org Interactive Fly, Drosophila




The function of knirps-related (knrl) is still unknown; however, a possible gap gene function in the abdominal region of the embryo can be excluded. Both kni and knrl are initially expressed in three identical regions of the blastoderm embryo: in an anterior cap domain, in an anterior stripe and in a posterior broad band linked to the kni gap gene function (Rothe, 1994). knirps and knrl are both functional in the head anlage. The lack of one gene activity can be overcome by the activity of the other. Whereas kni is also required for abdominal segmentation, knrl is nonfunctional in its posterior expression domain. Thus, the kni/knrl pair of genes provides a region-specific buffering system, rather than a case of global functional redundancy (Gonzalez-Gaitan, 1994).

Different combinations of gap repressors for common stripes in Anopheles and Drosophila embryos

Drosophila segmentation is governed by a well-defined gene regulation network. The evolution of this network was investigated by examining the expression profiles of a complete set of segmentation genes in the early embryos of the mosquito, Anopheles gambiae. There are numerous differences in the expression profiles as compared with Drosophila. The germline determinant Oskar is expressed in both the anterior and posterior poles of Anopheles embryos but is strictly localized within the posterior plasm of Drosophila. The gap genes hunchback and giant display inverted patterns of expression in posterior regions of Anopheles embryos, while tailless exhibits an expanded pattern as compared with Drosophila. These observations suggest that the segmentation network has undergone considerable evolutionary change in the dipterans and that similar patterns of pair-rule gene expression can be obtained with different combinations of gap repressors. The evolution of separate stripe enhancers in the eve loci of different dipterans is discussed (Goltsev, 2004).

In Drosophila, different levels of the Hunchback and Knirps gap repressor gradients define the limits of eve stripes 3, 4, 6, and 7, while Giant and Kruppel establish the borders of stripes 2 and 5. In situ hybridization probes were prepared for Anopheles orthologues of all four of these gap genes, as well as a fifth gap gene, tailless. hunchback displays a broad band of expression in the anterior half of the Anopheles embryo, encompassing both the presumptive head and thorax. This pattern is similar to that observed in Drosophila, although there are a few notable deviations: (1) there is no obvious maternal expression seen in early Anopheles embryos, whereas maternal hunchback mRNAs are strongly expressed throughout early Drosophila embryos; (2) there is a significant change in the posterior staining pattern. The Drosophila gene displays a strong posterior stripe of expression that is comparable in intensity to the anterior staining pattern. In Anopheles, this staining is significantly weaker than that of the anterior domain, and the posterior pattern is shifted anteriorly into the presumptive abdomen (Goltsev, 2004).

The Kruppel and knirps staining patterns are similar in Anopheles and Drosophila embryos. In both cases, the principal sites of expression are seen in the presumptive thorax and abdomen, respectively. However, the remaining two gap genes, giant and tailless, exhibit distinctive staining patterns. In Anopheles, giant exhibits a continuous band of staining in anterior regions, whereas the Drosophila gene is excluded from the anterior pole. Moreover, there is a prominent band of staining in the presumptive abdomen of Drosophila embryos that is not seen in Anopheles. Finally, tailless is expressed in a narrow stripe in the posterior pole of Drosophila embryos, whereas Anopheles embryos display a dynamic pattern that (transiently) extends throughout the presumptive abdomen (Goltsev, 2004).

The combinations of gap repressors that define the borders of eve stripes 2 to 7 are known in Drosophila. Stripes 2 and 5 are formed by the combination of Giant and Kruppel repressors, while distinctive borders for stripes 3, 4, 6, and 7 are established by the differential repression of the stripe 3/7 and stripe 4/6 enhancers in response to distinct concentrations of the Hunchback and Knirps repressor gradients. Double-staining assays provide immediate insights into the likely combination of gap repressors that are used for any given stripe. For example, the giant and Kruppel expression patterns abut the borders of eve stripes 2 and 5. Double-staining assays were done to determine the potential regulators of the Anopheles eve stripes. These experiments involved the use of digoxigenin-labeled hunchback, Kruppel, knirps, and giant hybridization probes along with an FITC-labeled eve probe. Different histochemical substrates were used to separately visualize the two patterns (Goltsev, 2004).

The anterior hunchback pattern extends through eve stripe 2 and approaches the anterior border of stripe 3. While the posterior pattern extends through stripes 6 and 7, this pattern is quite distinct from the posterior hunchback pattern seen in Drosophila, which abuts the posterior border of eve stripe 7. The anterior giant pattern extends from the anterior pole to eve stripe 2, while the posterior pattern abuts the posterior border of eve stripe 7. In Drosophila, the posterior giant pattern extends from eve stripe 5 to stripe 7. The Kruppel pattern may be somewhat narrower in Anopheles than Drosophila. It encompasses eve stripe 3 in Anopheles but includes both stripes 3 and 4 in Drosophila. Finally, knirps exhibits the same limits of expression in Anopheles as Drosophila. In both cases, the staining pattern extends from eve stripes 4 to 6. In Anopheles, the anterior knirps pattern straddles the anterior border of eve stripe 1. Some of the eve stripes are associated with the same combinations of gap repressors in flies and mosquitoes (e.g., stripes 2, 3, and possibly 4), whereas others show distinctive combinations of gap repressors (e.g., stripes 5, 6, and 7 (Goltsev, 2004).

In Drosophila, eve stripes 6 and 7 are regulated by different concentrations of Knirps and Hunchback. Low levels of Knirps define the anterior border of stripe 7, while higher levels are needed to repress eve stripe 6. Conversely, low levels of Hunchback establish the posterior border of eve stripe 6, while higher levels regulate stripe 7. The position of the knirps expression pattern is consistent with the possibility that it defines the anterior limits of stripes 6 and 7, just as in Drosophila. However, the posterior borders of these stripes are probably not regulated by Hunchback. The expanded pattern of tailless expression seen in Anopheles might permit it to establish the posterior border of eve stripe 6 and possibly stripe 7. An alternative candidate for the posterior stripe 7 border is giant, which is expressed in a tight domain within the posterior pole. Consistent with this possibility is the observation that the posterior giant pattern comes on relatively late, and the posterior stripe 7 border is the last to form among the seven eve stripes. The reversal of the posterior hunchback and giant expression patterns, along with the expanded tailless pattern, strongly suggests that different combinations of gap repressors are used to define eve stripes 5, 6, and 7 in Drosophila and Anopheles (Goltsev, 2004).

An implication of the preceding arguments is that each of the seven eve stripes is regulated by a separate enhancer in Anopheles. Only five enhancers regulate eve in Drosophila since four of the seven stripes (3, 4, 6, and 7) are regulated by just two enhancers (3/7 and 4/6) that respond to different concentrations of the opposing Hunchback and Knirps repressor gradients. The change in the posterior hunchback pattern virtually excludes the use of this strategy in Anopheles. Thus, stripes 3 and 7 are probably regulated by separate enhancers since different combinations of gap repressors appear to define the stripe borders. Similar arguments suggest that stripes 4 and 6 are also regulated by separate enhancers (Goltsev, 2004).

Why do some enhancers generate two stripes, while others direct just one? Consider the eve stripe 2 and stripe 3/7 enhancers in Drosophila. The stripe 3/7 enhancer is activated by ubiquitous activators, including dSTAT, and the two stripes are 'carved out' by the localized Hunchback and Knirps repressors. Knirps establishes the posterior border of stripe 3 and anterior border of stripe 7, while Hunchback establishes the anterior border of stripe 3 and posterior border of stripe 7. The stripe 2 enhancer directs just a single stripe due to the localized distribution of the stripe 2 activators, particularly Bicoid. In principle, a ubiquitous activator would cause the stripe 2 enhancer to direct two stripes, stripes 2 and 5. Opposing Giant and Kruppel repressor gradients would carve out the borders of the two stripes, similar to the way in which Hunchback and Knirps regulate the stripe 3/7 and stripe 4/6 enhancers. Presumably, the eve stripe 5 enhancer directs a single stripe of expression because it is regulated by a localized activator, possibly Caudal (Goltsev, 2004).

It is suggested that ancestral dipterans contained an eve locus with separate enhancers for every stripe. Anopheles eve might represent an approximation of this ancestral locus. The consolidation of enhancers that generate multiple stripes was made possible by cross-repression of gap gene pairs. In Drosophila, there are mutually repressive interactions between Hunchback and Knirps, as well as between Giant and Kruppel. The use of these interacting gap pairs along with ubiquitous activators permits the formation of two stripes from a single enhancer. It is possible to envision two ways in which mutual cross-repression of these gap genes helps to establish the precise patterns of pair-rule gene expression: (1) it ensures that there are zones free of repressor activity on both sides of Kruppel (for the Kruppel and Giant pair) and Knirps (for the Knirps and Hunchback pair) domains; (2) it protects the patterns of pair-rule gene expression from mutations that could potentially shift the domains of gap gene expression. For example, a mutation that could shift the expression of Kruppel would simultaneously shift the expression of Giant always leaving a repressor-free zone where Eve stripes would be established. Therefore, the evolution of the eve locus depends on the changes in the preceding tier of the segmentation network: refinement in gap gene cross-regulatory interactions (Goltsev, 2004).

Finally, it is easy to imagine that certain dipterans have a single enhancer for stripes 2 and 5, rather than the separate enhancers seen in Drosophila. Perhaps, the symmetric repression of Giant and Kruppel is a relatively recent occurrence, only now creating the opportunity for consolidated expression of stripes 2 and 5 (Goltsev, 2004).

Drosophila long-chain acyl-CoA synthetase acts like a gap gene in embryonic segmentation

Long-chain acyl-CoA synthetases (ACSLs) convert the long chain fatty acids to acyl-CoA esters, the activated forms participating in diverse metabolic and signaling pathways. dAcsl is the Drosophila homolog of human ACSL4 and their functions are highly conserved in the processes ranging from lipid metabolism to the establishment of visual wiring. This study demonstrates that both maternal and zygotic dAcsl are required for embryonic segmentation. The abdominal segmentation defects of dAcsl mutants resemble those of gap gene knirps (kni). The central expression domain of Kni transcripts or proteins was reduced whereas the adjacent domains of another gap gene Hunchback (Hb) were correspondingly expanded in these mutants. Consequently, the striped pattern of the pair-rule gene Even-skipped (Eve) was disrupted. It is proposed that dAcsl plays a role in embryonic segmentation at least by shifting the anteroposterior boundaries of two gap genes (Zhang, 2011).

In Drosophila embryo, a hierarchy of maternal, gap, pair-rule and segment polarity genes which encode transcription factors establish the anteroposterior axis and the embryonic segmentation. The spatially restricted transcription factors determine the complex gene expression patterns in the early embryo. Along with the maternal determinants, the gap gene products specify the boundaries of the adjacent gap gene expression domains and the downstream pair-rule gene stripes. Among them, Knirps (Kni) and Hunchback (Hb) form their expression patterns partly through mutual repression (Zhang, 2011).

The known maternal effectors are not sufficient to establish the gap domains and it is likely that unidentified maternal molecules exist and modulate the gap gene expression. The abundant maternally-deposited lipids in embryos have been recognized as an energy source for early embryo development. These molecules also have important functions in diverse signaling pathways during larval development such as shaping morphogen gradients. However, it remains unclear whether lipids participate in any way in the establishment of embryonic segmentation (Zhang, 2011).

Long chain acyl-CoA synthetase (ACSL) is a family of enzymes which adds Coenzyme A to the long chain (C12-20) fatty acids. As the activated form of fatty acids, the Acyl-CoA participates in various cellular processes including lipid metabolism, vesicle trafficking and signal transduction. ACSL4 is a member of the mammalian ACSL family and its mutations have been associated with non-syndromic X-linked mental retardation (MRX). The Drosophila gene dAcsl encodes the homolog of human ACSL4 and they are functionally conserved ranging from building visual circuitry to lipid homeostasis (Zhang, 2009). However, the developmental function of dAcsl at the embryonic stages remains unexplored (Zhang, 2011).

This report illustrates that dAcsl is required for embryonic segmentation both maternally and zygotically. The impaired segmentation caused by dAcsl mutations is similar to that of gap gene kni mutants. In dAcsl mutants, the domain of Kni transcripts or proteins was reduced whereas the domain of another gap gene Hb protein was correspondingly expanded. Consequently, the pair-rule gene expressions were perturbed in these embryos. It is proposed that dAcsl participates in embryonic segmentation by spatially modulating gap gene expression (Zhang, 2011).

The segmentation defects of dAcsl mutants resemble those of gap gene kni. The posterior domain of Kni transcripts or proteins was narrowed whereas the adjacent domains of another gap gene Hb correspondingly expanded in these mutants. The findings reveal the connection between long-chain acyl-CoA synthetase and embryonic segmentation in Drosophila. It is proposed that dAcsl functions in embryonic segmentation by modulating gap gene expression (Zhang, 2011).

The similarity in mutant phenotypes uncovers the possible link between this enzyme and kni. Although the strong genetic interaction exists between dAcsl and kni, two observations suggest that the function of dAcsl in segmentation seems not limited to kni. Firstly, the anterior Eve stripes were also affected in some mutant embryos where Kni is not expressed. Secondly, dAcsl also genetically interacted with Kr. The alteration of gap gene expression is consistent with the genetic interaction results, in which kni or Kr reduction enhanced dAcsl segmentation defects whereas hb did not. Since the anterior zygotic Hb domain was expanded posteriorly in dAcsl mutants, this Hb shift could affect the anterior boundaries of both Kr and Kni domains. Accordingly, certain degree of rescue of the dAcsl mutant phenotype was expected when hb gene dosage was lowered by half. However, an obvious effect was seen, which could simply be that one zygotic dosage of the Hb products along with the maternal contribution is enough to fulfill its normal function at this stage (Zhang, 2011).

Also, the early zygotic expression of Hb was somehow expanded more posteriorly, indicating a spatial increase in response to Bcd activity. No corresponding increase of Bcd was detected at protein levels though the Bcd gradient seemed less steep in the mutants. Additionally, the effects not limited to kni-like phenotype would have been seen if there were a posterior-ward shift due to a major change in Bcd. Further, because removing zygotic copy of dAcsl contributed ~ 4% more occurrence of segmentation defects than the maternal mutation alone (~ 11%), alteration in the gap gene functions cannot explain the defects developed post-zygotically unless dAcsl is also zygotically activated before cellularization (Zhang, 2011).

How can the gap gene-like phenotype in dAcsl mutants be explained or how does dAcsl act on gap gene expressions/activities? One possibility is that the altered distribution of the upstream maternal factors since kni transcripts were spatially reduced in the dAcsl maternal mutants. There are abundant lipid droplets which participate in the vesicle transport and store maternal proteins in the early embryo. Since dAcsl is predicted as an enzyme mobilizing fatty acid and required for neutral lipids formation in larval tissues (Zhang, 2009), the aberration of lipid droplets formation was anticipated in dAcsl mutant embryos. Consequently, the distribution of certain maternal determinants may be affected because of the compromised membrane trafficking, altered protein localization, etc. If this hypothesis is true, then other mutations such as Lsd2 which disrupt lipid droplets transport and neutral lipids storage in embryo should give similar phenotype as dAcsl mutations. However, only very minor segmentation defects were observed in Lsd2 mutant cuticles. Does the lipid storage decrease more in dAcsl than in Lsd2 mutants? The triglyceride levels were examined in early embryos and no significant difference could be detected between the wild type and dAcsl or Lsd2 mutant embryos. The relationship between the lipid-droplets formation and embryonic segmentation remains elusive. Nonetheless, as a lipid metabolism-related enzyme, dAcsl's effect in segmentation is specific and intriguing. However, the details of the connection between dAcsl and embryonic segmentation require more intensive investigations (Zhang, 2011).

Effects of Mutation or Deletion

During the early phase of embryonic development nascent zygotic transcripts longer than about 6 kilobases are aborted between the rapid mitotic cycles. Resurrector1 (Res1) and Godzilla1 (God1), two newly identified dominant zygotic suppressor mutations, and a heterozygous maternal deficiency of the cyclin B locus, complement the partial loss of function of the segmentation gene knirps by extending the length of mitotic cycles at blastoderm. The mitotic delay caused by Res1 and God1 zygotically and by the deficiency of the cyclin B locus maternally allows the expression of a much longer transcript of a kni cognate gene that would normally be aborted between the short mitotic cycles; consequently thesekni mutant progeny survive (Ruden, 1995).

In strong kni mutants, abdominal segments A1-A7 are fused and replaced by a single segment that shows a broad denticle field on the ventral side. Segment A8 is normal. (Nauber, 1988).

Cell migration during embryonic tracheal system development in Drosophila requires Dpp and Egf signaling to generate the archetypal branching pattern. Two genes encoding the transcription factors Knirps and Knirps related are shown to possess multiple and redundant functions during tracheal development. knirps/knirps related activity is necessary to mediate Dpp signaling that is required for tracheal cell migration and formation of the dorsal and ventral branches. The expression of kni and Knrl appears during stage 10 in the tracheal placodes. During primary branch formation, expression of kni and Knrl decreases and restricts to the dorsal- and ventral-most cells as well as visceral branch cells. kni and Knrl expression persists in the cells of dorsal, visceral, lateral trunk and ganglionic branches. Thus, kni and Knrl are expressed in the same spatio-temporal patterns, suggesting that kni and knrl may also share redundant functions during tracheal development (Chen, 1998).

Dpp signaling is required for the directed migration of dorsal and ventral tracheal cells. It activates kni expression and has been proposed to control target gene expression via Kni. To elucidate kni function in dorsal and ventral tracheal cells, tracheal formation was examined in kni mutant embryos and in embryos mutant for a deficiency, which uncovers both kni and knrl. In contrast to wild-type embryos, which develop ten tracheal metameres, kni and deficiency mutant embryos develop only five. This result reflects the lack of five abdominal segments in both kni and deficiency mutant embryos. However, while the remaining tracheal metameres in kni mutant embryos develop many aspects of a wild-type branching pattern, deficiency mutant embryos develop only rudimentary tracheal metameres, which invaginate but lack primary branching and interconnections. This suggests a functional back-up by knrl activity in kni mutant embryos. Furthermore, the lack of primary branching in deficiency mutant embryos suggests that kni/knrl activity participates in early primary branch outgrowth and hence hampers the analysis of a potential kni/knrl function during later stages of branch formation. To overcome both segmentation and primary tracheal branch defects, kni and deficiency embryos were rescued by a kni transgene that provides both kni segmentation gene function and kni placode expression. kni mutant embryos bearing the kni transgene develop a normal number of tracheal metameres as well as a wild-type-like tracheal branching. In deficiency embryos bearing the kni transgene, dorsal trunk formation is similar to wild-type, whereas dorsal branch outgrowth is lacking and lateral trunk fusion occurs only partially. In addition, visceral and ganglionic branches fail to contact the gut and the central nervous system, respectively. Thus, the region-specific kni/knrl tracheal expression in the dorsal, ventral and visceral branches is required for their formation. The wild-type-like branch outgrowth in the remaining tracheal anlagen of kni mutant embryos suggests that knrl can substitute for kni activity in such embryos. kni/knrl are shown to act independently of Fgf and Egf signaling key components. Region-specific kni/knrl expression is not controlled by Fgf or Egf signaling and kni/knrl activity does not affect key components of these pathways (Chen, 1998).

Dpp signaling is required for dorsal and ventral tracheal branch formation and for kni expression. The tracheal mutant phenotypes of embryos lacking the Dpp receptors Tkv and Put are reminiscent of the kni tracheal phenotype, suggesting that kni is necessary to mediate functional aspects of Dpp signaling. To link kni activity and Dpp signaling more directly, kni was expressed in a tkv mutant background by using the tracheal-specific driver. Since tkv mutant embryos lack the dorsalmost patches of branchless expression that are necessary for dorsal branch outgrowth, the analysis was focused on ventral branch formation in the presence of kni expression. These embryos develop a rudimentary ventral tracheal system that is indistinguishable from the branching of tkv mutants. Thus, the activation of kni expression by Dpp is necessary but not sufficient for ventral branch formation. This result also suggests that Dpp signaling controls additional genes different from kni/knrl that are necessary for branch outgrowth. Ectopic Dpp expression in all tracheal cells leads to dorsoventral cell migration, which causes the lack of dorsal trunk and visceral branches that are normally formed by anteroposterior migration. It also leads to ectopic kni expression in all tracheal cells. The finding that ectopic kni expression also interferes with dorsal trunk formation suggests a role of kni activity in mediating ectopic Dpp signaling. Thus, the tracheal phenotypes generated by either ectopic dpp or ectopic kni expression were examined. Ubiquitous tracheal dpp expression causes the lack of anterioposterior branch formation and the dorsal migration of supernumerary cells. Ubiquitous tracheal expression of one copy of kni leads to a reduced dorsal trunk and an increased number of cells migrating dorsally, whereas ubiquitous expression of two copies of kni results in the absence of the dorsal trunk and the migration of supernumary cells towards dorsal positions. Thus, kni activity leads to a dorsal tracheal cell migration, as observed for Dpp. In summary, these results indicate that the role of Dpp in directing tracheal cells to adopt a dorsoventral migration behaviour is mediated by kni/knrl activity, but kni/knrl is not sufficient to mediate Dpp-dependent branch formation (Chen, 1998).

In dorsal tracheal cells knirps/knirps related activity represses the transcription factor Spalt; this repression is essential for secondary and terminal branch formation. However, in cells of the dorsal trunk, spalt expression is required for normal anteroposterior cell migration and morphogenesis. spalt expression is maintained by the Egf receptor pathway and, hence, some of the opposing activities of the Egf and Dpp signaling pathways are mediated by spalt and knirps/knirps related. Furthermore, evidence is provided that the border between cells acquiring dorsal branch and dorsal trunk identity is established by the direct interaction of Knirps with a spalt cis-regulatory element (Chen, 1998).

It has been proposed that the Dpp and Egf signaling generates three different cell fates in the developing placode. This signaling confers the capacity of cells to migrate in distinct directions. kni/knrl activity have been shown to be necessary to mediate Dpp signaling for dorsal and ventral cell migration. In addition, repression of the Egfr signaling target sal by kni/knrl establishes a border between the dorsally and anteroposteriorly migrating dorsal branch and dorsal trunk cells, respectively. However, the repression of sal is not necessary for normal dorsoventral tracheal cell migration but rather for morphogenetic processes that occur independent of cell migration. Thus, tracheal cells that express sal and kni/knrl still adopt a dorsoventral migration behavior. Ectopic expression of kni/knrl in dorsal trunk cells has two consequences: (1) it represses Sal, which results in the lack of anteroposterior migration of dorsal trunk cells, and (2) ectopic kni/knrl leads primordial dorsal trunk cells to adopt a dorsoventral migration behavior. Thus, the observation that ectopic Dpp causes altered tracheal cell migration and lack of dorsal trunk formation is consistent with the proposal that these processes are mediated in part via kni/knrl. However, in contrast to ectopic Dpp, which inhibits visceral branch formation, ectopic kni/knrl tracheal expression does not affect anterior outgrowth of visceral branches. This observation is not unexpected since kni/knrl is expressed in visceral branch cells and is necessary for normal visceral branch morphogenesis. Thus, kni/knrl act within the genetic circuitry of visceral branch cell fate determination in a different way from the way these genes act during dorsal branch development. No mediation of dorsoventral cell migration is involved. kni/knrl may be part of a patterning system for visceral branch development within the Egfr signaling domain, whereas sal activity is necessary for dorsal trunk development (Chen, 1998 and references).

Endoreduplication cycles that lead to an increase of DNA ploidy and cell size occur in distinct spatial and temporal patterns during Drosophila development. Only little is known about the regulation of these modified cell cycles. Fore- and hind-gut development have been investigated and evidence is presented that the knirps and knirps-related genes are key components to spatially restrict endoreduplication domains. Lack and gain-of-function experiments show that knirps and knirps-related, which both encode nuclear orphan receptors, transcriptionally repress S-phase genes of the cell cycle required for DNA replication and that this down-regulation is crucial for gut morphogenesis. Furthermore, both genes are activated in overlapping expression domains in the fore- and hind-gut in response to Wingless and Hedgehog activities emanating from epithelial signaling centers that control the regionalization of the gut tube. These results provide a novel link between morphogen-dependent positional information and the spatio-temporal regulation of cell cycle activity in the gut Fuß, 2001).

In situ hybridization and antibody stainings reveal co-expression of both knirps and knirps-related initially at stage 10 in the ectodermally derived primordia of the esophagus in the foregut and the small intestine and the rectum in the hindgut, respectively. With the beginning of germband retraction, an additional co-expression domain appears in two lateral cell rows on each side of the large intestine. The expression of kni and knrl persists in these four domains in the gut epithelium throughout embryogenesis. The loss-of-function analysis using kni mutant embryos and embryos mutant for the deficiency Df(3L)riXT1 (which uncovers both the kni and knrl transcription units) reveals that only in the deficiency is gut organogenesis strongly affected from stage 14 onwards. Crumbs (Crb) was used as a marker for ectodermal epithelial cells that also visualizes the subdivision of the hindgut into the small intestine, large intestine and the rectum. The developing small intestine and the rectum epithelia in the hindgut and the esophagus epithelium in the foregut start to lose their integrity in the mutant. Expression studies indicate that the activity of the pro-apoptotic gene reaper is upregulated in many gut epithelial cells from late stage 10 onwards, indicating that the gut cells most likely undergo apoptosis. This eventually leads to a disconnection of the midgut from the hindgut and foregut epithelia at stage 15. The mutant gut phenotype in the deficiency can be rescued using a kni transgene that provides both kni segmentation and gut function. Similarly, the small intestine becomes rescued when kni or knrl are misexpressed in all the hindgut cells of Df(3L)riXT1 embryos using the 14-3fkh-Gal4 driver and the UAS-Kni or UAS-Knrl effectors. In summary, the data point toward a redundant role for kni and knrl during gut development, as has been observed for other kni/knrl dependent aspects of organogenesis Fuß, 2001).

kni and knrl are redundant regulators of cell fate in the stomatogastric nervous system and the wing and as regulators of cell migration in specific tracheal cell populations. In these studies, however, kni and knrl target genes, which regulate cell behavior (such as cell shape changes, cell adhesion, or cell migration), have not been identified and understanding how both genes control cell biological processes has remained elusive. Therefore, the cause for the disconnection of the fore- and hind-gut from the midgut in Df(3L)riXT1 mutant embryos was investigated. A test was performed to see whether kni/knrl are involved in the establishment of epithelial polarity in the gut cells. The localization of the polarity determinant Discs-lost, which marks the apical margins of epithelial cells, and the septate junction markers Fasciclin III and Neurexin IV were analyzed in wild-type and Df(3L)riXT1 mutant embryos. Anti-Discs lost (now redefined as Drosophila Patj) and anti-Fasciclin III double stainings reveal that the apical region and the septate junctions of the hindgut cells are still formed normally in Df(3L)riXT1 mutant embryos. However, double stainings of Neurexin IV and betaGal visualizing the nuclear reporter gene expression pattern of an enhancer trap line reveals that hindgut tissue of Df(3L)riXT1 mutant embryos contains much bigger nuclei and cells than the corresponding wild-type tissue. This suggested that an increase in DNA ploidy might have occurred in the kni;knrl double mutant condition and prompted an investigation of the pattern of endoreduplication cycles in the hindgut Fuß, 2001).

The development of the gut epithelium is accompanied by a stereotyped pattern of cell cycle regulation. The fore- and hind-gut primordia undergo a fixed number of postblastodermal cell divisions until late stage 10/early stage 11. Endoreduplication cycles have been described to occur at stages 13/14 in the hindgut. BrdU incorporation studies have shown that the hindgut epithelium displays a subdivision into replicating tissues (such as the developing large intestine) and quiescent tissues (such as the developing small intestine) and the rectum from stage 11 onwards. The replicative activity is reflected by a specific BrdU incorporation pattern in the hindgut: no incorporation is observed in the small intestine and rectum and but high incorporation is observed in the large intestine primordia in between. Notably, the kni/knrl expression pattern in the hindgut of wild-type embryos is complementary to the BrdU incorporation pattern. This complementarity also applies to the foregut in which kni/knrl are ubiquitously expressed. Endocycles have not been described for the developing foregut and BrdU is not incorporated from stage 11 onwards Fuß, 2001).

In Df(3L)riXT1 mutant embryos, the analysis of the BrdU incorporation pattern reveals a tissue and time specific defect of cell cycle activity in the hindgut epithelium. An ectopic domain of DNA replication in the rectum and a slight expansion of DNA replication into the small intestine is detectable using the BrdU incorporation assay in stage 13 mutant embryos. The appearance of a G1 phase in the endoreduplicative cycle and the transition from G1 to S phase is accompanied by a molecular network controlling the coordinate transcription of cycE. CycE in turn regulates the activity of the S-phase genes Polalpha, PCNA and RNR2. Since cycE is only weakly expressed in the hindgut whether its expression is changed in Df(3L)riXT1 mutant embryos could not be analyzed (both kni and knrl are unchanged in cycE mutants). On the contrary, the Polalpha, PCNA and RNR2 genes which are activated in response to CycE activity, indeed do have a strong expression pattern in the hindgut that parallels the BrdU incorporation pattern in wild-type embryos. In line with the BrdU experiments, loss of kni/knrl function in Df(3L)riXT1 mutant embryos leads to an ectopic expression of RNR2, PCNA and Polalpha in the rectum and an upregulation of these genes in the small intestine prior to the upcoming defect in these gut regions. To further investigate this, gain-of-function experiments were performed using the UAS-Gal4 system. Ectopic expression of either kni or knrl in the entire hindgut using the 14-3fkh-Gal4 driver and the UAS-Kni or UAS-Knrl effector lines merely leads to a mild reduction of the BrdU incorporation domain in the large intestine. Ectopic expression of both kni and knrl has a strong effect on DNA replication in the hindgut. The BrdU domain is completely abolished, suggesting a combinatorial function of both genes in the suppression of endoreduplication cycles. The expression of various cell cycle components was analyzed. Ectopic kni and knrl activities in the entire hindgut are able to completely repress the transcription of RNR2, PCNA and Polalpha in the large intestine. Notably, cycE mutants in which no endoreduplication occurs in the large intestine, display a mutant phenotype that is similar to the one obtained when kni and knrl are ubiquitously expressed in the hindgut Fuß, 2001).

Since endoreduplication usually has an impact on cell size, an investigation was carried out to see whether upon ectopic kni and knrl expression in the hindgut, changes in cell size occur. Anti-Neurexin IV antibody stainings reveal that many of the large intestine cells are indeed much smaller in these embryos as compared to wild type. The large intestine region becomes reduced in size under these conditions, although the overall cell number seems not to be affected. These results are consistent with the argument that the lack of endocycles in the large intestine region leads to a reduction of the cell sizes in this area. In summary, these results suggest that kni and knrl down-regulate endoreduplication activity in the gut by repressing S-phase genes of the cell cycle Fuß, 2001).

The kni and knrl expression domains in the developing foregut and hindgut partially overlap with the expression domains of wingless and hedgehog, which define signaling centers that control morphogenetic movements during the regionalization of the gut. To investigate whether kni/knrl expression and consequently also the restriction of the endoreduplication pattern in the gut is coordinated the Wg and Hh signaling cascades, expression studies in various lack and gain-of-function situations were performed. In hh mutants, kni expression is only mildly reduced in the developing fore- and hind-gut expression domains. In early wg mutants, kni fails to be expressed in the esophagus primordium and is strongly reduced in the developing small intestine and rectum. wg mutant embryos lack a foregut at later stages and have a strongly reduced hindgut. Ectopic expression of hh in all the hindgut cells using the UAS-Hh effector and the 14-3fkh driver line does not alter the kni or knrl expression domains in the hindgut, even when the Hh dose is increased by using effector lines with multiple UAS-Hh transgene insertions. However, if the same experiment is carried out in engrailed mutants, kni/knrl can be induced ectopically in all the hindgut cells. In wild-type embryos, engrailed is expressed in the dorsal part of the large intestine and exerts a repressing function on kni/knrl expression that apparently cannot be overcome by ectopic Hh activity. However, ectopic wg expression in all the hindgut cells using the UAS-Wg effector and the 14-3fkh driver line does result in ubiquitous induction of kni and knrl expression. engrailed expression in the hindgut of these embryos is repressed under these conditions. To investigate whether ectopic Wg expression in the hindgut interferes with DNA replication activity required for endoreduplication, BrdU incorporation was examined. BrdU incorporation is absent in the hindgut of such embryos. Consistent with this result, S-phase genes such as RNR2 are transcriptionally repressed upon ectopic Wg expression in all the hindgut cells using the 14-3fkh-Gal4 driver and UAS-Wg. As has been observed for ectopic kni/knrl expression in the hindgut, the size of the hindgut cells are reduced in these embryos Fuß, 2001).

Knirps was identified in a genome-wide analyses for transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites

Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, RNA interference (RNAi) was used to screen 730 transcriptional regulators and 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons were identified in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation (Parrish, 2006; Full text of article).

To assay for the stereotyped dendrite arborization pattern of class I da neurons (hereafter referred to as class I neurons) in RNAi-based analysis of dendrite development, a Gal4 enhancer trap line (Gal4221) was used that is highly expressed in class I neurons and weakly expressed in class IV neurons during embryogenesis. Because of the simple and stereotyped dendritic arborization patterns of the dorsally located ddaD and ddaE, the studies of dendrite development focused on these two dorsally located class I neurons (Parrish, 2006).

To establish that RNAi is an efficient method to systematically study dendrite development in the Drosophila embryonic PNS, it was demonstrated that injecting embryos with double-stranded RNA (dsRNA) for green fluorescent protein (gfp) is sufficient to attenuate Gal-4221-driven expression of an mCD8::GFP fusion protein as measured by confocal microscopy. Next whether RNAi could efficiently phenocopy loss-of-function mutants known to affect dendrite development was tested. Similar to the mutant phenotype of short stop (shot), which encodes an actin/microtubule cross-linking protein, shot(RNAi) caused routing defects, dorsal overextension, and a reduction in lateral branching of dorsally extended primary dendrites. Likewise, RNAi of sequoia or flamingo resulted in overextension of ddaD and ddaE, RNAi of hamlet resulted in supernumerary class I neurons, and RNAi of tumbleweed resulted in supernumerary class I neurons and a range of arborization defects, consistent with the reported mutant phenotypes. Thus, RNAi is effective in generating reduction of function phenotypes in embryonic class I dendrites (Parrish, 2006).

In contrast to the genes that coordinately affect dorsal dendrite outgrowth and lateral branching/outgrowth, a group of 21 genes (group B) were identified that have opposing effects on dendrite outgrowth and branching, suggesting that dendrite outgrowth and branching might partially antagonize one another. RNAi of 19 of these genes resulted in dorsal overextension of primary dendrites and a reduction in lateral branching/lateral branch extension. In the most severe cases, such as RNAi of the transcriptional repressor snail, dorsal overextension of almost completely unbranched dendrites was found. Like snail(RNAi), RNAi of the nuclear hormone receptor knirps, the transcriptional repressor l(3)mbt, as well as 15 other genes, all caused dorsal overextension of primary dendrites. As in the case of genes that normally limit arborization, RNAi of these genes rarely caused dendrites to cross the dorsal midline (Parrish, 2006).

In addition to the effects on primary dendrite extension, RNAi of each of these 18 genes limits the number and length of lateral dendrite branches. RNAi of some genes such as snail or knirps almost completely blocked dendrite branching, whereas RNAi of other genes such l(3)mbt had more modest effects on dendrite branching. In addition, a significant reduction of branching was noticed at the distal tip of the dorsally projected primary dendrite. In control treated stage 17 embryos, branchpoints are distributed along the primary dendrite, with the most distal branchpoint usually located within a few microns of the distal tip of the dendrite. In contrast, branching is rarely observed within 10 microns of the distal dendritic tip following RNAi of these group B genes. In some cases, such as snail(RNAi), knirps(RNAi), or l(3)mbt(RNAi), the most distal branchpoint is located 25 microns or further from the distal tip of the primary dendrite. Therefore, these TFs inhibit primary branch extension but promote lateral branching and lateral branch extension (Parrish, 2006).


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knirps: Biological Overview | Regulation | Targets of Activity and Protein Interactions | Developmental Biology | Effects of Mutation

date revised: 30 October 2015

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