knot/collier

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of kn at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

The 3.9 kb transcript first accumulates after 8 hours of embryogenesis, peaks in first instar larvae and is present at low levels in third instar larvae and pupae. Both col transcripts are detected at very low levels in male and female adults and are absent from dissected ovaries (Crozatier, 1996).

collier expression is detected at the beginning of mitotic cycle 14 in two laterally symmetrical stripes whose posterior limit is one or two rows of cells anterior to the postion where the cephalic furrow forms. This restricted expression persists during early gastrulation, when the cephalic furrow demarcates the head from the trunk and the posterior gnathal region (maxillary and labial primordia). During formation of the ventral furrow, the stripe of col expression widens from one to four cells. Comparison with the blastoderm fate map indicates that col expression extensively overlaps the mandibular segment anlage but is slightly displaced anteriorly. The col expression domain corresponds with the 'Engrailed intercalary spot' as well as in cells directly posterior to it. Thus col appears to be specifically expressed in posterior cells of the intercalary segment (parasegment -1) and in anterior cells of the mandibular segment [the first pregnathal (in front of the mouth) segment], a region that possibly corresponds to parasegment 0 (Crozatier, 1996).

By stage 11, col transcripts are still present in the mandibular bud region but additional expression is seen in a few cells of the procephalon and in segmentally repeated groups of cells in the trunk. In stage 13 and 14 embryos, col is expressed in a segmentally reiterated pattern in the ventral nerve cord and in lateral and dorsal groups of cells, including cells of the peripheral nervous system, as well as in patches of cells in the brain (Crozatier, 1996).

It has been hypothesized that the hypopharyngeal lobe is the ventral aspect of the intercalary segment, in which only the dorsal component expresses lab during stages 10 and 11. The anterior expression boundary of most homeotic genes differs in the ventral ectoderm compared to the dorsolateral ectoderm, so the expression of lab in only the dorsolateral intercalary segment would not be unusual. However, kn ectodermal expression, which appears to mark the hypopharyngeal lobe, is separated by hh-expressing cells from the intercalary anterior compartment in which lab is expressed (the short stripe of hh-expressing cells extends from the ventral margin of the ectoderm where it meets the endoderm in the stomatodeal opening to the dorsal limit of kn ectodermal expression). Furthermore, while the ventral portion of the stripe of kn expression gives rise to the hypopharyngeal lobe, the dorsolateral portion does not appear to give rise to any ectodermal derivatives, but instead to cephalic mesoderm. Thus, the hypopharyngeal lobe is not thought to represent the ventral component of the intercalary segment, but rather is a separate domain posterior to it. Because the anterior compartment cells of the hypopharyngeal lobe primordium are not separated by intervening hh- or en-expressing cells from the anterior mandibular segment, the hypopharyngeal lobe should be considered nominally part of the mandibular segment, although it appears to be defined independently at blastoderm stages, as judged by kn activation. This is in keeping with the mandibular defects found in kn mutants that eliminate the hypopharyngeal lobe (Seecoomar, 2000 and references therein).

Ample evidence indicates, however, that in other insects the hypopharyngeal lobe is part of the intercalary segment. Unlike Drosophila, lab is expressed in the hypopharyngeal lobe in the flea and other insects. In all insects, en-expressing cells marking the posterior compartment of the intercalary segment, form on the posterior margin of the lab-expressing cells. Perhaps this difference in lab expression indicates that what is called the 'hypopharyngeal lobes' in Drosophila are not homologous to the hypopharyngeal lobes of other insects, but rather reflect a novel proturberance in the mandibular segment. Alternatively, it is possible that the Drosophila 'hypopharyngeal lobes' are indeed homologous to the hypopharyngeal lobes of other species, but there has been a concerted shift in Drosophila relative to the head morphology of both lab expression and posterior compartment of the intercalary segment, such that the hypopharyngeal lobe no longer resides in the intercalary segment. Because the hypopharyngeal primordium (via activation of kn) appears to be set aside independently at blastoderm stage, much earlier than the segmentation of the cephalic segments, such a shift in segmentation vis-a-vis the hypopharyngeal lobe seems plausible. Further analysis of the expression of the homeotic genes (such as lab, Dfd and cnc) and the upstream patterning genes (such as kn, btd and other gap/pair-rule genes) in a number of closely and distantly related insects will be necessary to resolve these hypotheses (Seecoomar, 2000 and references therein).

Effects of Mutation or Deletion

Hedgehog (Hh) plays an important role in Drosophila wing patterning by inducing expression of Dpp, which serves to organize the wing globally across the A-P axis. Hh signaling also plays a direct role in patterning the medial wing through the activation of the Hh-target gene, knot (kn). kn is expressed in Hh-responsive cells near the A-P compartment boundary, where its expression is dependent on fu, a component of Hh signaling. kn is required for the proper positioning of veins 3 and 4 and to prevent ectopic venation between them. Furthermore, the anterior expansion of the normal kn expression domain causes an associated anterior shift in the position of vein 3 in the resultant wing. Ectopic expression of kn elsewhere in the wing imaginal disc results in the failure to properly activate the vein initiation genes, rho and Dl. Expression of the gene encoding the EGF-receptor (Egfr), which is required for vein initiation and subsequent differentiation, is normally depressed in the 3-4 intervein region. This downregulation of Egfr in the medial portion of the imaginal disc is dependent on kn activity and ectopic expression of kn inactivates Egfr elsewhere in the wing primordium. It is proposed that kn expression in Hh-responsive cells of the wing blade anlagen during the late third instar creates a zone of cells in the medial wing in which vein primordia cannot be induced. The primordia for veins 3 and 4 are laid down adjacent to the kn-imposed vein-free zone, presumably by a signaling factor (such as Vn) also synthesized in the medial region of the wing (Mohler, 2000).

The original viable kn allele causes veins 3 and 4 to form closer together and produces a corresponding shift in their primordia (as detected by rho expression) in the late third instar disc (Sturtevant, 1995). Mosaic analysis of strong, embryonic kn alleles by Nestoras (1997) indicates that kn is important in suppressing vein formation between veins 3 and 4, but not in other regions of the wing. Because vein 4 runs just posterior to the A-P compartment boundary, the region affected by kn mutant clones corresponds approximately to the Hh-responsive cells along the A-P compartment boundary. kn is also required for suppressing vein formation in ptc minus clones in the anterior compartment; these ptc minus clones mimic Hh-responsive cells in which Hh has bound to the Ptc receptor. Nestoras (1997) proposed that kn functions to separate veins 3 and 4 by imposing a vein-free region in response to Hh signaling. Mosaic analysis of strong, pupal lethal alleles of fu, a serine/thronine kinase required in Hh-responsive cells for a normal response, shows a similar requirement for fu in preventing ectopic vein induction between veins 3 and 4, suggesting a direct role for Hh signaling in controlling the 3-4 intervein space (Mohler, 2000 and references therein).

In situ hybridization of the kn cDNA to wing imaginal discs indicates that strong kn expression is limited primarily to a stripe in the middle of the wing blade region of the imaginal disc. Weaker kn expression can also be detected in more posterior regions of the wing pouch and in portions of the hinge region of the disc. Significantly, kn medial expression is reduced in fu mutants, which encode a ser-thr kinase that functions in many aspects of Hh signaling (Mohler, 2000).

Colabelling of KN mRNA expression with a ptc-lacZ reporter construct, reveals that the medial expression of kn is located within the ptc-expressing cells (the Hh-responsive cells). The posterior margin of kn expression matches the posterior margin of ptc-lacZ expression at the anterior-posterior compartment boundary, although kn expression does not extend quite as far anterior as the ptc-lacZ reporter. In contrast, most of the kn expression is posterior to the expression domain of a dpp-lacZ reporter construct (marking a region of strong dpp expression), overlapping this domain only slightly. Thus, kn, like en and ptc is expressed close to the A-P boundary, whereas strong dpp expression is displaced slightly to the anterior, with respect to kn and the A-P boundary (Mohler, 2000).

Expression of the rho gene has been demonstrated to be a determinant for the vein primordia laid down in the late third instar wing disc. Double-labelled in situ hybridizations reveal that kn is expressed precisely between the vein primordia for veins 3 and 4. Thus, KN mRNA expression matches the region determined by mosaic analysis for kn function: between veins 3 and 4 in the Hh-responsive cells of the wing blade (Mohler, 2000).

No alteration of vein expression is found in knot mutant discs or in discs in which kn has been ectopically expressed, indicating that the expression of vn in this region is not controlled by kn. Egfr expression, however, is regulated by knot. Although transcription of Egfr is initially uniform throughout the wing blade, by the end of the third instar Egfr transcription has been repressed along the wing margin and in a medial stripe across the wing blade region. Double in-situ hybridization reveals that the medial region of Egfr downregulation coincides with the region of kn gene expression. To determine whether downregulation of Egfr is necessary for the formation of the 3-4 intervein region, Egfr was ectopically expressed in the region in which it is usually downregulated. Ecoptic expression of EGFR in the medial wing driven by the ptc-GAL4 driver generates wings with fusion of veins 3 and 4 in the proximal portion of the wing. Ectopic expression of Egfr driven throughout the wing blade dorsally does not cause formation of ectopic veins between veins 3 and 4, although a significant amount of ectopic vein material is induced anterior to vein 3 and posterior to vein 4. This suggests that the creation of a vein-free zone between veins 3 and 4 is not likely to be explained solely by downregulation of Egfr by kn (Mohler, 2000).

These results indicate that a second mechanism, in addition to downregulation of the Egf-receptor, must exist to position veins 3 and 4 and prevent vein formation between them, at least in the distal portion of the wing. This secondary mechansim for blocking vein formation between veins 3 and 4 may reflect the fact that Vn is not the only vein-promoting factor synthesized in the medial wing: local Dpp signaling may also play a significant role in the activation of vein 3. Dpp signaling in concert with Hh signaling is required for the activation in the medial wing of the iroquois-complex genes, araucan and caupolican, which in turn are required for the formation of vein 3. The expression of the iro genes is not sufficient to activate vein 3 initiation, since kn overexpression in this region does not affect iro gene expression, but does eliminate expression of the vein initiation genes. The following hypothesis is favored: whereas vein 4 is activated primarily by Vn signaling onto the adjacent Egfr-active domain, vein 3 is combinatorily activated by Vn signaling and by Dpp signaling mediated through iro complex activation. While the Vn and Dpp-inducers may be expressed over a fairly broad domain in the medial wing, because of the block on vein initiation provided by kn activity, vein 3 must form just outside the kn expression domain. The gaps that form in vein 3 when the kn expression domain is expanded to the anterior may be caused by moving the border of the kn expression domain out of range of Vn signaling and either high-level Dpp signaling or the resultant iro expression domain. This process is most likely to produce gaps in the distal portion of vein 3, since the expression of both vn and iro is not as wide in the distal regions of the disc as in the proximal regions (Mohler, 2000 and references therein).

Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. The phenotypic analysis is reported of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap'n'collar. The parasegmental register of col activation is controlled by the combined activities of the head-gap genes buttonhead and empty spiracles and the pair-rule gene even skipped; col therefore integrates inputs from both the head and trunk segmentation systems, which were previously considered to be essentially independent. After gastrulation, positive autoregulation of col is limited to cells of anterior PS0. Conversely, heat-pulse induced ubiquitous expression of Col leads to disruption of the head skeleton. Together, these results indicate that col is required for establishment of the PS(-1)/PS0 parasegmental border and formation of the intercalary segment. These data support neither a simple combinatorial model for segmental patterning of the head nor a direct activation of segment polarity gene expression by head-gap genes, but rather argue for the existence of parasegment-specific second order regulators acting in the head, at a level similar to that of pair-rule genes in the trunk (Crozatier, 1999b).

Three independent alleles of collier (designated as col¹, col² and col³) display lethality at the late embryonic/first instar larval stage with specific defects in the head skeleton similar to those observed upon heat-shock induced expression of col antisense RNA in early gastrula embryos. These defects are a complete lack of the ventral arms (VA) and a strong reduction of the lateral gräten (LG), two structures thought to be derived from the intercalary/mandibular segment anlagen. The T-ribs in the floor of the pharynx and the antennal, maxillary or hypopharyngeal sensory organs are present, as well as a normal pattern of internal sensory structures, detected by 22C10 antibody staining. The mutant embryos that hatch give rise to larvae that do not grow and tend to crawl out of the medium, suggesting that they are unable to feed. That these defects result from col mutations was further substantiated by the lack of a somatic dorsal muscle in which col is specifically expressed. The same head and muscle phenotypes are observed for the 3 alleles in homozygous or hemizygous combinations, suggesting that they are strong hypomorphic or null alleles. Further support was provided by sequence analysis of the col¹ allele, where a G to A transition (amino-acid position 228) eliminates a splice acceptor site. This lesion should result in the non-removal of intron 6 and the production of a truncated Col protein; indeed, no Col protein can be detected in col¹ mutant embryos using anti-Col polyclonal antibodies directed against the divergent carboxy-terminal end of the protein (Crozatier, 1999b).

The embryonic head phenotype of col¹ hemizygous mutant embryos indicates a loss of skeletal structures derived from the intercalary, and possibly mandibular, segments without transformation toward another segment identity. To investigate this segmentation phenotype in more detail, col expression was compared with that of the segment polarity genes hh and wg. At the blastoderm stage, the posterior limit of col expression is parasegmental (PS0/PS1), since it precisely abuts the mandibular stripe of hh-expressing cells. Whether its anterior limit is also parasegmental cannot be answered at this stage because the expression of segment polarity genes in pre-gnathal segments is not yet established at this stage. Examination of early stage 11 embryos shows that col expression overlaps the intercalary hh stripe and abuts the intercalary Wg spot, indicating a parasegmental anterior border for col expression. At this stage however, col expression has been lost from the posterior part of PS0, since it does not overlap mandibular Wg expression. The cnc gene, which codes for a b-ZIP transcription factor, has been postulated to act as a segment identity gene in the mandibular segment. Consistent with col being expressed in PS0, col and cnc expression only partly overlap, in the region corresponding to the anterior mandibular segment. Together, these data indicate a parasegmental register of col expression at the blastoderm stage, which is subsequently restricted to anterior PS0 (Crozatier, 1999b).

A determination was made of whether col mutations affect the expression of wg and En, which mark the anterior and posterior compartments of each segment, respectively. In col¹ hemizygous embryos, both the intercalary stripe of En and the spot of wg expression are missing. Since col expression does not overlap the intercalary Wg spot, the loss of this spot in col mutant embryos suggested that col does not regulate wg expression directly but possibly by an hh-dependent mechanism. It has indeed been found that in col mutant embryos, the intercalary stripe of hh is also absent, or much reduced. Together, these results show that col controls hh, en and wg expression in the intercalary segment and is required for establishing the PS(-1)/PS0 parasegmental border. The head skeleton structures ventral arm (VA) and lateral-gräten (LG), which are, respectively, either missing or reduced in col mutant embryos, are also affected in two other head mutants: crocodile (croc), which codes for a forkhead-domain protein, and cnc. These structures are also affected in embryos mutant for the homeotic genes Dfd and lab, which are expressed, respectively, in the mandibular and maxillary segments, and in the intercalary segment. col expression was examined in embryos mutant for croc, cnc, Dfd or lab. In none of these embryos was there a change in col transcription. Conversely, no changes could be detected for croc, Dfd or Lb expression in col¹ hemizygous embryos, indicating that expression of each of these three genes is independent of col. In contrast, col is required for cnc transcription in the posterior intercalary segment at stage 9-10. Because this region is anterior to the region of overlap between col and cnc expression at the blastoderm stage, it is concluded that this region corresponds to a secondary site of cnc expression initiated at stage 9, under control of col activity. In cnc mutant embryos, intercalary hh expression is normal, indicating that hh and cnc are regulated by col, independent of one another (Crozatier, 1999b).

col mutations provoke cell death in the forming hypopharynx. The normal pattern of Lab expression and morphology of the intercalary lobe (sometimes referred to as hypopharyngeal lobe) at stage 11 indicate that lack of hh and cnc expression in col¹ hemizygous embryos are probably not linked to cell death. In order to address this question more directly, the distribution of reaper (rpr) mRNA, which marks cells fated to undergo apoptosis, was examined. In wild-type embryos at stage 11, there are two invariant sites of rpr expression: inside the epidermal layer of the gnathal region where the primordium of the salivary gland invaginates, and near the caudal tip of the extended germ band. In col¹ embryos, an additional site of rpr expression is observed, which does not correspond to cells of the intercalary lobe but rather to cells of the hypopharyngial epithelium (floor of the forming pharynx). These cells have been previously shown to derive from the ventral side of the intercalary segment primordium and lineage tracing analysis has linked them to MD2. This specific cell death is consistent both with the domain of col expression at the onset of gastrulation and the head skeleton phenotype of col mutant larvae (Crozatier, 1999b).

col expression is first detected during the interphase of mitotic cycle 14, when expression of head-gap genes has already resolved from initial broad domains into defined stripes. The stripe of col expression is included in that of btd, overlaps that of ems, and is restricted both dorsally and ventrally to neuroectodermal cells. Examination of dorsal (dl) mutant embryos shows that Dl is required for col repression in the mesodermal plate. The ectopic expression of col observed in twist (twi) and snail (sna) mutant embryos suggests that Dl target genes, rather than Dl itself, are involved. Embryos lacking ems function also show a ventral derepression of col expression. Further, at stage 10, ems mutant embryos show an abnormal pattern of col mRNA accumulation, with a mandibular stripe in addition to intercalary stripe of col-expressing cells. This suggests a second role for ems in regulating col. In btd mutant embryos, there is a complete loss of col expression, whereas there is no change in embryos lacking both slp (slp1 and slp2) genes, consistent with previous data establishing that btd but not slp is required for intercalary en and wg expression (Crozatier, 1999b).

Together, these results confirm that col acts downstream of head gap genes in the transcriptional cascade patterning the head. The fact that col is expressed in a parasegment immediately anterior to the trunk, raises the possibility of a regulation by components of the trunk segmentation system such as pair rule genes. In paired (prd) mutant embryos, col expression starts normally but, soon after gastrulation, becomes restricted to a few cells located anterior to the abnormal cephalic furrow which forms in these embryos. col mis-expression might thus be a rather indirect effect of the prd mutation. In addition to prd, the only pair rule gene whose mutations appear to affect col expression is eve. At the beginning of cycle 14, the stripe of col expression is located immediately anterior to Even-skipped (Eve) stripe 1, these two domains becoming separated by a single row of cells during the process of cellularization. col is ectopically expressed in eve mutant embryos, in a region roughly corresponding to PS1, indicating that Eve acts as a repressor of col in this parasegment. During the interphase of cycle 14, the broad band of ectopic col activation resolves into a distinctive stripe, separated from the normal PS0 stripe by one to three cells going from ventral to dorsal. col expression precisely overlaps the expression of string (stg), in the region prefiguring mitotic domain 2. Expression of stg, which triggers the G2/M transition, is unchanged in embryos deficient for col and vice versa, arguing that MD2 cells undergo a concerted mitotic and differentiation program, set upstream of both col and stg. This led to an examination of whether eve was also involved in defining the position of MD2, using antibodies against the phosphorylated form of histone H3 as a marker of mitosis. Like col transcription, MD2 expands posteriorly in eve mutant embryos at early cycle 14 to form a second, ectopic, stripe of mitotic cells at the beginning of gastrulation. Together, these results show that col expression and MD2 position integrate inputs from both the head and trunk segmentation systems, which were previously considered as being essentially independent (Crozatier, 1999b).

Late control of col expression involves restriction to the anterior PS0 and positive autoregulation. At the onset of gastrulation (stage 7), col is expressed in the entire PS0. At stage 10, the two separate patches of cells that keep expressing col correspond to lateral cells of the intercalary lobe and ventral cells invaginating within the atriopharyngeal cavity, respectively, indicating a restriction of col expression to anterior PS0. To determine when this restriction occurs, use was made of the greater stability of the beta-gal protein compared to lacZ (or col) mRNA and the two patterns were compared in P[col5-lacZ] embryos. In order to follow col transcription rather than transcript accumulation, a col intron probe was used that labels nascent nuclear transcripts. Before mitosis 14, the lacZ and col mRNA and beta-gal protein patterns completely overlap. After completion of mitosis 14, all the ectodermal cells derived from the MD2 domain can be visualized by beta-galactosidase immunostaining. Only the most anterior cells continue to transcribe col-lacZ (or col). This observation suggests that, at cycle 14, an asymmetric cell division occurs with respect to the maintenance of col transcription. After stage 11, col transcription is only maintained in a subset of the cells of the intercalary lobe. In col mutant embryos, this transcription is lost, indicating a direct or indirect, positive auto-regulation (Crozatier, 1999b).

To investigate further the role of col transcriptional regulation in head morphogenesis, the functional consequences of ubiquitous expression of the Col protein were examined, using hs-col transgenic lines. One 45 minute heat shock was applied at different times during embryonic development. Whereas heat-shock treatment of hs-col embryos later than 5 hours after egg laying (AEL) has no apparent effect on embryonic development and viability, heat-shock treatment between 3 and 5 hours AEL (stages 6 to 9) causes embryonic death. Cuticle preparations of dead embryos display a normal segmental array of thoracic and abdominal denticles, but a disrupted head skeleton. The H piece, which originates in part from the maxillary, and partly from the labial segment anlagen, is consistently missing and the LG are also affected. Because col activity is required for maintaining its own expression in the head ectoderm, it was asked whether ubiquitous expression of the Col protein was altering endogenous col expression. In situ hybridization to hs-col embryos with a col intron probe, which allows hs-col and endogenous col transcripts to be distinguished, revealed that col transcription is ectopically activated in mandibular cells. Whether this ectopic col transcription results from activation and/or maintenance in all MD2-derived cells after mitosis 14 remains to be established. No specific change in the expression pattern of En, cnc, Dfd and Lab could be detected, however, suggesting that ectopic activation of other Col targets is responsible for the induced phenotype (Crozatier, 1999b).

This paper summarizes the regulatory cascade controlling formation of the intercalary segment. Activation of col in PS0 at the blastoderm stage (stage 6) is strictly dependent upon btd. Col is ventrally repressed by ems and the mesodermal genes sna and twi; by the trunk pair rule gene eve in the posterior, and by a yet unknown factor in the anterior. At stage 10, col expression has become restricted to anterior PS0, where it activates intercalary expression of hh and cnc, and possibly en. Loss of hh activity leads to the non cell-autonomous loss of wg expression in anterior adjacent cells [PS(-1) cells]. btd activation of eve in PS1 creates a regulatory loop allowing for differential gene expression between PS0 and PS1. cnc expression in the mandibular segment is dependent upon btd (Crozatier, 1999b).

The knot gene of Drosophila is required for the formation of the hypopharyngeal lobe in the germ-band stage embryo and the ventral arms and lateralgräte of the larval head skeleton. Rearrangement mutations of knot disrupt the previously characterized gene, collier. kn is required for the activation of cnc (cap'n'collar, a bZIP homeotic selector gene) in the progenitors of the hypopharyngeal lobe. kn is also required for the activation of CK01299, an EST marker for the cephalic mesoderm. Based on the relative expression of kn and the posterior compartment-specific gene hedgehog, additional evidence is provided that in Drosophila, unlike many other insects, the hypopharyngeal lobe arises from part of the mandibular segment (Seecoomar, 2000).

knot is expressed in early embryogenesis from stage 7 to 11 in a single domain corresponding to mitotic domain 2 within the mandibular segment. Comparison of the expression of kn in the mandibular segment with cnc, a homeotic gene required for labral and mandibular segment identity, in late stage 10 reveals that kn is expressed in the anterior-most region of the mandibular segment, whereas cnc is expressed throughout the anterior compartment of the mandibular region. Unlike cnc, kn is also significantly expressed in the cephalic mesoderm. Injecting antisense COL RNA into embryos prior to blastoderm formation has revealed a role for kn in mandibular development. This results in mandibular defects, particularly the shortening of the lateralgräte of the head skeleton. Embryos mutant for kn^SA1, the rearrangement allele broken within the transcription unit, or for any of four kn mutations isolated with EMS show similar disruptions of head skeleton. In zygotically mutant kn embryos the observed defects of mandibular structures are somewhat variable. However, for each of the four EMS mutations, kn mutant embryos derived from homozygous germ-line clone mothers had consistently stronger head skeleton defects. For the two strongest EMS alleles germ-line clone-derived zygotic mutant embryos ('zygotic + maternal') consistently lack the ventral arms and have foreshortened lateralgräte, but retain the adjacent ventral plate, ventral T-ribs and posterior pharyngeal wall. The variability in the zygotic mutant embryos is primarily due to variation in the extent of the lateralgräte. Because the difference between the zygotic alone and zygotic + maternal mutant embryos is simply in the extension and shape of the lateralgräte, it is not clear whether the difference is due to loss of maternal kn expression or to other potential differences in genetic background between these two lines. No significant maternal expression can be detected by in situ hybridization to early embryos, although low levels of RNA may not be easily discerned from background staining and the potential for maternal protein stores exists. Due to the possibility of maternal contribution to kn early embryonic function, subsequent molecular analysis of kn function was conducted in homozygous embryos derived from germ-line clones mutant for a strong allele (Seecoomar, 2000).

The development of multicellular organisms requires the establishment of cell populations with different adhesion properties. In Drosophila, a cell-segregation mechanism underlies the maintenance of the anterior (A) and posterior (P) compartments of the wing imaginal disc. Although engrailed (en) activity contributes to the specification of the differential cell affinity between A and P cells, recent evidence suggests that cell sorting depends largely on the transduction of the Hh signal in A cells. The activator form of Cubitus interruptus (Ci), a transcription factor mediating Hh signaling, defines anterior specificity, indicating that Hh-dependent cell sorting requires Hh target gene expression. However, the identity of the gene(s) contributing to distinct A and P cell affinities is unknown. A genetic screen based on the FRT/FLP system has been to search for genes involved in the correct establishment of the anteroposterior compartment boundary. By using double FRT chromosomes in combination with a wing-specific FLP source, 250,000 mutagenized chromosomes were screened. Several complementation groups affecting wing patterning have been isolated, including new alleles of most known Hh-signaling components. Among these, a class of patched (ptc) alleles was identified exhibiting a novel phenotype. These results demonstrate the value of this setup in the identification of genes involved in distinct wing-patterning processes (Végh, 2003).

A total of 250,000 mutant chromosomes covering the X chromosome and both major autosomes were screened. Four complementation groups were identified that affected wing patterning similar to mutations in smo. The largest of these groups represents alleles in smo itself. Two groups exhibiting a subset of smo phenotypes represent new alleles of fused and collier/knot. Fused is a positive regulator of Hh signaling, and collier/knot is an Hh target gene required for the formation of the L3/L4 intervein region. Surprisingly, the remaining complementation group turned out to consist of novel ptc alleles with striking characteristics. Molecularly, they represent point mutations causing an amino acid substitution in either the first or the second large extracellular loop. In contrast to ptc null alleles, homozygous mutant clones failed to upregulate Hh target genes even in the presence of Hh. Together these findings suggest that the mutant proteins repress Smo constitutively, most likely because they fail to bind Hh. Animals mutant for trans-heterozygous combinations of these new ptc alleles with ptc^S2 are fully viable. The ptc^S2 product lacks the ability to repress Smo but is able to sequester, and hence bind to, Hh. The intragenic complementation that was observed suggests that both functions of Ptc, binding of Hh and repression of Smo, can be provided by individual proteins that possess only one of each. Recently, it was shown that a combination of two proteins, one consisting of the N- and the other the C-terminal half of Ptc, reconstitutes Ptc function. Although these experiments cannot be directly compared with the findings in this study, together they do suggest that Ptc function can be separated intramolecularly into independent modules of N- vs. C-terminal and extra- vs. intracellular domains. One possible scenario that could explain the intragenic complementation would be if Ptc proteins act in a multimeric complex (Végh, 2003).

Cellular immune response to parasitization in Drosophila requires the EBF orthologue Collier

Drosophila immune response involves three types of hemocytes ('blood cells'). One cell type, the lamellocyte, is induced to differentiate only under particular conditions, such as parasitization by wasps. This study investigated the mechanisms underlying the specification of lamellocytes. collier (col), the Drosophila orthologue of the vertebrate gene encoding early B-cell factor (EBF), is expressed very early during ontogeny of the lymph gland, the larval hematopoietic organ. In this organ, Col expression prefigures a specific posterior region recently proposed to act as a signalling centre, the posterior signalling centre (PSC). The complete lack of lamellocytes in parasitized col mutant larvae revealed the critical requirement for Col activity in specification of this cell type. In wild-type larvae, Col expression remains restricted to the PSC following parasitization, despite the massive production of lamellocytes. It is therefore proposed that Col endows PSC cells with the capacity to relay an instructive signal that orients hematopoietic precursors towards the lamellocyte fate in response to parasitization. Considered together with the role of EBF in lymphopoiesis, these findings suggest new parallels in cellular immunity between Drosophila and vertebrates. Further investigations on Col/EBF expression and function in other phyla should provide fresh insight into the evolutionary origin of lymphoid cells (Crozatier, 2004).

Col is expressed in Drosophila lymph glands at the end of embryogenesis. In the absence of a specific molecular marker, the embryonic anlage of lymph glands has been mapped to the thoracic lateral mesoderm by lineage analysis of transplanted cells. By histochemical staining, it was observed that Col is expressed in two discrete clusters of cells in the dorsal mesoderm of thoracic segments T2 and T3, starting at the germ-band extension, when lymph gland hemocyte precursors become specified (stage 11). These clusters of Col-expressing cells grow closer during germ-band retraction before coalescing to form the paired lobes of the lymph glands (early stage 13). Double staining for Col and Odd-skipped, a lymph gland marker expressed from that stage onward, confirmed that Col-expressing cells are lymph gland precursors. Thereafter, only three to five cells located at the posterior tip of each lobe maintain high levels of Col expression, although low levels are still detected in the other cells of the lymph glands and in some pericardial cells. Col expression thus identifies a few cells of the thoracic dorsal mesoderm as the lymph gland primordium and distinguishes a specific posterior region of this hematopoietic organ. The embryonic hematopoietic primordium has been defined as the cephalic domain of Srp expression at the blastoderm stage. Srp is not detected, however, in lymph gland precursors prior to stage 12. Consistent with this result, larval hematopoietic progenitors expressing Col are observed in srp^6G (an amorphic allele) mutant embryos, indicating that the specification of the embryonic and larval lymph gland progenitors may involve different processes (Crozatier, 2004).

B- and T-lymphocytes mediate adaptive immunity, a phylogenetically recent component of the immune system as it is found only in gnathostomes. How adaptive immunity emerged during evolution, and was built on top of the innate immune system by which it is controlled and assisted, remains a fascinating question. The requirement for Col function in the Drosophila cellular immune response, and EBF function in B-cell development in vertebrates, suggests that Col/EBF function was co-opted early during the evolution of cellular immunity. A puzzling question remains, however, of how the cell-autonomous function of EBF in B-cell development, and the non-cell-autonomous function of Col in lamellocyte development, could relate to an ancestral Col/EBF function. It is proposed that the ancestral expression of Col/EBF in a subset of hematopoietic cells conferred on these cells the ability to respond to signals from circulating immune supervisors (generically designated as macrophages) and provide a secondary line of defence against specific intruders. This cell-specific property in turn laid the ground for the emergence of the vertebrate lymphoid cells on one side and the Drosophila PSC on the other. Although admittedly highly speculative, this proposal takes into account the following considerations. B-cell development represents the default fate of lymphoid progenitors. Although specification of B-cells critically depends on EBF (and the basic helix-loop-helix protein E2A), commitment depends on another gene, Pax5. The Pax5^-/- pro-B-cells retain the ability to generate a whole range of both 'innate' myeloid and lymphoid cells. Thus, the ontogeny of the B-cell lineage from preexisting myeloid cell types has occurred through several steps, one key event being the co-opting of Pax5, acting downstream of EBF, for which there is no known counterpart in Drosophila hematopoiesis. Second, the co-opting of Col activity for lamellocyte differentiation in larval hematopoiesis most likely came on top of a preexisting hematopoietic system, such as that operating in Drosophila embryos. Further investigation of Col/EBF functions in intermediate phyla should provide more insight into the diversity of myeloid lineages and ontogeny of the lymphoid lineages during evolution (Crozatier, 2004).

Control of multidendritic neuron differentiation in Drosophila: the role of Collier

Proper sampling of sensory inputs critically depends upon neuron morphogenesis and expression of sensory channels. The highly stereotyped organisation of the Drosophila peripheral nervous system (PNS) provides a model to study neuronal determination and morphogenesis. This study reports that Collier/Knot (Col/Kn), the Drosophila member of the COE family of transcription factors, is transiently expressed in the subset of multidendritic arborisation (da) sensory neurons that display an highly branched dendritic arborisation, class IV neurons. When lacking Col activity, class IV da neurons are formed but display a reduced dendrite arborisation. Col control on dendrite branching is distinct from that exerted by Cut, another transcription factor expressed in class IV neurons and necessary for proper dendrite morphogenesis. Col is also required for the class IV da-specific expression of pickpocket (ppk), which encodes a degenerin/epithelial sodium channel subunit required for larval locomotion. Characterisation of the col upstream region identified a 9-kb cis-regulatory region driving col expression in all class IV md neurons, even though these originate from two types of sensory precursor cells. Altogether, these findings indicate that col is required in at least two distinct programs that control the morphological and sensory specificity of Drosophila md neurons (Crozatier, 2008).

Each individual cell of the Drosophila embryonic PNS has been described, providing a unique model system to investigate the mechanisms of coding of neural identity. This study shows that expression of the COE transcription factor Collier/Knot is specific to a subset of md neurons, the class IV neurons. Col is required both for the specific expression of ppk, a gene encoding a subunit of a sodium-gated channel involved in locomotion and aspects of the elaborate dendritic branching pattern of class IV md neurons. It should be noted that an independent study of col function in md neurons came to very similar conclusions (Hattori, 2007: Crozatier, 2008).

Systematic screens for transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites have revealed that a number of distinct transcription factors are involved in different aspects of dendrite extension, lateral branching and arborisation as well as in restriction of the dendritic coverage. However, to date the expression pattern of very few of these transcription factors is known during embryonic and post embryonic development. Col is the only known transcription factor whose expression is restricted to a subtype of md neurons. One particularly intriguing feature is that the three class IV md neurons found per abdominal hemisegment originate from at least two different types of lineages, the md-es (v'ada) or md-solo lineages (ddaC and vdaB), pointing to the existence of a convergence process towards the same neuronal phenotype. Since col remains transcribed in these three neurons in embryos mutant for col, it suggests that they are specified independently of col activity. The regulation of Col expression and the function in class IV md neurons provide a unique paradigm to study how the transcriptional control of md specification operates in independent lineages (Crozatier, 2008).

col transcription is first observed in the pII cells at the origin of class IV md neurons, indicating that it becomes restricted to these neurons following asymmetric division of the pIIb cell. Two or three Col-positive cells in place of each dorsal md neuron were often detected in embryos mutant for sanpodo, which encodes a four-pass transmembrane protein which interacts with the Notch receptor. Conversely, md-specific col transcription is often lost in embryos mutant for numb (numb¹, a null allele), which down regulates Notch signalling in one of the pIIb daughter cells. It is therefore concluded that col transcription is repressed by Notch signalling. Previous studies on col requirement for the formation of the DA3 embryonic muscle showed that col transcription in the sibling DO5 muscle lineage is also repressed by Notch. It thus appears that col repression by Notch is used in several independent cell lineage decisions. Whether the same effectors of the Notch pathway are involved both in the md neuronal and DA3 muscle lineages remains to be determined. One intriguing feature of col transcription in post-mitotic md neurons is that it is only transient. This could be due to a direct control by maternal and/or early zygotic transcription activators that dilute away during embryogenesis. Alternatively, it could involve the progressive accumulation in md neurons of a repressor able to shut down col transcription. Preliminary dissection of the Drosophila col upstream region mapped the cis-regulatory information necessary for col transcription in md neurons to a fragment located between − 9 and − 5 kbp upstream of the transcription start site (Dubois, 2007). Expression of the reporter gene was not detected, however, in the neurons innervating the dh2 and vp5 es organs, indicating that Col expression in md neurons and es neurons is under the control of separate cis-elements. Whether (partly) different cis-elements are also involved in md-solo, versus md-es col activation remains an open question. An identical pattern of Col expression in Class IV md neurons is observed in Drosophila virilis, indicating that the transcriptional regulatory network controlling this expression has been conserved between these two Drosophila species. The − 9- to − 5-kbp fragment of col upstream DNA contains many sequences, between 20 and 80 bp in length, which are identical between D. melanogaster and D. virilis and represent as many potential regulatory sequences. Further dissection of this fragment should allow identifying which combinations of transcription factors are involved in the specific activation and temporal restriction of col transcription in class IV md neurons (Crozatier, 2008).

Activation of ppk transcription in class IV md neurons requires Col activity. Unlike Col, ppk remains expressed in these neurons until the end of larval development, showing that once activated, ppk expression is maintained independently of Col. This suggests the occurrence of a relay mechanism. The several hours delay between Col accumulation and ppk activation, in both normal embryos and upon pan-neuronal expression of Col, also indicates that the ability of Col to activate ppk depends upon another md-specific factor. Whether this factor(s) is itself a target of Col only activated in md neurons or an md-specific factor whose activity is potentiated by Col remains to be investigated. Finally, whether and how the delay and maintenance mechanisms are coupled is also unknown, at present. The transient character of Col expression does not favour a feed-forward mechanism such as that proposed for the specification of two lineage-related neurons in the CNS, where Col and its downstream targets act together to activate lineage-specific neuropeptides. The cis-regulatory region driving ppk expression in class IV md neurons has been identified. Parallel studies on col and ppk cis-regulation should now allow deciphering more precisely the transcriptional control of class IV md neuron differentiation (Crozatier, 2008).

In addition to ppk expression, class IV md neurons differ from the other md neurons by the length and degree of arborisation of their dendrite network. This dendritic arborisation is unchanged in ppk mutant larvae, showing that ppk expression and the dendritic network of class IV md neurons are specified by two independent programs. Formation of the primary branches of the md dendrite network starts to form at the end of embryogenesis and continues to elongate during larval development. At the same time, a more elaborate pattern of secondary arbors develops. The recent identification of no less than 76 transcription factors influencing (class I, in that case) dendrite formation suggests that the formation and maintenance of the dendritic network is regulated at many different, likely successive levels. One of these factors is Cut, which regulates distinct dendrite branching patterns in different md classes in a dose-dependent manner. It is therefore proposed that Col plays a dual function in implementing the class IV md neuron identity. According to this model, at least one unidentified class IV md neuron-specific TF is required for activating Col expression and regulating the level of Cut (Cut^medium) expression. On one side, Col cooperates with other md-specific TFs to activate ppk transcription specifically in class IV md neurons, independent of Cut. On the other, Col and Cut^medium are involved in establishing the secondary complex dendrite network, typical of these neurons that develops in larvae. Since col transcription is not maintained beyond embryogenesis, its role in secondary dendrite branching and maintaining ppk transcription in larvae must be indirect and likely involves (an) intermediate TF(s). Systematic identification of col and cut targets in embryonic class IV md neurons should allow to better understand their respective roles. col involvement in two parallel regulatory networks (or dual function) links two salient features of class IV md neurons, an extended dendritic field and ppk expression. Dendrites act as information-integrating centers as they receive sensory or synaptic inputs. How expression of the Na+ channel subunit Ppk and extended dendrite arborisation are physiologically linked is a next question (Crozatier, 2008).

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