (Lindsley and Zimm, 1992) (http://flybase.bio.indiana.edu/), and Df(2R)GC8 and enbGC1 (Gertler et al., 1995) are siblings generated from the same parental strains. The GAL4/UAS system (Brand and Perrimon, 1993) was used to misexpress bnl, using the 69B-GAL4 driver, which is expressed broadly in the embryo, and UAS-bnlB4-2 (Sutherland et al., 1996).
Mutagenesis
Isogenized dp cn bw males (aged 1-3 days after eclosion) were treated with 25 mM ethyl methane sulfonate (EMS) overnight and mated to sco/CyO females for 3-5 days (L. Messina, B. Lubarsky and M. A. K., unpublished). dp cn bw [*]/CyO progeny were tested: two out of 2086 mutagenized chromosomes screened failed to complement the lethality of ribex12, rib1 and rib2, and were named ribP7 and ribP16.
Antibodies and immunostaining
Embryo fixation and immunostaining were as described (Samakovlis et al., 1996). Primary antisera were TL1 and mAb 2A12 against tracheal lumenal antigens (Samakovlis et al., 1996), mAb 2-161 against DSRF (1:1000 dilution, from M. Gilman), anti-diphosphorylated-ERK (1:500, Sigma) and anti-ß-galactosidase (1:1500, Cappel). Tracheal cells were visualized by staining embryos carrying the 1-eve-1 P[lacZ] insertion (Perrimon et al., 1991) with anti-ß-galactosidase. Embryos were visualized with a Zeiss Axiophot microscope under Nomarski optics or a Molecular Dynamics MultiProbe 2010 laser scanning confocal microscope. For confocal fluorescence microscopy, 1-eve-1 embryos were stained with anti-ß-galactosidase and anti-Crumbs mAb Cq4 (1:1000) (Tepass et al., 1990). Signal was developed by horseradish peroxidase (HRP)-catalyzed deposition of either cyanine 3- or fluorescein-labeled tyramide reagent (NEN Life Science Products).
Polyclonal rabbit anti-peptide antisera were raised against synthetic peptides in Rib (Research Genetics, Huntsville, AL). Antisera (Ab21802K) generated against the C-terminal peptide P3 (LKRELMEGEDAQARAD, Fig. 5) from two separate rabbits gave a similar staining pattern in whole-mount embryos. Antisera (Ab21083M) from a rabbit immunized with two peptides, P1 (TKSQEPEILRTAKELQVK) and P2 (EKHEQNIIKRERDQDEREDA), gave a similar pattern. The -Rib antisera were used at 1:1000 dilution and developed by HRP immunohistochemistry or HRP-catalyzed fluorescein tyramide reagent deposition and counterstained with DAPI (1 µg/ml in glycerol mounting medium) (Patel, 1994).
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Genetic mapping of rib
Meiotic recombination in a cora2,+/+,rib1 transheterozygote was scored. Forty-two cora+rib+ chromosomes were generated out of 70,073 chromosomes screened. DNA sequencing in the genomic interval between cora and rib identified five polymorphisms (PM1-5) between the cora2 and rib1 chromosomes (rib1 chromosome sequence shown in bold): PM1 (at 21 kb in Fig. 4C) GC(A/G)CGAATTTCTA (HhaI site underlined); PM2 (at 28 kb) TTAATGTTAAAGACTTAG (bold region deleted in cora2 chromosome, DdeI site underlined); PM3 (at 40 kb) CT(T/G)CAGGTCAAGGG (PstI); PM4 (at 70 kb) GAATCT(C/T)TT(T/C)TTGCACA (XmnI); and PM5 (at 92 kb) GAG(A/C)TCCACTCGAG (SacI). cora+rib+ recombinant chromosomes were scored for the polymorphisms by restriction digestion with the indicated enzymes of PCR fragments containing the polymorphic sites.
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Molecular biology
A full-length rib cDNA (LD16058; Research Genetics) was sequenced (GenBank Accession Number, AF416603). NCBI tBLASTn homology searches (www.ncbi.nlm.nih.gov), Baylor College of Medicine BEAUTY post-processing (searchlauncher.bcm.tmc.edu) and InterPro pattern prediction searches (www.ebi.ac.uk) were used to identify BTB/POZ and Pipsqueak motifs.
To identify mutations in rib EMS alleles, DNA fragments (0.6-1 kb) spanning the rib-coding sequence and intron-exon junctions were amplified by PCR using genomic DNA isolated from rib1/CyO, rib2/CyO, ribz1/CyO, ribP7/CyO and ribP16/CyO adult flies as template. PCR products were sequenced and polymorphisms between the rib and CyO chromosomes were identified as overlapping peaks on the sequence chromatogram. Because rib1, rib2 and ribz1 were derived from isogenic parents, as were ribP7 and ribP16, polymorphisms unique to a particular rib allele were assumed to be EMS-induced mutations. Putative EMS-induced mutations were confirmed by sequence of two independent PCR products.
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RESULTS |
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The basal surface of the tracheal epithelium continues to migrate in rib mutants
The TL1 tracheal antiserum used in the above experiments stains the apical surface and lumen of the developing tracheal epithelium. To further characterize the tracheal migration defects, we examined rib1, rib2, and ribex12 mutants using the tracheal cytoplasmic marker 1-eve-1, a viable P[lacZ] insert in trachealess. We found that despite the severe defect in movement of the apical tracheal surface described above, the basal surface continued to extend actively in the mutants.
A schematic diagram of a migrating tracheal branch is shown in Fig. 2A. In wild type, the basal surface of the tracheal epithelium is broad and smooth with an occasional pseudopodium extending from cells at the growing tip, much like those seen at the leading edge of migrating fibroblasts (Fig. 2C,G,H,M). As the basal surface extends toward the Bnl FGF signaling centers, the cell bodies and apical surface follow (Fig. 2B,F), and basal cytoplasmic extensions are never very prominent. In rib mutants, pseudopodia still extended from the basal surface (Fig. 2E,J,K,O), and were more numerous and pronounced than in wild type (asterisks in Fig. 2E,K,O), occasionally forming extremely long processes that appeared to reach their normal targets (arrowheads in Fig. 2E,K). This dissociation of the migration of the apical and basal tracheal surfaces was evident at stage 12 and continued for several hours into stage 14 (data not shown). At this later stage, the apical surface began to deform in the direction of the basal cytoplasmic extensions, but even at this later stage the net distance traveled by the apical side was far less than in wild type. The apical defect is unlikely to reflect a general defect in apical-basal polarity of the tracheal epithelium because the apical determinant Crumbs (Tepass et al., 1990), the apical marker TL1, and an apically localized mRNA (K. S. and M. A. K., unpublished) were expressed and localized properly at the apical tracheal surface in rib mutants (Fig. 2D,I and data not shown). We conclude that rib mutations selectively affect movement and morphogenesis of the tracheal cell bodies and apical surface.
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rib encodes a BTB domain protein
rib maps meiotically to 2-88 (Nusslein-Volhard et al., 1984). Complementation tests with deficiencies in the region placed rib in cytological interval 56B5 to 56C1 (Fig. 4A). The left and right endpoints of this interval were defined molecularly (Fig. 4B). This was done by scanning the region for single nucleotide polymorphisms (SNPs) that distinguished sibling deficiency chromosomes from the CyO balancer chromosome. Polymorphisms that were missing in Df(2R)GC8 or Df(2R)P34 chromosomes over the balancer were assumed to be due to deletion of that region in the deficiency chromosome. This localized rib to an 150 kb interval between 5-HT1A receptor gene and predicted gene CG7230 (Fig. 4B).
The position of the rib1 mutation was refined by meiotic recombination mapping with respect to cora2 and five nearby polymorphic markers including PM1 and PM2 (Fig. 4C). rib1 mapped 0.12 cM to the right of cora2, 0.07 cM to the right of PM1 and 0.03 cM to the right of PM2. This localized rib1 to a 9 kb region (+28 to +37 kb in Fig. 4C) at the extreme right end of the 150 kb interval defined by deficiency mapping. CG7230 is the only transcription unit in the 9 kb region, and extrapolation of the recombination mapping data predicted that rib1 lies within the CG7230 transcription unit (Fig. 4C,D).
To confirm that CG7230 is rib, we sequenced the CG7230-coding region and intron-exon junctions of the five rib EMS alleles. CG7230 encodes a protein with a BTB (Bric à brac, Tramtrack, Broad) domain and a Pipsqueak (Psq) DNA-binding motif (see below). All five rib alleles contain mutations that remove or alter a conserved element in the protein (Fig. 5A). rib1 and ribP7 carry nonsense mutations that truncate the predicted protein at residues 22 and 283, respectively, removing half or more of the protein and one or both functional motifs, consistent with their assignment as amorphic alleles. rib2 and ribz1 carry an identical missense mutation which changes a highly conserved arginine (Arg58) in the BTB domain (Ahmad et al., 1998) to histidine. This change is apparently highly deleterious because both alleles behave as amorphic alleles. ribP16, a hypomorphic allele, carries a missense mutation that changes Ser389 in the predicted Psq DNA-binding motif to cysteine. We also showed by whole-mount in situ hybridization that CG7230 transcript is absent in Df(2R)P34 embryos (data not shown), so although the deficiency does not impinge on CG7230 coding sequence (Fig. 4D), it must remove essential regulatory elements. The coding regions of the two genes flanking CG7230 (CG7229 and CG11906) were also sequenced in the ribP7 and ribP16 alleles and no mutations were found. We conclude that CG7230 is rib.
An EST representing CG7230 was sequenced and compared with the Drosophila genomic sequence (Adams et al., 2000). Fig. 4D shows the deduced gene structure. There is a single predicted open reading frame that encodes a 661 residue (71 kDa) protein (Fig. 5A). There are stop codons in all three reading frames upstream of the coding sequence, implying that this is a complete coding sequence.
Blast sequence homology searches with the deduced Rib protein sequence identified a region similar to BTB (or POZ for Poxvirus and zinc finger) domains, a protein-protein interaction domain found in zinc finger transcription factors and actin-associated proteins of the Drosophila Kelch family (Bardwell and Treisman, 1994; Zollman et al., 1994). Like other BTB domains, the BTB domain in Ribbon is 120 residues and located close to the N terminus. It shares 35-38% identity (
60% similarity) with three founding members of the BTB family, the Drosophila developmental regulatory proteins Bric à brac, Tramtrack and Broad-complex transcription factor (Fig. 5B). The BTB domain of Rib is slightly more similar (40% identity, 63% similarity) to the BTB domain of longitudinals lacking (lola), a zinc-finger nuclear factor implicated in axon growth and targeting in the Drosophila nervous system (Giniger et al., 1994). Other BTB domains show less identity, such as Kelch (25% identity) and human promyelocytic leukemia zinc finger (PLZF) protein (24% identity).
Many BTB domain proteins also contain a zinc-finger DNA-binding domain, but we were unable to identify a zinc finger motif in Rib using PROSITE. However, Blast/BEAUTY searches with the Rib-coding region revealed a 49 residue region (residues 367 to 415) with similarity to the DNA-binding domain of Psq, a protein involved in patterning the early Drosophila egg, embryo and adult eye (Weber et al., 1995; Horowitz and Berg, 1996). The Psq DNA binding domain consists of four tandem repeats of an 50 amino acid motif (Lehmann et al., 1998), each of which is 30-48% identical to each other and 28-36% identical to the region in Rib (Fig. 5C). The Psq repeats also show similarity to the helix-turn-helix DNA-binding domain of a number of prokaryotic recombinases including E. coli Hin Invertase (Lehmann et al., 1998). A single Psq motif is found in Drosophila Tyrosine Kinase Related (Tkr) protein (Haller et al., 1987). Interestingly, both Psq and Tkr also contain N-terminal BTB domains (36% identity between the Psq and Rib BTB domains; 32% between Tkr and Rib). Psq, Rib, and Tkr thus define a new subtype of BTB domain proteins with a Psq DNA-binding motif.
rib is expressed in epithelia undergoing morphogenetic movements
rib expression during development was analyzed by in situ hybridization of whole-mount embryos (Fig. 6). rib transcript was detected in the developing tracheal system, as the tracheal precursor cells invaginate to form tracheal sacs and primary branches bud and grow out from the sacs (Fig. 6G and data not shown). rib was also expressed in a variety of other tissues, in a complex and dynamic pattern that coincides with various morphogenetic movements (Fig. 6C-J). For example, rib was first detected at stages 5-8 (2.5-3.5 hours AEL) in the primordia of the anterior and posterior midgut as they invaginate to form tubes (Fig. 6B-D); expression turned on again in the tubes a couple hours later (stages 10-14,
5-11 hours AEL) as they extend and form the central portion of the gut (Fig. 6H-J). rib expression was also seen in the developing mesoderm at or just after its ectodermal invagination and epithelial-to-mesenchymal transition, as the mesodermal cells begin their dorsal migration (
4 hours AEL; Fig. 6C,E). rib was also expressed in the stomodeum and proctodeum, as they invaginate to form the foregut and hindgut, and as the Malpighian tubules bud (Fig. 6E, H). rib was expressed broadly in the epidermis from stage 10 to 15 (
5-13 hours AEL) as it migrates during dorsal closure (Fig. 6I). Some of these sites of rib expression are also associated with morphological defects in rib mutants (see Discussion).
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Ribbon is a nuclear protein
Two polyclonal antisera were raised against non-overlapping peptides in Rib (Fig. 5A). In stains of whole-mount embryos, both antisera showed the same pattern of Rib protein expression, which followed the pattern of rib transcripts (Fig. 7A-D and data not shown). Staining is specific for Rib because it was absent in ribex12 (Fig. 7F) and in ribP7, a nonsense mutation that truncates the protein N-terminal to the epitopes. Rib staining showed a cellular distribution consistent with nuclear localization (Fig. 7A,C,E). This was confirmed by co-staining with the nuclear dye DAPI, which showed coincidence of Rib and DAPI staining except in regions near the centromere which stain intensely with DAPI (Hiraoka et al., 1990) but excluded Rib (Fig. 7G-I).
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DISCUSSION |
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Rib is a putative transcription factor
rib encodes a 71 kD protein with a BTB motif most similar to those found in other developmental regulatory proteins, and a separate motif similar to the DNA binding domain of Psq. The presence of two motifs found in transcription factors, together with the immunolocalization data showing that Rib is nuclear, suggest that Rib is a DNA-binding transcription factor that promotes tracheal migration and tubulogenesis by regulating the expression of target genes. Because BTB domains are known to serve as protein-protein interaction domains (Bardwell and Treisman, 1994), Rib may act as a homo- or heterodimer or higher order complex. Although its targets in the trachea are unknown, rib mutants affect the expression of the zipper myosin heavy chain in migrating epidermis during dorsal closure (Blake et al., 1998). Thus, zipper could be a target of Rib, at least in epidermis.
The genetic analysis indicates that rib functions downstream or parallel to the Bnl FGF pathway. If Rib functions downstream in the FGF pathway, for example, if its transcriptional regulatory activity is regulated by RAS/MAPK signaling like the Yan (Rebay and Rubin, 1995; Hacohen et al., 1998), DSRF (Guillemin et al., 1996) and possibly Pointed (Brunner et al., 1994) transcription complexes, then this would provide a natural way of coupling morphogenetic events in the tracheal cell bodies and apical surface to events at the basal surface, where the Bnl signal is received. When the Bnl pathway is active, the receptor would directly stimulate outgrowth of the basal tracheal surface. But it would also indirectly stimulate migration and morphogenesis of the cell body and apical surface via activation of Rib and induction of its target genes.
Rib, together with the Drosophila proteins Psq and Tkr, defines a new subfamily of BTB proteins containing Psq DNA-binding motifs. Although most other BTB domain proteins are also believed to function as transcription factors, this much larger subfamily of BTB domain proteins all contain zinc-finger (ZF) DNA-binding motifs (Zollman et al., 1994; Albagli et al., 1995; Read et al., 2000). The BTB domains of BTB/Psq proteins are no more similar to each other than they are to those of BTB/ZF proteins, although they are more similar to each other than they are to the BTB domain of Kelch, a cytoplasmic protein. The reason for the common coupling of BTB domains with two structurally unrelated DNA binding motifs, but not other DNA binding motifs, is unclear.
rib functions in a variety of epithelial migration and morphogenesis events
In addition to the tracheal system, rib is expressed in a number of other developing tissues, in a complex and dynamic pattern that coincides with various morphogenetic movements. Several sites of expression coincide with epithelial invaginations or evaginations to form tubes or sacs, as occurs during tracheal branch budding. These sites include the anterior and posterior midgut invaginations, salivary gland primordia as they invaginate and extend, stomodeum and proctodeum as they invaginate to form the foregut and hindgut, and Malpighian (renal) tubules as they bud. rib is also expressed during spreading of a planar epithelium, the epidermis, during dorsal closure. rib is expressed during or just after a number of mesenchymal-epithelial transitions, including the midgut mesenchyme as it reorganizes into an epithelium and forms the central portion of the gut.
Several of these morphogenetic events are defective in rib mutants. Indeed, rib mutants were first identified by their dorsal closure defect (Nusslein-Volhard et al., 1984). rib mutants also have defects in a number of other tissues including the Malpighian tubules, salivary glands and hindgut (Jack and Myette, 1997; Blake et al., 1998; Blake et al., 1999). Each of these are epithelia, and there are defects in the cell shape changes that underlie their morphogenesis. For example, rib mutant epidermal cells fail to elongate properly, and salivary gland cells fail to constrict apically (Blake et al., 1998).
Almost all epithelia undergoing morphogenetic movements face similar challenges as the tracheal epithelium during primary branching different parts of the epithelium must move coordinately although they are exposed to different environments. Based on our analysis of the tracheal function of rib, as well as its expression and activity in a variety of other morphogenetic processes, we propose that Rib is a key regulator of the movement and morphogenesis of epithelia, particularly events in the cell bodies or apical surface during budding of tubular epithelia. In some migrating epithelia, such as the spreading epidermis during dorsal closure, it is not clear that rib mutations specifically disrupt an apical process, so Rib may also influence epithelial migration and morphogenesis in other ways. Insight into the mechanisms and molecules that execute these morphogenetic events may come through the identification of transcriptional targets of Rib.
Another characterization of rib appeared after review of this manuscript (Bradley and Andrew, 2001).
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ACKNOWLEDGMENTS |
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