The lin gene produces a single mRNA species, which is expressed broadly, at low levels in the embryonic epidermis during the time of epidermal patterning and at higher levels elsewhere in the embryo (Hatini, 2000)
In addition to their roles in patterning and morphogenesis of the hindgut, the Drosophila genes drumstick (drm) and brother of odd with entrails limited (bowl) are required in the foregut for spatially localized gene expression and the morphogenetic processes that form the proventriculus. drm and bowl belong to a family of genes encoding C2H2 zinc finger proteins; the other two members of this family are odd-skipped (odd) and sob. In both the fore- and hindgut, drm acts upstream of lines (lin), which encodes a putative transcriptional regulator, and relieves the lin repressive function. In spite of its phenotypic similarities with drm, bowl was found in both foregut and hindgut to act downstream, rather than upstream, of lin. These results support a hierarchy in which Drm relieves the repressive effect of Lin on Bowl, and Bowl then acts to promote spatially localized expression of genes (particularly the JAK/STAT pathway ligand encoded by upd) that control fore- and hindgut morphogenesis. Since the odd-family and lin are conserved in mosquito, mouse, and humans, it is proposed that the odd-family genes and lin may also interact to control patterning and morphogenesis in other insects and in vertebrates (Johansen, 2003).
By following the spatially localized expression domains of Wg, Dead ringer (Dri), and Connectin (Con), it is possible to identify at least four processes that contribute to morphogenesis of the three-layered valve of the proventriculus. During stages 13-15, a bulge referred to as the 'keyhole' forms in the foregut ectoderm, at its junction with the endoderm of the anterior midgut; this appears to be a process of evagination, similar to that described for formation of the vertebrate optic cup. The keyhole can be distinguished from the anterior ectoderm of the esophagus and from the anterior endoderm of the anterior midgut, since it is not surrounded by visceral mesoderm and is flanked by two domains of Wg expression. During stage 16, the anterior hemisphere of the keyhole reverses its curvature, and is approached by the posterior hemisphere; this has been described as a folding process. During stage 17, the posterior portion of the keyhole moves interiorly over the anterior lip of the developing proventriculus, a movement that appears similar to involution over the dorsal lip of the blastopore in Xenopus. Also during stage 17, the most posterior portion of the esophagus, i.e. that portion just anterior to the keyhole, inserts posteriorly into the pocket consisting of the most anterior endoderm of the anterior midgut. During this step of insertion, there is significant elongation of the most interior cells, which are derived from the anterior of the keyhole. At the conclusion of proventricular morphogenesis, the domain of Wg expression initially at the anterior of the keyhole is now inserted most distally into the proventriculus, while the domain of Wg initially posterior to the keyhole is now just interior, at the most anterior of the proventriculus (Johansen, 2003 and references therein).
drm and bowl mutants, which exhibit similar defects in hindgut elongation, are also similar in that they have related effects on proventriculus folding; in both mutants, rather than undergoing the first step of evagination to form the keyhole, the entire foregut remains as a narrow tube (Johansen, 2003).
In both foregut and hindgut, localized expression of signaling molecules is required for gut morphogenesis; in the hindgut, this patterned expression has been shown to depend on drm and bowl. In the foregut, drm and bowl are also required for patterned gene expression, specifically for the two stripes of Wg that bracket the keyhole primordium, as well as for expression of upd and dri in the posterior hemisphere of the keyhole primordium. Another indicator of foregut patterning is its investment with visceral musculature, which surrounds the foregut epithelium with the exception of the keyhole region. In both drm and bowl mutants, the entire foregut (up to the anterior midgut) is surrounded by visceral mesoderm, indicating that the keyhole region is not established in these mutants. The odd mutant has no detectable foregut or hindgut defect. In a screen of over 10,000 chromosomes, no lethal mutation was isolated in sob; it therefore seems unlikely that sob plays a significant role in gut morphogenesis (Johansen, 2003).
The similar sequence, phenotype, and partially overlapping expression of drm and bowl suggest that these genes might play redundant roles in foregut and hindgut morphogenesis. To address this possibility, double, triple, and quadruple mutants were constructed. Proventriculus and hindgut morphologies, patterned gene expression, and investment with foregut visceral musculature indicate that the drm bowl double mutant has hindgut and foregut phenotypes similar to those of drm and bowl single mutants. Further, drm sob odd (drmP2) and drm sob odd bowl (drmP2 bowl) mutants also have foregut and hindgut phenotypes that resemble those of drm and bowl single mutants. Since no additional phenotypes were revealed when drm, sob, odd, and bowl mutants were combined, it is concluded that, in the gut, members of the odd family, in particular drm and bowl, do not have redundant or overlapping function (Johansen, 2003).
One odd-family member, drm, has previously been shown to interact with lin; like drm, lin is required for both hindgut patterning and the cell rearrangement that elongates the hindgut (Green, 2002 and Iwaki, 2001). In the foregut, the processes of folding and involution that form the proventriculus fail to occur in both drm and lin mutant embryos; the resulting phenotypes, however, are distinct: the drm foregut is long and narrow, while that of lin is short and bloated. Consistent with what has been observed for the hindgut, the foregut phenotype of the drm lin double mutant is very similar to that of the lin single mutant. In addition, the region that will become the keyhole, i.e., that portion of the foregut bracketed by Wg expression and lacking Con-expressing visceral musculature, is expanded in both lin and drm lin embryos. Since the foregut in the drm lin double mutant (both in terms of gene expression and morphology) is similar to that seen in the lin single mutant, it is concluded that in the foregut, as has been shown in the hindgut (Green, 2002), lin is epistatic to (acts downstream of) drm (Johansen, 2003).
Since the bowl phenotype is similar to that of drm in both foregut and hindgut, the epistatic relationship between bowl and lin in both fore- and hindgut was investigated. Strikingly, while the drm lin phenotype is similar to that of lin, the bowl lin mutant phenotype, in both fore- and hindgut, appears the same as that of bowl. Further, the hindgut and foregut of drm bowl lin and drm sob odd bowl lin (drmP2 bowl lin) embryos are indistinguishable from those of bowl embryos. These results are consistent with observations that odd and sob are not required for gut morphogenesis (Wang, 1996). Most importantly, they show that in both hindgut and foregut, bowl is epistatic to (acts downstream of) lin (Johansen, 2003).
More detailed analysis of gene expression confirms the epistasis of bowl to lin in both the hindgut and foregut. upd expression, which is observed throughout the small intestine (anterior portion of the hindgut) in wild-type (Iwaki, 2001), is barely detectable in bowl and bowl lin, but greatly expanded in lin hindguts. Similarly, hh expression, seen in both small intestine and rectum, is greatly reduced in the anterior of bowl and bowl lin, but expanded in lin hindguts. En expression, seen on the dorsal side of the large intestine (Iwaki, 2002), is expanded to both dorsal and ventral sides in bowl and bowl lin, but missing from lin hindguts. dri, expressed strongly in the two boundary cell rows and at a lower level in all cells of the small intestine (Iwaki, 2001), is expressed in duplicated boundary cell rows in bowl and bowl lin hindguts, but at a low level throughout the lin hindgut. All of these results confirm the epistasis of bowl to lin in the hindgut (Johansen, 2003).
Characterization of gene expression also supports the epistasis of bowl to lin in the foregut. The expression of upd in the foregut epithelium, in a region that will become the posterior keyhole, is not seen in bowl or bowl lin mutant foreguts, but is greatly expanded in lin mutants. hh expression, which extends throughout the keyhole and part of the foregut anterior to it, is significantly reduced in bowl and bowl lin, while it appears to be expanded in lin embryos. dri, expressed in a narrow ring in the posterior keyhole region, is missing in bowl and bowl lin, but expanded in lin embryos. It is concluded that in both foregut and hindgut, lin is epistatic to drm, and bowl is epistatic to lin; in other words, lin acts downstream of drm, and bowl acts downstream of lin (Johansen, 2003).
The byn-GAL4 construct drives posterior gut-specific expression, specifically, a uniformly high level of expression in the hindgut starting at stage 8 and continuing through embryogenesis (Iwaki, 2002). When this construct is used to drive ectopic expression of drm throughout the hindgut, a lin-like phenotype, i.e., expansion of small intestine, is observed, while ectopic expression of lin throughout the hindgut results in a drm-like defect, i.e., loss of small intestine (Green, 2002). This is demonstrated by morphology, by expanded expression of upd and hh, and by absence of expression of En (Johansen, 2003).
In contrast to the dramatic effect of ectopic drm, ectopic expression of bowl throughout the hindgut has little effect on morphology and patterning. The morphology of the hindgut is altered only modestly: the small intestine is somewhat wider, the large intestine shorter, and the rectum a little longer. Similarly, patterning of the hindgut does not appear different: expression of upd, hh, and En is normal. Therefore, although bowl is required to specify the small intestine and for normal hindgut elongation, it functions differently from drm (Johansen, 2003).
A genetic hierarchy of drm, lin, and bowl activity controls the localized expression of upd in both foregut and hindgut. Previous work has shown that, most likely by establishing a gradient of JAK/STAT activity, localized expression of upd is required for hindgut morphogenesis, specifically the cell rearrangement that drives hindgut elongation (Green, 2002 and Johansen, 2003a). upd is expressed in the region of the foregut that will become the posterior hemisphere of the keyhole, and remains restricted to this region through the remainder of embryogenesis. Stat92E, a transcriptional target of JAK/STAT signaling in the embryo, is expressed in the foregut in a domain that overlaps with, but extends beyond (both anteriorly and posteriorly), the ring of upd expression in the keyhole. Analysis of anti-Crb stained embryos shows that, in upd mutants, the evagination and folding steps of proventriculus morphogenesis occur, but the involution and insertion steps do not take place. It is concluded that upd is required for late steps in proventricular morphogenesis, and that Upd is likely to affect cells beyond the domain in which it is expressed (Johansen, 2003).
For each of the Drosophila odd-family genes drm, sob, odd, and bowl, a specific ortholog could be identified in the Anopheles gambiae genome; this was possible because of the high similarity in amino acid sequence between specific zinc fingers. Three of the four Anopheles odd-family genes (the fourth was not mapped at the time of this submission) are, as in Drosophila, clustered on one chromosome arm (Johansen, 2003).
There is a high degree of identity not only between the sequence of each of the five zinc fingers in the Drosophila and Anopheles Sob and Bowl proteins, but also between these and the five zinc fingers encoded by a splice variant of mouse Odd-skipped related 2. Remarkably, these proteins share 88% identity over the 135 amino acids that comprise five zinc fingers, implying that this group of fingers may have conserved molecular function (Johansen, 2003).
Like the odd-family genes, lin is highly conserved between Drosophila and Anopheles, showing 43% identity over 858 amino acids, and a striking 76% identity in the 165 amino acid 'Lines homology domain' at the carboxy terminus (Johansen, 2003 and references therein).
From the data presented here and previously, it is concluded that drm, lin, and bowl have the same relationship to each other in both the foregut and the hindgut. drm and bowl in one case, and lin in the other, affect specification of the small intestine in different ways: bowl, expressed throughout the hindgut, and drm, expressed at the anterior of the hindgut, are both required to establish the small intestine, while lin, expressed throughout the hindgut, represses the small intestine (Green, 2002; Iwaki, 2001). Similarly, in the foregut, drm and bowl are both required to establish the keyhole, while lin, expressed throughout the foregut, represses formation of the keyhole region. For mutants in all three genes, failure to establish the keyhole results in an early failure in morphogenesis of the proventriculus (Johansen, 2003).
These observations can be integrated with the results of epistasis and ectopic expression experiments presented in this study to yield a hierarchical model for the mechanism by which drm, lin, and bowl gene activities interact to specify discrete domains in the gut. The epistasis of bowl to lin as demonstrated in this study means that the repressive activity of lin acts through bowl. Bowl promotes specification of the small intestine and keyhole, while Lin represses specification of these regions. Drm, expressed in the small intestine and in the keyhole, relieves repression of the small intestine and keyhole by repressing Lin, thereby allowing Bowl to function (Johansen, 2003).
Although bowl is necessary to specify small intestine fate, overexpression studies suggest that it is not sufficien to specify this fate in the context of gene activity in the hindgut. At least two possible reasons can be imagined for this: either the level of bowl activity generated by the byn-GAL4 driver is not sufficient to overcome the repressive effect of lin, or another activity in addition to bowl (and present only in the anterior hindgut) is required to promote small intestine fate (Johansen, 2003).
The drm-lin-bowl hierarchy that patterns the epithelial foregut and hindgut tubes functions by a distinctly different mechanism from the segmentation hierarchy that patterns the blastoderm embryo. During embryonic segmentation, a cascade of transcription factor-encoding genes (gap, pair-rule, and segment polarity) is sequentially expressed in more and more restricted domains. During gut development, in contrast, the activator Bowl and the repressor Lin are expressed throughout both foregut and hindgut; it is the spatially localized expression of Drm that is required for patterning of the distal foregut and hindgut (at the junction with the midgut). Drm, presumably by its direct binding to Lin (Green, 2002), relieves the repression of Bowl by Lin. The Drm-Lin-Bowl genetic hierarchy defined in this study is thus based, not on transcriptional regulatory interactions, but on protein-protein interactions (Johansen, 2003).
Foregut and hindgut comprise the most distal portions of the gut tube, connecting the endodermal midgut to the exterior; both are ectodermal in origin, arising from the invagination of the stomodeum and proctodeum, respectively. Although the proventriculus undergoes a different type of morphogenesis than the hindgut (evagination, folding, involution, and insertion, versus elongation by cell rearrangement), it is significant that the drm-lin-bowl hierarchy in the hindgut obtains in the foregut as well. The drm-lin-bowl hierarchy acts in both ectodermal gut tissues to specify a small domain at the most interior position of the tube (i.e., the most posterior of the foregut and the most anterior of the hindgut) (Johansen, 2003).
Specification of these domains results in the localized expression of upd, encoding the Drosophila JAK/STAT pathway ligand. In the hindgut, upd is required for morphogenetic behavior (rearrangement) of cells distant from the site of its expression, suggesting that a gradient of Upd may orient cell rearrangement (Johansen, 2003a). Since upd is required for proventriculus formation, and a domain of Stat92E expression is observed in the foregut extending beyond the domain of localized upd expression, there may also be a gradient of Upd in the foregut that plays a role in the morphogenetic processes that form the proventriculus (Johansen, 2003).
These results support the proposal that foregut and hindgut morphogenesis are controlled in parallel by the some of the same genes. In addition to the drm-lin-bowl-upd pathway delineated here, the transcription factor encoded by forkhead (fkh) is required for expression of wg, hh, and decapentaplegic (dpp), which each contribute to aspects of both foregut and hindgut morphogenesis. Thus, in Drosophila, a number of molecular pathways are similarly deployed to promote both fore- and hindgut morphogenesis; some of this pathway conservation may extend to other organisms (Johansen, 2003).
The one-to-one correspondence among each of the four members of the Drosophila and Anopheles (both members of the order Diptera) Odd family proteins, indicates that the four members were present prior to the divergence of the suborder Cyclorrhapha (which include Drosophila) from the suborder Nematocera (which include Anopheles), approximately 250 MYA. Drosophila chromosome 2L and Anopheles chromosome 3R, on which the odd family of each species is located, respectively, are the most conserved pair of chromosome arms between the two species. Based on their high sequence similarity to Drosophila drm and bowl, the Anopheles gambiae orthologs are likely to control similar developmental processes, in particular, gut morphogenesis (Johansen, 2003 and references therein).
odd-like genes are present in mammals and in Ciona intestinalis, a hemichordate. The mouse Osr2 and human OSR1 proteins display 65% and 70% respective identity to Drosophila Odd, Sob, and Bowl over their first three zinc fingers. The Ciona Odd-family protein, with two zinc fingers, has approximately 85% identity to the first and second zinc fingers of Drm, Sob, Odd, and Bowl. Intriguingly, human OSR1 is expressed in the adult colon, the mammalian equivalent to the Drosophila hindgut, and the Ciona homolog shows expression in the esophagus of the young adult. An important question to be addressed, therefore, is whether mammalian and Ciona odd-like genes also play roles in gut development (Johansen, 2003).
The presence of lin-like genes in other genomes suggests that the interaction between odd family members and lin demonstrated for Drosophila may also obtain in other organisms. It is striking that the Lin ortholog in Anopheles is 45% identical overall, and 76% identical over 165 amino acids, to Drosophila Lin. Since each of the four members of the Anopheles odd family has an ortholog in the Drosophila odd family, the epistatic relationships observed between Drosophila drm, lin, and bowl are also likely to obtain in Anopheles. It is proposed that drm, lin, and bowl may play similar roles in Drosophila and mosquito gut development; analysis of expression of the Anopheles orthologs will be an important step toward testing this hypothesis. The human WINS1 and mouse Wins2 proteins show a much lower, although significant, 29 and 27% respective identity to Drosophila Lin. Since these genomes do not contain 1:1 orthologs of each of the odd family members, it is unlikely that there is a drm-lin-bowl pathway in these species. Nevertheless, interactions between Osr1/2 and Lin proteins may very well play important roles in embryonic patterning and morphogenesis (Johansen, 2003).
Genetic evidence shows that lines is required for the function of the Abdominal-B protein. In lines mutant embryos Abdominal-B protein expression is normal but is incapable of promoting its normal function: formation of the posterior spiracles and specification of an eighth abdominal denticle belt. The tail and A8 segment of lin embryos are highly abnormal. The A8 denticle belt is replaced by naked cuticle that occasionally forms a few denticles less pigmented than the normal ventral denticles. This abnormal A8 cuticle does not resemble the cuticle of any region of the wild-type or of the lin mutant embryo. The absence of anal pads and the abnormal hindgut suggests abnormal development of abdominal segment 11, however, other aspects of the tail development are normal, such as the formation of an anal tuft. In lin embryos the sensory organs are formed at roughly correct positions but have an abnormal shape (Castelli-Gair, 1998).
The Abd-B gene directs the formation of the posterior spiracles by controlling downstream target genes. The defects associated with lines mutation arise because in lines mutant embryos the Abdominal-B protein cannot activate its direct target empty spiracles (ems) or other downstream genes, such as cut (ct) and spalt (sal), while it can still function as a repressor of Ultrabithorax and abdominal-A. empty spiracles is one gene required for the formation of posterior spiracles. ems expression in the posterior spiracles is regulated by Abd-B. In lin embryos the transcription of ems is not activated in the posterior spiracles, showing that lin is required for Abd-B to activate its direct downstream target. The other putative Abd-B downstream targets cut and spalt are also required for the normal development of the posterior spiracles. The activation of ct and sal in the anlage of the posterior spiracles requires Abd-B function but their activation remains independent of one another and of ems, suggesting that all three genes are independently controlled by Abd-B. In lin mutants neither ct nor sal are activated in the anlage of the posterior spiracles. These results show that in lin mutant embryos, Abd-B is incapable of activating some of its targets. The requirement of lines for Abd-B function is not a specific property of the A8 segment. In wild-type embryos, ectopic Abd-B expression using the GAL4 targeting system results in the formation of ectopic posterior spiracles in segments anterior to A8. In contrast, ectopic Abd-B expression in lin mutants does not form ectopic posterior spiracles, showing that no matter where the Abd-B protein is expressed in the embryo it requires lines to be fully functional (Castelli-Gair, 1998).
The effect of lin on Abd-B can be explained at the molecular level if lin is required for protein posttranscriptional modification or as a transcriptional cofactor of Abd-B. There is some evidence that the Abd-B protein is posttranslationally modified. If Lin were mediating this process, it would imply that such posttranscriptional modification is functional in vivo. Alternatively if Lines is a transcriptional cofactor of Abd-B, Lines would be interacting with Abd-B in a similar way to that proposed for Extradenticle with Ubx and Abd-A, or Ftz-F1 with Ftz. It is interesting that Exd does not have any effect on Abd-B protein binding or function, and that lin is specific for Abd-B but not for the other Hox genes tested. This suggests that different HOX proteins use different cofactors that contribute to the DNA binding specificity of the HOX proteins (Castelli-Gair, 1998).
The Drosophila embryonic hindgut is a robust system for the study of patterning and morphogenesis of epithelial organs. In a period of about 10 h, and in the absence of significant cell division or apoptosis, the hindgut epithelium undergoes morphogenesis by changes in cell shape and size and by cell rearrangement. The epithelium concomitantly becomes surrounded by visceral mesoderm and is characterized by distinct gene expression patterns that forecast the development of three morphological subdomains: small intestine, large intestine, and rectum. At least three genes encoding putative transcriptional regulators, drumstick (drm), bowl, and lines (lin), are required to establish normal hindgut morphology. The defect in hindgut elongation in drm, bowl, and lin mutants is due, in large part, to the requirement of these genes in the process of cell rearrangement. Further, drm, bowl, and lin are required for patterning of the hindgut, i.e., for correct expression in the prospective small intestine, large intestine, and rectum of genes encoding cell signals (wingless, hedgehog, unpaired, Serrate, dpp) and transcription factors (engrailed, dead ringer). The close association of both cell rearrangement and patterning defects in all three mutants suggest that proper patterning of the hindgut into small intestine and large intestine is likely required for its correct morphogenesis (Iwaki, 2001).
Focusing on hindgut elongation that occurs after stage 10, neither apoptosis nor cell proliferation contribute significantly to the process. Thus, hindgut morphogenesis occurs normally in the apoptosis-deficient DfH99 mutant, and the only cell proliferation occurring in the hindgut after stage 10 is in a small domain at the anterior of the small intestine. The morphogenesis of the hindgut after stage 10, in particular its elongation and narrowing, must therefore be driven by changes in cell size, shape, and rearrangement (Iwaki, 2001).
After the cessation of the postblastoderm mitoses, an endoreplication cycle increases the size of the cells of the large intestine (but not small intestine or rectum). Inhibition of this endoreplication by different genetic manipulations results in a shorter large intestine with a smaller cell size, but roughly normal diameter. Endoreplication thus appears to be required to bring the large intestine to its full length, but not to play a critical role in reducing hindgut diameter. There is a change in cell shape, from columnar to cuboidal, as the hindgut elongates; such a change increases epithelial surface area and thus could contribute to hindgut elongation, but not to a reduction in its diameter (Iwaki, 2001).
The threefold elongation of the hindgut is accompanied by a three- to four-fold reduction of circumferential cell number, but not by appreciable cell proliferation or apoptosis. The major process driving this stereotypic elongation and narrowing must therefore be cell rearrangement. Elongation by cell rearrangement is a morphogenetic process of broad significance: it has been shown to drive gastrulation and embryonic axis elongation, and elongation of various tissues, throughout the bilateria. To date, few molecules required for this process have been identified. Elongation by cell rearrangement of the Drosophila germband, ovarian terminal filaments, and stigmatophore requires the Evenskipped homeodomain, Bric a brac BTB, and Grain GATA proteins, respectively, while that of the C. elegans dorsal epidermis requires the DIE-1 zinc finger protein. The genetic pathways in which these presumed transcriptional regulators function have not yet been determined. Only the Xenopus Brachyury transcription factor has been shown to affect cell rearrangement by controlling expression of a specific target, Wnt11, which acts via the planar cell polarity pathway to orient cell intercalation. A fuller understanding of the molecular basis of oriented cell rearrangement clearly depends on the identification of additional required genes and genetic pathways (Iwaki, 2001).
Since the hindguts of their mutant embryos are shorter and wider than normal, drm, bowl, and lin have been identified as possible regulators of the cell rearrangement that drives hindgut elongation. Analysis of hindgut morphology and gene expression patterns in mutants indicates that drm, bowl, and lin function in hindgut development after the primordium has already been established and internalized by gastrulation. No massive apoptosis in the hindgut (as seen in fkh, cad, and byn) is observed in drm, bowl, or lin hindguts. The number of cells in the hindgut epithelium of drm, bowl, or lin mutants is within 20% of wild type, demonstrating that cell proliferation is roughly normal in these mutants. The byn and fkh genes are expressed normally throughout drm, bowl, and lin hindguts, and otp is expressed throughout drm and bowl hindguts. The hindgut visceral mesoderm, on the basis of its expression of Connectin, appears to be established normally in drm, bowl, and lin mutants. Taken together, these results indicate that early events in hindgut development, namely the establishment and maintenance of the primordium (including initiation of gene activity, and cell proliferation throughout the primordium), its internalization during gastrulation, and its investment with visceral mesoderm, all occur more or less normally in drm, bowl, and lin mutants. The shorter overall length, and the two- to three-fold greater circumferential cell number seen in drm, bowl, and lin hindguts, must therefore be a result of a failure to complete the cell rearrangement that elongates and narrows the wild-type hindgut (Iwaki, 2001).
Patterning of the Drosophila hindgut serves as a microcosm of the complex anteroposterior and dorsoventral patterning that takes place during vertebrate gut development. In the Drosophila hindgut, patterning along the anteroposterior axis gives rise to the small intestine, large intestine, and rectum; patterning along the dorsoventral axis gives rise to the large intestine ventral and large intestine dorsal domains, and the boundary cells. Previous studies described gene expression patterns in the different domains of the developing Drosophila hindgut (as well as the requirement of fkh for these expression patterns), but did not identify any genetic activity that distinguished among or specified the different domains (Iwaki, 2001).
This study shows that drm, bowl, and lin are required for the gene expression patterns that distinguish these three domains: lin is required for expression characteristic of large intestine (dpp, dri, and en) and rectum (Ser, hh, and wg); drm and bowl are required for expression characteristic of small intestine (hh and upd). By both morphological criteria (cell shape, presence or absence of boundary cells) and gene expression patterns (expanded expression of genes expressed in the small intestine), lin hindguts appear to consist of a greatly expanded small intestine and to lack the large intestine and rectum. In contrast, both morphological and gene expression characteristics of drm and bowl hindguts indicate that they lack most or all of the small intestine, and consist only of large intestine (which remains unelongated) and rectum. A model consistent with these data is that lin functions in the hindgut to repress small intestine fate and to promote large intestine and rectum fate, while establishment of the small intestine requires the activity of drm and bowl. The requirement for drm (but not bowl) for wg expression at the most anterior of the hindgut could be explained if the domain of bowl function in the small intestine does not extend to the most anterior of the hindgut (consistent with the expression of bowl). Since they have opposite effects on Ser expression, bowl and drm may function in different ways, possibly in different pathways, to promote small intestine fate (Iwaki, 2001).
The function of lin as both an activator and repressor of gene activity in the developing hindgut is consistent with molecular and genetic characterization of its function in other embryonic tissues. In the developing dorsal epidermis, lin is required for transcriptional regulation (both activation and repression) of targets downstream of wg signaling. In the developing posterior spiracles, lin is required for the activation by Abd-B of its transcriptional targets. lin encodes a novel protein that is expressed globally throughout the embryo, including the developing hindgut. When ectopically expressed, Lin protein is detected in nuclei of cells signaled by Wg. The early expression of wg throughout the hindgut primordium, starting at the blastoderm stage and continuing to stage 10, might, analogous to its effect in the dorsal epidermis, activate Lin. This might be required for Lin to carry out its function, demonstrated here, of promoting expression of genes characteristic of large intestine identity (otp, dpp, en, and dri), and repressing expression of genes characteristic of small intestine identity (hh, upd, and Ser).
It has been shown by genetic analysis that bowl and drm function to establish the small intestine. bowl encodes a zinc finger protein related to Odd-skipped and is expressed strongly in the hindgut primordium starting at the blastoderm stage and continuing through stage 11. Although the Bowl protein has not been shown to be nuclear or to bind DNA, the fact that it encodes five tandem zinc fingers suggests that it is a transcription factor. Thus, Bowl might function in the hindgut as an activator or coactivator of transcription of genes characteristic of small intestine fate. Finally, drm encodes a zinc finger protein related to Bowl and Odd-skipped and is expressed during stage 10 in the anterior of the developing hindgut, consistent with its required role in establishing the small intestine. drm, like bowl, is required for gene expression characteristic of small intestine fate. The drm protein may, like Bowl, function as a transcriptional regulator in the small intestine primordium (Iwaki, 2001).
Thus drm, bowl, and lin are required for both patterning and cell rearrangement of the hindgut. At least one other putative transcriptional regulator expressed in the hindgut has similar properties: Dichaete encodes a Sox protein required for en, hh, and dpp expression in and elongation of the hindgut. The question therefore arises whether any of the genes expressed in different hindgut domains are mediators of the required role of drm, bowl, lin, or Dichaete in hindgut morphogenesis (Iwaki, 2001).
The phenotypes described for wg, hh, dpp, dri, Ser, and en do not suggest a role for these genes in hindgut elongation by cell rearrangement. Mutations in Ser, dri, and en do not appear to affect overall hindgut morphology. The hindgut in wg mutants is extremely small, suggesting that the critical function of wg in hindgut development is in establishing and maintaining the primordium, but not in elongation. dpp mutant hindguts are shorter, consistent with the role of dpp in endoreplication in the large intestine; nevertheless, dpp hindguts have a roughly normal diameter. In hh mutant embryos, the rectum degenerates and hindgut length is reduced, but the overall morphology, particularly the narrowing of the large intestine, appears normal (Iwaki, 2001).
Only in upd embryos is a defect in both elongation and narrowing of the hindgut observed; significantly, upd is expressed only in the small intestine, the same domain that is largely missing from drm and bowl mutants. Shorter and wider hindguts are seen in younger upd embryos, but the majority of hindguts in mature upd embryos appear normal. Thus, while upd may at least partially mediate drm and bowl function in the hindgut, there must be other targets of these genes that are required for cell rearrangement in the hindgut (Iwaki, 2001).
It is concluded that, if correct patterning of the hindgut is a prerequisite for its elongation by cell rearrangement, either all the targets of drm, bowl, and lin that are the essential components of the necessary patterning have not been identified, or the genes presently identified have overlapping or redundant function. Consistent with the idea that cell rearrangement in the Drosophila hindgut requires its correct patterning, convergent extension during vertebrate gastrulation has been shown to depend on patterning of cell fates along the dorsoventral axis of the embryo. It is, of course, possible that hindgut patterning and cell rearrangement, although closely associated both temporally and in the drm, bowl, and lin mutant phenotypes, do not have a necessary relationship to each other. A number of genes are known that, without affecting patterning, control cell rearrangement by directly affecting morphogenetic movements. This is a property of the Drosophila GATA transcription factor-encoding grain in stigmatophore elongation and of the zebrafish trilobite locus in body axis elongation. Thus, drm, bowl, lin, and Dichaete, in addition to patterning the hindgut, might be regulating other genes that independently control cell rearrangement. Nonetheless, the relationship between patterning of both small intestine and large intestine, on the one hand, and cell rearrangement, on the other hand, is striking. drm and bowl hindguts have a substantial cohort of large intestine cells, yet fail to complete cell rearrangement, presumably due to absence of the small intestine. lin hindguts have an excess of small intestine cells and also fail to complete cell rearrangement, presumably due to absence of the large intestine. The connection between hindgut patterning and cell rearrangement observed in drm, bowl, and lin mutants supports the idea that interaction between two correctly patterned anteroposterior subdomains, the small and large intestine, is a requirement for cell rearrangement in the hindgut tubule (Iwaki, 2001).
The pattern defect in dorsal epidermis suggests that lin mutations affect only late-stage Wg signaling. In contrast, in the ventral epidermis, the major lin mutant phenotype does not reflect a defect in Wg signaling. In wild type, the denticle belt is composed of six rows, each with distinct shape, size, and polarity. In lin mutants, the first three rows are replaced with smooth cuticle. Normally, patterning in this portion of the denticle belt is controlled by epidermal growth factor-receptor (EGF-R) and not Wg signaling. Thus, the ventral pattern defect in lin mutants suggest a role for Lin within the EGF-R pathway. Note also that EGF-R function plays no role in patterning the dorsal epidermis, where lin function is linked with Wg signaling. Thus, while in some tissues, lin may cooperate with a different signaling pathway, in dorsal embryonic epidermis, lin is specifically necessary for late, Wg-dependent cell-type specification. Thus, it is hypothesized that lin encodes a factor necessary for stage-specific, Wg-dependent decisions (Hatini, 2000).
There are only subtle changes in hh gene expression in lines mutants, and these occur only after the period during which Hh activity specifies fates. However, lines mutations could affect the domain of Hh protein expression, or increase the sensitivity of cells to Hh activity. In both of these cases the specification of 3° cell fates in lines mutants would still be dependent on Hh activity. To test this, the cuticle phenotype of lines;hh double mutant embryos was analyzed. In embryos carrying only the hhts mutation, no 3° cell types are specified if Hh function is removed after 6 hours AEL (1° and 2° cell types are also absent). However, in the lines; hhts double mutant, it appears that 3° cell types are restored. The 4° cell types are missing and small denticles similar to 3° cell types are produced. Thus, in the absence of lines, 3° cell types are formed independent of hh activity. This implies that hh signaling normally antagonizes the activity of lines and that their regulatory relationship in specifying the 3° cell types is similar to that of hh and ptc in specifying the 1° and 2° fates (Bokor, 1996).
Wg activity is required between 6-9 hours AEL for 4° cell types. Thus, the deletion of 4° cell types observed in lines mutants suggests that lines might normally regulate wg expression or function during this interval. In wild-type embryos between 6 to 9 hours AEL, wg is expressed in a patch of cells dorsally within each parasegment. As the dorsal epidermis stretches over the amnioserosa the patch becomes a stripe. In lines mutants, wg expression is normal through most of the 6-9 hours AEL period. It decays prematurely at about 9 hours AEL, and expression is absent during dorsal closure. It is concluded that lines activity is required to maintain the expression of wg. However, this requirement comes only after the time that wg signaling specifies most of the 4° cells. Thus, the loss of all 4° cell types in lines mutants cannot be attributed to loss of wg gene expression. It remains possible that lines activity is required for Wg signaling. However, the mutant phenotypes are different in that there are extra 3° cell types in lines mutants but not in wg mutants. Thus, lines must be involved in some other non-Wg-dependent process, or must act independent of Wg signaling (Bokor, 1996).
hh and wg act in two apparently exclusive domains of cells across each segment of the dorsal epidermis. By examining how the different cell types are established it has been found that lines plays a significant role at the interface of these two domains, repressing the differentiation of 3° cell types while also promoting the differentiation of 4° cell types. Thus it is important to consider how lines activity might regulate or be regulated by the other two pathways. lines does not simply regulate wg gene expression. It is also unlikely that lines activity mediates wg signaling because the wg and lines phenotypes are different. When wg is inactivated cells die since the segment is much shorter than in wild type. In lines mutants segment length is the same as wild type. Thus, in lines mutants, either 4° cells are transformed into 3° cells, or 4° cells die and 3° cells are stimulated to divide. Regardless of the mechanism, the differences between the lines and wg phenotypes suggest that these two activities are independently required for 4° cell survival and differentiation. For example, late wg input may be essential for cell viability and lines activity may assign cell identity. Another observation argues for independent roles of Wg and lines. In wg null mutants, where both Wg and Hh signaling is lost early, most cells die, but the few surviving cells differentiate as 4° cells. This appears to be due to lines function, since in lines wg double mutants these few cells take on 3° rather than 4° fates (Bokor, 1996).
The role of Hh in patterning involves the selective antagonism of Ptc activity to allow the differentiation of 1° and 2° cell types and the antagonism of lines activity to allow the differentiation of 3° cell types. Thus, lines may define a novel pathway under the influence of Hh signaling. These regulatory relationships leave open the question of what molecule(s) specify the fates of the 1°, 2° and 3° cell types (Bokor, 1996).
Between stages 10 and 11 pannier loses expression in the A8 segment. Expectedly, it is under the control of Abd-B; in Abd-B mutants the gap in A8 does not appear. However, none of the known Abd-B target genes (sal, ems and grn) is involved in the regulation, since their mutations do not affect pnr expression. The finding that lin, which is considered as a co-factor of Abd-B, is involved, suggests that downregulation of pnr in the A8 segment is mediated either by an unknown Abd-B target or directly by interaction between the Abd-B and Lin products. It is not clear why pnr activity has to be eliminated precisely in the A8 segment. This segment gives rise to the spiracles, protruding structures that are very different from those differentiated by the other abdominal segments where pnr remains active. In fact, there are several Abd-B target genes specifically activated in the spiracles. It is possible that the formation of these structures demands that the pnr activity, which specifies larval epidermis of very different morphology, be turned off (Heranz, 2001).
Central to embryonic development is the generation of molecular asymmetries across fields of undifferentiated cells. The Drosophila wing imaginal disc provides a powerful system with which to understand how such asymmetries are generated and how they contribute to formation of a complex structure. Early in development, the wing primordium is subdivided into a thin layer of peripodial epithelium (PE) and an apposing thickened layer of pseudostratified columnar epithelium (CE), known as the disc proper (DP). The DP gives rise to the wing blade, hinge and dorsal mesothorax, whereas the PE makes only a minor contribution to the ventral hinge and pleura. The mechanisms that generate this major asymmetry and its contribution to wing development are poorly understood. The Lines protein destabilizes the nuclear protein Bowl in ectodermal structures. This study shows that Bowl accumulates in the PE from early stages of wing development and is absent from the DP. Broad inhibition of Bowl in the PE resulted in the replacement of the PE with a mirror image duplication of the DP. The failure to generate the PE severely compromised wing growth and the formation of the notum. Conversely, the activation of bowl in the DP (by removal or inhibition of lines function) resulted in the transformation of the DP into PE. Thus, this study provides evidence that bowl and lines act as a binary switch to subdivide the wing primordium into PE and DP, and assigns crucial roles for this asymmetry in wing growth and patterning (Nusinow, 2008).
The wing PE can be identified molecularly and morphologically as a thin epithelial sheet overlying the thickened DP epithelium. Mapping studies show that the distribution of Bowl and Lines correlates with the establishment of this asymmetry. The wing primordium inherits its subdivision into en-expressing cells that form the posterior compartment and adjacent anterior compartment cells from the embryonic epidermis. Bowl accumulates in the posterior en-expressing cells in the embryonic epidermis, suggesting that the wing primordium also inherits the PE/DP subdivisions from pre-existing asymmetries across this tissue (Nusinow, 2008).
lines and odd-skipped genes act as a switch to specify alternative cell fates across fields of cells. bowl and lines specify the alternative 1°-3° and 4° cell fate across the dorsal embryonic epidermis. bowl and lines also specify alternative cell fates in the developing gut, leg and eye imaginal discs. The asymmetric distributions of Bowl and odd-skipped genes, and the reciprocal distribution of Lines in the wing primordium are also used to specify the alternative PE and DP fates. Indeed, functional studies show that ectopic lines expression or the inhibition of bowl function in the PE transforms PE into DP fate. Reciprocally, the removal of lines function from the DP transforms DP into PE fate. The data further suggest that lines exerts its function by controlling the stability of the Bowl protein. Thus, lines and bowl act as a switch to specify alternative DP and PE fates across the wing primordium. The distribution of Lines and Bowl correlates with the subdivision of the wing primordium into a thin squamous and a thickened columnar epithelial sheet. The activation of EGF receptor and Wg signaling in the DP may specify the formation of a columnar epithelial morphology. The pathways that specify the squamous morphology of the PE downstream to bowl remain to be elucidated (Nusinow, 2008).
Previous studies that relied on surgical and genetic ablations of the PE, and on inhibition of certain signaling pathways within the PE, suggested important roles for the PE in disc growth and patterning. It is now possible to examine wing development in discs lacking PE. These discs were significantly smaller than wild type, and the notum was dramatically reduced in size relative to the pouch and hinge. Progenitor cells that originate in the PE may stream laterally to populate the growing notum, and the loss of this progenitor cell population may account for the severe reduction in notal growth. The reduction in wing growth could have resulted from the disruption of Wg or Dpp signaling activities, as these morphogens control cell survival and cell proliferation in the wing. Indeed, a block to Dpp or Wg signaling results in formation of tiny wing rudiments. However, the expression of Wg and Dpp and their target genes was normal in these discs, indicating that the reduction in wing growth was not a consequence of the loss of wg or dpp expression, or of signaling activities. These findings instead suggest that the PE acts in parallel to the AP, DV and PD patterning systems, in part, by promoting cell survival in the DP, and in part by promoting the growth of the notum (Nusinow, 2008).
The results presented in this study argue that lines and bowl function as field-specific selector genes to specify the identity and the behavior of the DP and the PE of the wing primordium, respectively. Selector genes control cell fate, cell affinity and the competence to send and respond to patterning signals in a cell autonomous manner. lines and bowl act cell autonomously to control the fate, affinity and the interaction between the PE and the DP. As a putative transcription factor, Bowl may regulate a developmental program to control the identity and the behavior of the PE. By inhibiting Bowl accumulation, lines may allow the execution of an alternative developmental program in the DP. These studies define a new system to identify, in a systematic way, these developmental programs (Nusinow, 2008).
The regulatory Lines/Drumstick/Bowl gene network is implicated in the integration of patterning information at several stages during development. This study shows that during Drosophila wing development, Lines prevents Bowl accumulation in the wing primordium, confining its expression to the peripodial epithelium. In cells that lack lines or over-expressing Drumstick, Bowl stabilization is responsible for alterations such as dramatic overgrowths and cell identity changes in the proximodistal patterning owing to aberrant responses to signaling pathways. The complex phenotypes are explained by Bowl repressing the Wingless pathway, the earliest effect seen. In addition, Bowl sequesters the general co-repressor Groucho from repressor complexes functioning in the Notch pathway and in Hedgehog expression, leading to ectopic activity of their targets. Supporting this model, elimination of the Groucho interaction domain in Bowl prevents the activation of the Notch and Hedgehog pathways, although not the repression of the Wingless pathway. Similarly, the effects of ectopic Bowl are partially rescued by co-expression of either Hairless or Master of thickveins, co-repressors that act with Groucho in the Notch and Hedgehog pathways, respectively. It is concluded that by preventing Bowl accumulation in the wing, primordial Lines permits the correct balance of nuclear co-repressors that control the activity of the Wingless, Notch and Hedgehog pathways (Benítez, 2009).
The Drosophila wing is a discrete organ that has been used to study the coordination of signaling pathways during development. The developing wing disc is a sac-like structure composed of the columnar epithelium or disc proper cells (DP), the cuboidal marginal cells (MC) and the overlying squamous cells (SC); MC and SC constitute the peripodial epithelium (PE). During larval development, imaginal cells proliferate extensively and are patterned. After metamorphosis, the DP cells differentiate into the cuticle that forms the adult wing and notum, whereas PE cells contribute little to these structures (Benítez, 2009).
The Lin/Drm/Bowl cassette is emerging as an important molecular mechanism with which to coordinate various pathways in different developmental contexts. In all cases, the steady-state accumulation of Bowl is regulated by the relative levels of Drm and Lin proteins. High levels of Drm impede binding of Lin to Bowl and, thus, this transcriptional repressor becomes stabilized in the nucleus. In this study it was found that regulatory interaction Lin/Drm/Bowl also functions during wing development. In lin- or Drm GOF cause ectopic expression of Bowl and dramatic overgrowths within the wing disc. These overgrowths frequently showed altered cell identity, resembling more proximal disc margin cells. Some of the effects can be explained by the ability of Bowl to interact with Gro co-repressor through the eh-1 motif, forming a complex that sequesters Gro from other repressors complexes such as Su(H)/H/Gro and Mtv/Gro (Benítez, 2009).
Although Bowl is ubiquitously transcribed in the wing disc, Bowl protein is present only in the SC and MC, being normally absent from the DP cells. The spatial distribution of nuclear Bowl is dependent on Drm, which causes Lin to relocalize to the cytoplasm. Drm is absent from most of the DP cells and, therefore, Lin turns down the steady-state accumulation of Bowl protein in these cells. In the absence of Lin, Bowl accumulates in the DP cell nuclei and elicits the dramatic alterations observed in lin- mutant cells. Therefore, the main function of Lin is to prevent Bowl accumulation in the DP cells, restricting Bowl protein to MC and SC of the PE (Benítez, 2009).
The main alterations in lin-, Drm GOF or Bowl GOF clones can be classified according to the signaling pathways temporally affected. The earliest defect observed is the repression of Wg pathway responses and the evidence suggests that Bowl functions as a repressor of the Wg pathway. However, activated forms of nuclear Wg pathway components, such as ArmS10 or dTcf, cannot restore the expression of the proximal-distal markers owing to repression of the Wg targets in lin-, indicating that Bowl must act in parallel to or downstream of Arm and dTcf (Benítez, 2009).
Bowl is a zinc-finger protein that can interact with the co-repressor Gro directly through the eh-1 motif. The results indicate that this mechanism is also important under conditions where Bowl accumulates in the wing disc. Most of the alterations observed in lin- or Drm GOF clones can be explained by Bowl sequestering Gro from other repression complexes (causing activation of N targets and Hh). Several results support this model. First, the strong genetic interaction between lin and gro alleles, where trans-heterozygous combinations between lin and gro alleles result in dramatic phenotypes, argue that Gro is a limiting factor. Second, removal of eh-1 motif that recruits Gro, eliminates the effects of Bowl on the Hh and N pathways. Third, ectopic expression of Gro, H or Mtv partially suppress the phenotypes of ectopic Drm or Bowl. These observations imply a 'tug of war' between Bowl, H and Mtv for Gro. Increased H or Mtv would shift the balance back in favor of N target repression and Hh repression (Benítez, 2009).
By contrast, the repression of Wg pathway observed in lin- cells appears to involve a different mechanism. Although the effect is Bowl dependent, repression of Wg targets also occurs with Bowleh1-, indicating that Gro sequestration is not required. Similarly, co-expression of Bowl with H or Mtv cannot re-establish the repression of the Wg targets. These results show that Bowl is able to repress Wg targets independently of Gro and the observation that Bowleh1- VP16 can cause some ectopic expression of Sens suggests that this may involve a direct effect of Bowl on Wg targets (Benítez, 2009).
Wnt/Wg, N and Hh signaling represent major conserved signaling channels to control cell identity and behavior during development. An antagonistic interaction between the Wg and Hh has also been described in the embryo and at the intersection of the D/V and A/P compartment borders of the wing disc. Similarly, Wnt/Wg and N activities are closely entangled in many different systems. Mutual dependent interactions between N and Wnt signaling have been observed in vertebrate skin precursors, in rhombomere patterning and in somitogenesis. It has also been reported that orthologues of the Odd-skipped family, Osr1 and Osr2, function as transcriptional repressors during kidney formation. It is possible therefore that Lin/Bowl/Gro interaction is evolutionary conserved and it will be interesting to discover whether lin is an important regulatory factor in other systems (Benítez, 2009).
By analyzing lin- clones in the wing primordium, this study has uncovered the consequences of stabilizing Bowl in the DP cells. There are, however, two regions where Bowl accumulates normally, in the MC and SC within the PE. Removal of Bowl in the PE might lead to ectopic Wg protein and thus to ectopic activity of the Wg signaling to transform PE from squamous to columnar cells. In this context, recently, it has shown that Bowl inhibition by ectopic expression of Lin results in the replacement of the PE by a mirror image duplication of the DP cells. However, not much alteration has been observed in cell morphology nor in the expression of markers such as Ubx or Hth when Bowl was depleted in PE cells (bowl- clones and UAS-BowlRNAi). It could be that the recovered bowl- clones were not induced early enough or that the levels of Bowl-RNAi were not sufficient to completely eliminate the Bowl function in these cells. Nevertheless, these manipulations revealed that bowl- phenotypes in the proximal wing and notum are consistent with a functional role in MC. Therefore, it is concluded that Lin has an important role in restricting Bowl to the MC (and PE), delimiting a Bowl-free territory that forms the DP cells and enables their responsiveness to key signaling pathways such as Wg (Benítez, 2009).
To function properly, tissue-specific stem cells must reside in a niche. The Drosophila testis niche is one of few niches studied in vivo. Here, a single niche, comprising ten hub cells, maintains both germline stem cells (GSC) and somatic stem cells (cyst stem cells, CySC). This study shows that lines is an essential CySC factor. Surprisingly, lines-depleted CySCs adopted several characteristics of hub cells, including the recruitment of new CySCs. This led to an examination of the developmental relationship between CySCs and hub cells. In contrast to a previous report, no significant conversion was seen of steady-state CySC progeny to hub fate. However, it was found that these two cell types derive from a common precursor pool during gonadogenesis. Furthermore, embryos mutant for lines, an obligate antagonist of bowl function (Hatini, 2005), exhibited gonads containing excess hub cells, indicating that lines represses hub cell fate during gonadogenesis. In many tissues, lines acts antagonistically to bowl, and it was found that this is true for hub specification, establishing bowl as a positively acting factor in the development of the testis niche (Dinardo, 2011).
This analysis together with previous lineage-tracing shows that some hub cells and some CySCs are derived from the SGPs of PS11. The remaining CySCs could in principle derive from either PS10 or PS12. Currently, neither of those mesodermal parasegments can be uniquely lineage traced. However, the remaining hub cells probably derive from PS10 SGPs, as that would fit with the identification of receptor tyrosine kinase signaling as an antagonist of hub fate among posterior SGPs (Dinardo, 2011).
Aside from pathways known to repress hub fate, work is also beginning to identify positive functions necessary to specify these cells. This study found that bowl is one factor, as mutants had fewer hub cells, and those present appeared compromised for hub cell function. Still, the existence of residual hub cells suggests that Bowl is not the only factor required for hub cell specification, and, indeed, Notch signaling is a second positively acting component (Dinardo, 2011).
It is of interest that both Notch and bowl are positively required for hub cell specification, since these two genes act together in several other tissues. However, the exact epistatic relationship between bowl and the Notch pathway can be complex. There is some evidence that Notch activation leads to Bowl accumulation. Since it was found that Notch and also the relief-of-repression hierarchy consisting of drm/lines/bowl acts during hub cell specification, a simple model would be that Notch activation induces an antagonist of lines, for example, drm. This allows Bowl protein to accumulate in a subset of SGPs and to promote hub fate, while SGPs that retain functional Lines would adopt CySC fate. Attractive as this model is, testing some of its predictions was difficult. Attempts to visualize endogenous protein accumulation for Bowl and for Lines in the gonad has been frustrating. In addition, although drm mutants had reduced hub cell number, drm-expressing cells have not been identified within the forming gonad (Dinardo, 2011).
Thus, the relationship between Notch and the drm/lines/bowl cassette may be indirect, an outcome of the fact that both systems use the co-repressor Groucho. It has been suggested that conditions which alter the levels of available Bowl, such as in drm (down) or lines (up) mutants, could reciprocally affect the amount of Groucho available to Suppressor of Hairless, which requires this co-repressor to maintain repression of Notch target genes. Whether or not the relationship between Notch and Bowl for hub cell specification is direct, loss of Notch has a stronger phenotype than loss of bowl. Thus, the Notch pathway must also engage a separate pathway that specifies some hub cells (Dinardo, 2011).
During gonadogenesis, the current model suggests that Lines represses hub fate and promotes CySC fate. It is intriguing that a requirement for lines persists in CySCs during the steady-state operation of the testis. Analysis at this later stage suggests that lines plays a similar, though not identical, role. Although cells in gonads from lines mutant embryos fully adopt hub cell fate, in the testis the lines-depleted CySCs only partially adopt hub fate, as they do not recruit new GSCs. Thus, at steady-state, some additional regulation over the distinction between CySC and hub cell fate has been added on. Such a factor(s) remain to be identified (Dinardo, 2011).
Even the partial conversion of lines mutant CySCs into hub cells is an intriguing phenotype. Recently, a lineage relationship has been described for several stem cell-niche pairs, where stem cells can generate cells of their niche. These include production of Paneth cells in the mammalian intestine, the production of transient niche cells in the fruitfly intestine, and the repair of ependymal cells by neural progenitors of the sub-ventricular zone. In the steady-state testis, it was recently suggested that CySCs can efficiently generate new hub cells. Thus, it is considered whether lines might be deployed at steady state to govern this transition, but no increase was detected in conversion in flies with decreased lines gene dose. In fact, in wild type it was found that the conversion of cells into hub fate was insignificant compared with what has been reported. As one method used in this study was essentially identical to one used in the original report, the reason for the discrepancy is uncertain. Lineage-marking was very efficient. For example, two days after delivery of FLP by one heat-shock, 85% of testes possessed a labeled CySCs, with an average of 1.5 CySCs per testis. In the previous report, a similar regimen produced only 13% of testes with labeled CySCs. Still, it is not clear how an increase in marking efficiency could account for a decrease in apparent frequency of conversion of CySC progeny into hub cells (Dinardo, 2011).
Thus, since CySCs do not normally generate hub cells, why might lines function be maintained in CySCs so long after its embryonic requirement? The favored model is that lines is deployed during steady-state for a distinct purpose. For example, recent work on the lines/bowl cassette suggests that it assists in signal integration. This idea is appealing as the niche cells and their local environment are subjected to the action of a number of signaling pathways, such as Hh, Wnt, BMP, Jak/STAT and EGFR. Currently, it is not fully understood how these pathways function in the steady-state operation of the niche, nor how signals from distinct pathways integrate to produce a single outcome. Even the dogma of the heavily studied Jak/STAT pathway continues be challenged and refined by recent data. Perhaps as newer data uncovers the nuanced roles of several of these pathways, the lines/bowl cassette will figure into the integration of those signals (Dinardo, 2011).
Finally, the fact that lines-depleted CySCs recruited neighboring wild-type somatic cells to adopt CySC fate is striking. Although the imaging tools necessary to reveal which somatic cells are recruited to CySC fate are unavailable, the fact of their recruitment suggests that under these mutant conditions cyst cells can de-differentiate into CySCs. It has been elegantly shown that maturing germ cells can de-differentiate, creating new GSCs. As those maturing germ cells are encysted by the somatic cyst cells, during de-differentiation this grouping must break apart to release individual germline cells that repopulate the niche. Whether cyst cells de-differentiate to CySCs in these cases has not been directly assessed. If this happens under physiological conditions, it would be of great interest to study how cyst cells de-differentiation occurs, and testes harboring lines-deficient clones might aid in such studies (Dinardo, 2011).
The growth and patterning of Drosophila wing and notum primordia depend on their subdivision into progressively smaller domains by secreted signals that emanate from localized sources termed organizers. While the mechanisms that organize the wing primordium have been studied extensively, those that organize the notum are incompletely understood. The genes odd-skipped (odd), drumstick (drm), sob, and bowl comprise the odd-skipped family of C2H2 zinc finger genes, which has been implicated in notum growth and patterning. This study shows that drm, Bowl, and eyegone (eyg), a gene required for notum patterning, accumulate in nested domains in the anterior notum. Ectopic drm organized the nested expression of these anterior notum genes and downregulated the expression of posterior notum genes. The cell-autonomous induction of Bowl and Eyg required bowl, while the non-autonomous effects were independent of bowl. The homeodomain protein Bar is expressed along the anterior border of the notum adjacent to cells expressing the Notch (N) ligand Delta (Dl). bowl was required to promote Bar and repress Dl expression to pattern the anterior notum in a cell-autonomous manner, while lines acted antagonistically to bowl posterior to the Bowl domain. These data suggest that the odd-skipped genes act at the anterior notum border to organize the notum anterior–posterior (AP) axis using both autonomous and non-autonomous mechanisms (Del Signore, 2012).
In many developmental processes, signals that emanate from field borders play a crucial instructive role in patterning morphogenetic fields. The early Drosophila embryo is patterned by opposing gradients of Bicoid and Nanos that are generated from localized translation of corresponding mRNAs at the anterior and posterior poles of the embryo. In the embryonic epidermis, the pattern of cell differentiation across each segment is regulated by the secreted Wg and Hh signals that emanate from localized sources at the anterior and posterior borders of each segment. Similarly, the dorsoventral axis of the vertebrate spinal cord is organized by Shh ventrally, and BMP and Wnt signals that emanate from localized dorsal sources. By contrast, current models of notum AP patterning focus mainly on the organizing influence of Dpp, which is secreted from the posterior border of the notum. Previous work has found that odd-skipped genes are expressed along the anterior border of the notum, and that broadly inhibiting their function in early wing discs caused a severe reduction or complete loss of the notum. As this reduction occurred despite the maintenance of dpp expression (Nusinow, 2008), whether the odd-skipped genes might define a second organizing center within the developing notum was investigated. The current findings indeed suggest that signals that emanate from the anterior border of the notum act reciprocally to Dpp to promote expression of anterior notum genes and repress expression of posterior genes. Through loss- and gain-of-function clonal analyses, it was demonstrated that the odd-skipped genes pattern the notum AP axis both locally through regulation of Eyg, Bar, and Dl, and broadly through the regulation of Eyg and Tup. Finally, it was shown that lines acts antagonistically to bowl in this process (see Model of the role odd-skipped genes in notum AP patterning) (Del Signore, 2012).
drm overexpression was sufficient to promote Eyg accumulation non-autonomously within the notum. This activity suggests that drm controls expression of an unidentified signal that spreads from the drm domain to induce Eyg accumulation non-autonomously. Alternatively, drm could initiate the propagation of a cascade of local inductive interactions to induce Eyg at a distance. Recent studies have shown that recruitment of cells to the wing field is accomplished by the propagation of a feed forward signal from the DV compartment boundary. In this process signaling at the border between Vestigial (Vg) and non-Vg expressing cells is used to recruit non-Vg expressing cells to the expanding wing field, a process dependent on signaling through the Fat-Dachsous pathway. Though a functional relationship between odd-skipped genes and Ft-Ds signaling has yet to be characterized, it is interesting to note that Ds accumulates in a complex graded AP pattern across the notum, consistent with such a role (Del Signore, 2012).
In addition to the broad induction of Eyg accumulation, it was surprising to find that drm overexpression also induced Bowl in cells just adjacent to clones. Though the effect was subtle, it is noted that this pattern of activation recapitulated the endogenous nested pattern of drm and Bowl expression in the presumptive prescutum. It is unclear whether the nested expression of odd-skipped genes plays a functional role in notum AP patterning. Despite this, the concordance of endogenous and ectopic expression patterns supports the hypothesis that ectopic drm induces a physiologically relevant program of anterior gene expression in the notum. One possible clue as to the relevance of this nested pattern may come from the observation that only drm was able to promote Bowl non-autonomously. In contrast, lines−/−, odd+, and sob+ clones each induced only cell-autonomous accumulation of Bowl. Notably, these clones rounded up and segregated from the epithelium, while drm expressing clones remained integrated with the surrounding epithelium. One interpretation of these data is that abrupt discontinuities in the level of Odd-skipped proteins may alter epithelial morphology. This interpretation is further supported by the observation that bowl mutant clones within the Bowl domain adopt a compact, round morphology relative to clones outside the Bowl domain. It is hypothesized that drm promotes lower levels of Bowl in nearby cells to dampen otherwise sharp discontinuities in Bowl activity to regulate local buckling of the epithelium (Del Signore, 2012).
Alternatively, differences in the total levels or ratios of Odd family proteins along the anterior border of the notum could elicit different transcriptional outcomes. Since Odd and Bowl have been shown to interact with the transcriptional co-repressor Groucho, variation in the levels of the Odd-skipped proteins could titrate Groucho and affect Groucho-dependent transcriptional outputs. Alternatively, given their distinct structure outside the zinc finger domain, the Odd-skipped proteins could interact with distinct sets of target genes to pattern the anterior border of the notum. Though additional experiments will be required to ascertain whether such mechanisms are active in the prescutum, this study provides evidence that bowl is strictly required for the early autonomous induction of Eyg, the later expression of Bar genes, and the repression of Dl. These results provide evidence that odd-skipped genes act both independently and redundantly to organize the notum AP axis (Del Signore, 2012).
bowl is essential for patterning the prescutum, but not for broadly patterning the notum AP axis. Previous studies have revealed a variety of essential and redundant functions for odd-skipped family genes in patterning embryonic and larval tissues. In the embryo, drm and bowl antagonize lines function to pattern the dorsal embryonic epidermis, foregut, and hindgut, while odd functions as a pair rule gene to promote embryonic segmentation. In the leg imaginal disc, bowl is essential for patterning the tarsal proximodistal axis at early stages, but acts redundantly with other odd-skipped genes to control leg segmentation later in developmen. In the eye, bowl is essential for the initiation of retinogenesis from the eye margin, while odd and drm have been proposed to activate Bowl redundantly (Del Signore, 2012).
Loss-of-function analysis revealed that neither drm nor odd is necessary to stabilize Bowl. At present the possibility cannot be excluded that sob is necessary to promote Bowl accumulation because a null sob mutant is not yet available. Biochemical and genetic analysis demonstrates that not only Drm, but also Odd and Sob can each outcompete the interaction of Lines with Bowl and stabilize the Bowl proteins in S2 cells and in vivo. These results suggest that different combinations of Odd-skipped proteins could be used to activate bowl depending on context (Del Signore, 2012).
Previous work suggested reciprocal roles for lines and odd-skipped genes in subdividing the early wing disc into disc proper and peripodial epithelium. The loss-of-function analysis described in this study suggests that the odd-skipped genes act redundantly to control the early specification of the PE and the subsequent expansion of the notum, while revealing an essential role for bowl in specification of the anterior prescutum. Redundancy can increase the robustness of essential developmental processes and provide a buffer against fluctuations in activity of single genes. The redundant role of the odd-skipped genes in PE specification and notum expansion could therefore serve to ensure the optimal growth of the wing disc at early stages and that of the notum at later stages and protect these critical processes from perturbations (Del Signore, 2012).
It is concluded that the growth and patterning of the wing field are coordinated with the elaboration of the wing PD axis. The developing notum lacks an obvious PD axis, and instead is subdivided into a series of AP and mediolateral domains. The establishment of organizers that act antagonistically from opposing field borders is a robust strategy to subdivide the notum AP axis. This work demonstrates that the odd-skipped genes act autonomously at the anterior border of the notum to specify the prescutum, and non-autonomously at short and long range to control the expression of transcription factors that prefigure the differentiation of the notum AP axis. Though further experiments will be required to characterize the mechanism by which this putative organizer acts, these studies provide evidence that the anterior border of the notum exhibits the functional attributes of an organizer (Del Signore, 2012).
The Drosophila wing imaginal disc is a sac-like structure that is composed of two opposing cell layers: peripodial epithelium (PE, also known as squamous epithelia) and disc proper (DP, also known as pseudostratified columnar epithelia). The molecular mechanism of cell morphogenesis has been well studied in the DP but not in the PE. Although proper Dpp signalling activity is required for proper PE formation, the detailed regulation mechanism is poorly understood. This study found that the Dpp target gene spalt (sal) is only expressed in DP cells, not in PE cells, although pMad is present in the PE. Increasing Dpp signalling activity cannot activate Sal in PE cells. The absence of Sal in the PE is essential for PE formation. The ectopic expression of sal in PE cells is sufficient to increase the PE cell height. Down-regulation of sal in the DP reduced DP cell height. It was further demonstrated that the known PE cell height regulator Lines, which can convert PE into a DP cell fate, is mediated by sal mis-activation in PE because sal-RNAi and lines co-expression largely restores PE cell morphology. By revealing the microtubule distribution, it was demonstrated that Lines- and Sal-heightened PE cells are morphologically similar to the intermediate cell with cuboidal morphology (Tang, 2016).
The wing disc is a sac-like structure composed of PE, DP, and intermediate cells linking the PE and DP. To investigate the potential role of Dpp signalling in PE morphogenesis, the distribution of Dpp signalling activity in late 3rd instar (L3) wing imaginal discs was revealed in both the x-y and x-z views. Using an antibody of phospho-Mothers against dpp (pMad) to reveal Dpp signal transduction activity, it was found that Dpp signal transduction was ubiquitously present in both the PE and DP. The pMad level was relatively reduced in the central PE compared with the central DP. Dpp target gene expression patterns were detected in the PE. The main Dpp target genes are brinker (brk), omb, and sal in the L3 wing discs. brk was transcribed in both the PE and DP, with a relatively weaker level in the PE, as indicated by a brk-lacZ reporter. However, both omb and sal were transcribed only in the DP, not in the PE. These data indicate that the Dpp target genes omb and sal are asymmetrically expressed in the PE and DP. Brk is also a repressor of other Dpp target genes, including sal and omb, and thereby restricts their expression domains to the medial DP region. The presence of Brk in the PE might be a direct cause of the absence of Omb and Sal in the PE. To assess this possibility, brk-RNAi was expressed in the PE. Sal expression was not detectable in the central PE. The efficiency of brk-RNAi was demonstrated by the elevation of Brk targets Omb and Sal in lateral wing discs of C765>brk-RNAi. To further confirm that Dpp signalling cannot induce sal expression in the PE, a constitutive active form of the Dpp receptor tkvQD was expressed in the PE. Sal was not induced in central PE. When tkvQD clones were generated, Sal was induced only in clones within the DP and not in clones located in the PE). Similarly, Omb was not induced in clones located in the PE. Ubiquitous expression of tkvQD failed to induce Omb in the PE. These data demonstrate that Dpp signalling cannot activate omb and sal in PE (Tang, 2016).
The expression patterns of Dpp target genes are well studied in wing DP. Dpp controls target genes (sal and omb) expression indirectly through repression of the transcriptional repressor Brk. Dpp target gene expression patterns have not been studied in wing PE to date. The results revealed that pMad is ubiquitously present in both DP and PE. However, brk-lacZ was still present in PE. Either suppressing brk or elevating Dpp signalling by expressing tkvQD cannot induce Sal and Omb in the PE. Except for Lin, other factors, such as Bowl, Wg, and EGFR, cannot induce Sal in the PE. The factors that suppress sal in the PE require further investigation (Tang, 2016).
As omb and sal are expressed only in the DP, not the PE, it was therefore asked whether this asymmetric transcription of omb and sal is essential for correct PE formation. To test this possibility, sal was mis-expressed in the PE using the Gal4-UAS system. dpp-Gal4 line is expressed in narrow stripes in the lateral PE and in the middle DP. When sal was ectopically expressed in the dpp-Gal4 domain, the height of lateral PE was notably elongated to a height similar to that of intermediate cells. Then, sal was expressed driven by C765-Gal4, which is ubiquitously expressed in both DP and PE. A similar elongation phenotype was observed in the PE. The cell height of the central PE was elongated to a height similar to that of cuboidal cells. The extent of elongation as a result of dpp-Gal4 was stronger than that of C765-Gal4. This difference might be due to the differences in Gal4 activity because dpp-Gal4 is stronger than C765-Gal4. The quantification of cell height, using the ratio between PE and DP cells within one wing disc, revealed a significant increase in sal mis-expression discs. Consistently, the PE height ratio between sal mis-expression and control also revealed a significant increase. To confirm this result, sal over-expression clones were generated in PE. From the x-z cross view, the clonal height was apparently elongated to a height similar to that for cuboidal cells. Therefore, it is concluded that sal is sufficient to elongate PE height (Tang, 2016).
Unlike the effect of sal mis-expression, the elongation of PE height in case of omb ectopic expression was not apparent, however, the differences in the PE/DP ratio and normalized PE height for C765>omb wing discs are statistically significant. Strong overexpression of omb induces severe extrusion and basal delamination, and cell motility can thicken the wing disc. Although a relatively weaker UAS-omb line was used, the side effect from cell movement may remain, thus leading to the statistic difference in the PE measurement. A previous report demonstrates that if Dpp signalling is suppressed in the PE by Ubx-Gal4 driven dad, a portion of the PE cells are elongated to a cuboidal shape. Therefore, suppressing Dpp signalling in the PE and expressing sal in the PE exhibit similar effects. When carefully assessing the Ubx-Gal4 expression domain, a portion of the expressing cells were, surprisingly, located in the DP. Thus, a non-autonomous effect from a loss of Dpp signalling in the DP in the Ubx>dad wing disc is reasonable. Because when dad-expressing clones were generated within PE, the height of PE did not increase. When Omb-RNAi was driven by hh-Gal4 which is expressed in PE and the posterior compartment of DP, the posterior DP height was reduced. Interestingly, the height of opposite PE was increased. Thus, it is possible that there is a connection between DP and PE during cell morphogenesis. To directly confirm the non-autonomous effect on PE elongation, the DP height was shortened by expression of either dad or brk within the DP specific nub-Gal4 domain. Consistently, the PE height was apparently increased (Tang, 2016).
Previous studies have revealed that mis-expressing lin induces ectopic sal expression in the PE. Thus, sal may mediate lin’s role in PE elongation. First, the experiment of lin mis-expression in the PE was repeated and the elongation phenotype was consistently observed in the PE. Then, the transcription state of sal was revealed using a sal-lacZ reporter. sal was apparently transcribed in the PE. The sal gene complex is composed of two functionally redundant genes: spalt major (salm) and spalt-related (salr). A rescue experiment was performed by co-expressing lin and salm-RNAi. The morphology of the wing imaginal discs was rescued to an approximate normal state, and PE height was no longer elongated. The cell heights of the corresponding genotypes were also measured. sal down-regulation largely rescued the abnormal cell height induced by lin mis-expression. These results indicate that sal mediates the role of lin in promoting PE elongation. However, Lin elongated PE to a greater extent than Sal did, according to the statistic measurements. Other mediators may be involved downstream of Lin. Since that Ubx-Gal4 line is also expressed in part of the DP cells, potential non-autonomous effects between DP and PE can not be ruled out (Tang, 2016).
Given that the mis-expression of sal in the PE elongates cell height, whether down-regulating Dpp-Sal signalling in DP is sufficient to shorten the DP was assessed. nub-Gal4 is only expressed in the wing pouch region in the DP. When Dpp signalling was mildly inhibited by expressing a dominant negative form of the Dpp receptor, tkvDN, in the nub-Gal4 domain, DP cell height was reduced. Unlike the strong inhibition of Dpp signalling by expressing dad, the non-autonomous effect on PE height was not apparent in nub>tkvDN. The Dpp signalling activities in discs of nub>tkvDNand nub>dad were revealed by anti-Sal staining. The DP height was slightly reduced when sal was down-regulated either by salm-RNAi or salr-RNAi; however, the extent of this reduction was weaker than that of tkvDN. Then, sal mutant clones were generated, marked by the loss of GFP in the DP. The intensity of F-actin labelled by Phalloidin was much stronger in the clone regions. The x-z cross-section showed that the apical side of sal mutant clones in the DP was retracted toward the basal side. These data suggested that Dpp-Sal signalling is required to maintain DP elongation. Interestingly, a similar retraction phenotype was also observed in sal-overexpressing clones. Therefore, both sal loss- and gain-of-function clones induce an apical retraction phenotype in the DP. This phenotype is observed in both omb loss- and gain-of-function clones in the DP. Omb exhibits a graded distribution in the DP along the A/P axis and specifies unknown apically distributed adhesion molecules. A continuous Omb level is essential for maintaining the epithelial integrity of the wing disc. Therefore, sharp discontinuity in either Omb or Sal levels in the DP induces apical retraction of cells. To confirm this conclusion, a sharp discontinuity of Sal was generated in the DP using dpp-Gal4 driven UAS-sal. Sal continuity was disrupted at the A/P boundary and where deep apical folds were formed. The expression domain of sal in the DP is narrower than that of omb and vg. Beyond the sal domain, omb and vg can ensure the correct cell morphogenesis in the DP. Clones lacking Vg function are also extruded from the DP layer (Tang, 2016).
Microtubule cytoskeleton is polarized during cell morphogenesis in the wing imaginal disc. To reveal microtubule-based cytoskeleton changes induced by either lin or sal mis-expression and the microtubule dynamics during normal development, the microtubule level was monitored via antibody staining. In the 2nd instar, all cells were cuboidal shape, and microtubules were uniformly distributed. During the early 3rd instar, the cell shape begins to differentiate. PE cells were largely shortened, whereas DP cells were remarkably elongated. Correlating with DP elongation, the microtubule network was asymmetrically enriched to the apical side of the DP. When lin was mis-expressed in the PE by Ubx-Gal4, sal was activated in the PE. Both direct and indirect sal expression induced PE height elongation with an even microtubule distribution. The microtubule levels of PE were increased compared with the wild type PE. The microtubule distribution in Sal-elongated PE was similar to that in very lateral PE and intermediate cells in the L3 stage or undifferentiated cells in earlier larval stages. In the rescue experiment, lin and salm-RNAi were co-expressed. PE height was restored were similar to that in wild type PE. Therefore, based on the cell height and microtubule distribution, Sal mis-expression converts the PE into a cuboidal cell shape (Tang, 2016).
During tissue morphogenesis, cell-shape changes always accompany microtubule cytoskeleton rearrangement. Dpp signalling activity has been proposed to play a basic function in microtubule organization. Dpp signalling is graded in the DP along the A/P axis, with higher levels in the medial DP region, which is enriched in apical microtubules. Thus, a correlation is noted between Dpp signalling activity and microtubule levels in the DP. Clones with loss-of-function of Dpp receptors in the DP appear extruded (cell height is severely shortened) and exhibit reduced apical microtubule levels. Consistently, clones with both loss- and gain-of-function sal and omb in the DP consistently exhibit severe apical retraction with shortened cell height and loss of apical microtubule enrichment. Therefore, the data support the hypothesis that Dpp-Omb/Sal signalling activity plays a more general function in microtubule-based cell morphogenesis. Other transcription factors that induce reductions in DP cell height also correlate with the loss of apical microtubule enrichment. The Tbx6 subfamily gene cluster Dorsocross (Doc) initiates wing hinge/blade fold formation. In the Doc expression domain in the DP, cells are shortened from the apical side with severe loss of apical microtubules (Tang, 2016).
Benítez, E., Bray, S. J., Rodriguez, I. and Guerrero, I. (2009). Lines is required for normal operation of Wingless, Hedgehog and Notch pathways during wing development. Development 136(7): 1211-21. PubMed Citation: 19270177
Bokor, P. and DiNardo, S. (1996). The roles of hedgehog, wingless and lines in patterning the dorsal epidermis in Drosophila. Development 122(4): 1083-1092. PubMed Citation: 8620835
Castelli-Gair, J. (1998). The lines gene of Drosophila is required for specific functions of the Abdominal-B HOX protein. Development 125: 1269-1274. PubMed Citation: 9477325
Del Signore, S. J., Hayashi, T. and Hatini, V. (2012). odd-skipped genes and lines organize the notum anterior-posterior axis using autonomous and non-autonomous mechanisms. Mech Dev 129: 147-161. PubMed ID: 22613630
Dinardo, S., et al. (2011). lines and bowl affect the specification of cyst stem cells and niche cells in the Drosophila testis. Development 138(9): 1687-96. PubMed Citation: 21486923
Fagotto, F., Gluck, U. and Gumbiner, B. M. (1998). Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of beta-catenin. Curr. Biol. 8: 181-190. PubMed Citation: 9501980
Green, R. B., et al. (2002). Drumstick is a zinc finger protein that antagonizes Lines to control patterning and morphogenesis of the Drosophila hindgut. Development 129: 3645-3656. 12117814
Hatini, V., et al. (2000). Tissue- and stage-specific modulation of Wingless signaling by the segment polarity gene lines. Genes Dev. 14: 1364-1376. PubMed Citation: 10837029
Hatini, V., Green, R. B., Lengyel, J. A., Bray, S. J. and Dinardo, S. (2005). The Drumstick/Lines/Bowl regulatory pathway links antagonistic Hedgehog and Wingless signaling inputs to epidermal cell differentiation. Genes Dev. 19(6): 709-18. 15769943
Herranz, H. and Morata, G. (2001). The functions of pannier during Drosophila embryogenesis. Development 128: 4837-4846. 11731463
Iwaki, D. D., et al. (2001). drumstick, >bowl, and lines are required for patterning and cell rearrangement in the Drosophila embryonic hindgut. Dev. Biol. 240(2): 611-26. 11784087
Johansen, K. A., Green, R. B., Iwaki, D. D., Hernandez, J. B. and Lengyel, J. A. (2003). The Drm-Bowl-Lin relief-of-repression hierarchy controls fore- and hindgut patterning and morphogenesis. Mech. Dev. 120(10): 1139-51. 14568103
Nusinow, D., Greenberg, L. and Hatini, V. (2008). Reciprocal roles for bowl and lines in specifying the peripodial epithelium and the disc proper of the Drosophila wing primordium. Development 135: 3031-3041. PubMed ID: 18701548
Nusslein-Volhard, C. and E. Wieschaus. (1980). Mutations affecting segment number and polarity in Drosophila. Nature 287: 795-801. PubMed Citation: 6776413
Nusslein-Volhard, C., E. Wieschaus, and H. Kluding. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. I. Zygotic loci on the second chromosome. Roux's Arch. Dev. Biol. 193: 267-282
Tang, W., Wang, D. and Shen, J. (2016). Asymmetric distribution of Spalt in Drosophila wing squamous and columnar epithelia ensures correct cell morphogenesis. Sci Rep 6: 30236. PubMed ID: 27452716
date revised: 21 November 2016
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