The Interactive Fly
Genes involved in tissue and organ development
Joining Morphogenesis and Salivary Gland Development
Drosophila salivary glands consist of two major cell types: secretory cells and duct cells. Secretory cells are columnar epithelial cells that synthesize and secrete high levels of protein. Duct cells are cuboidal epithelial cells that form the simple tubes connecting the secretory cells to the larval mouth. Salivary glands arise from two ventral ectodermal plates of approximately 100 cells each, in the region of the presumptive posterior head. Salivary glands differentiate without further cell division and increase in size simply by increasing the volume of individual cells. Thus, all of the changes that occur during differentiation take place within and between pre-existing cells, greatly simplifying the analysis of organ development since it eliminates concerns about regulated control of cell division, potential unequal partitioning of cellular factors during mitosis, and programmed cell death (Andrew, 2000 and references therein).
During the earliest stages of salivary gland formation, the secretory cells of the salivary gland change shape from cuboidal to columnar, forming the salivary gland 'placode'. Following this shape change, cells in the dorsal-posterior region of the placode undergo apical constrictions as the nuclei move from the surface of the embryo to a more basal position within each cell. These wedge-shaped cells then begin to invaginate. As this initial population of cells continues to invaginate, the remaining primordial cells at the surface also change shape and internalize. A salivary gland tube forms and elongates dorsally, as additional cells invaginate and become internalized. After elongating dorsally, cells in the salivary gland tube migrate posteriorly so that about one-third of the tube is bent towards the posterior end. Towards the end of invagination, almost the entire salivary gland tube is directed to the posterior. By late embryogenesis, the salivary gland cells have reached the most posterior extent of their migration, reaching to the middle of the third thoracic segment, dorsolateral to the ventral nerve cord. The salivary duct cells, which arise from the most ventral regions of the salivary gland primordia, are the last cells to invaginate. These cells, which form both the two lateral individual ducts and a central common duct, connect the secretory cells to the larval mouth (Andrew, 2000 and references therein).
Concomitant with the cell movements necessary for embryonic salivary gland formation, future secretory cells also undergo the physiological changes required for high levels of secretion. Prior to invagination, genes that encode components of the secretory pathway start to be transcribed at much higher levels in the salivary gland secretory primordia than in other embryonic tissues. This high level of transcription continues throughout embryogenesis. By late embryogenesis, active secretion is evident by light and transmission electron microscopy. Also during invagination, the secretory cells initiate the multiple rounds of DNA replication without subsequent division (endoreduplication) that create the giant polytene chromosomes needed to meet the increased metabolic requirements of these cells. The developing salivary gland thus provides a simple system for studying the control of organelle position and size, cell shape changes, cell migration, tube formation, changes in cell physiology, the transition from euploidy to polyteny, and tissue-specific gene expression (Andrew, 2000 and references therein).
Why do organs form at a particular place within the developing embryo? What controls the number and types of cells that comprise a given tissue? These questions have been answered for the Drosophila salivary gland. The position of the salivary gland primordia, the number of cells committed to form salivary glands, and the distinction between the two major cell types (secretory cells and duct cells) are controlled by localized expression of transcription factors and by localized cell signaling. Two homeotic genes, Sex combs reduced (Scr) and Abdominal-B (Abd-B), and a third gene that encodes a zinc-finger protein, teashirt (tsh), collectively restrict the salivary glands to a distinct anterior-posterior position in the embryo, known as parasegment 2 (PS2). Within PS2, a signaling pathway initiated by the product of the decapentaplegic (dpp) gene limits which cells become committed to form salivary glands. Finally, epidermal growth factor (EGF) signaling in the most ventral cells of the salivary gland primordia specifies a duct cell fate. Salivary gland formation also requires the function of two more globally expressed transcription factors, encoded by extradenticle (exd) and homothorax (hth) (Andrew, 2000 and references therein).
The homeotic gene Scr is initially expressed in the entire ectoderm of PS2, including the cells that give rise to the salivary glands. In embryos missing Scr function, salivary gland expression of all tested salivary gland markers is either lost or reduced to the background levels of non-salivary gland tissues. Correspondingly, ectopic expression of Scr causes the expression of all tested salivary gland markers in new places in addition to PS2. Thus, Scr is not only necessary for the formation of salivary glands, but Scr can also induce salivary gland fates in cells that do not normally give rise to salivary glands (Andrew, 2000 and references therein).
Although global expression of Scr leads to the formation of extra salivary glands, these additional salivary glands only form in the more anterior parasegments, PS0 and PS1. In regions posterior to PS2, two different proteins block salivary gland induction by Scr. In the region from PS3 to PS13, the zinc-finger protein Tsh blocks salivary gland formation, and in PS14, the homeotic protein, Abd-B, blocks salivary gland gene induction. Even though Scr-induced salivary fates are limited to more anterior segments when Scr is expressed everywhere, some downstream genes, such as fork head (fkh), are also induced in more posterior segments. This observation suggests differences among salivary gland genes with respect to which anterior-posterior regulators limit their expression. How Abd-B and Tsh block the induction of salivary gland genes by Scr has not been determined, although genetic studies suggest that the mechanisms are different. When Scr is expressed to very high levels throughout a wild-type embryo, or is expressed to moderate levels throughout an embryo with only one functional copy of Abd-B, a few cells in PS14 express several salivary gland markers. Thus, the relative amounts of Scr and Abd-B determine whether or not salivary gland target genes are activated. When Scr is expressed throughout an embryo with only one functional copy of tsh, the expression of most salivary gland genes is still limited to anterior segments, suggesting that Tsh is more effective at blocking the induction of salivary glands by Scr. Interestingly, Tsh blocks salivary gland formation in PS3 at two levels: by repressing transcription of Scr itself in ventral cells of PS3, and also by blocking Scr activation of most salivary gland target genes. Regulation of Scr activity at two levels in PS3 may be important for the role of Tsh in specifying'trunk' identities, since the salivary glands are a 'head-specific' structure. These results indicate that Scr acts as a positive factor for salivary gland fates, whereas Tsh and Abd-B are negative regulators. The expression profiles of Scr, tsh and Abd-B determine the position along the anterior-posterior axis where salivary glands will form (Andrew, 2000 and references therein).
Salivary gland formation also requires the function of two genes, Exd and Hth, which have more global expression domains. Exd is a homeodomain-containing protein that coordinately binds and regulates the expression of genes downstream of other homeotic proteins. Hth is a homeodomain-containing protein that is necessary for the nuclear localization of Exd. Both Exd and Hth are required for salivary glands to form and they function at two levels. Exd and Hth, but not Scr, are required to maintain expression of Scr in the salivary gland primordia. Exd and Hth are also required to activate expression of salivary gland genes. Salivary gland genes are not expressed in exd mutants when Scr is expressed throughout the embryo using a heat-shock driven enhancer (Andrew, 2000 and references therein).
Scr is required to form salivary glands; however, both the Scr transcript and Scr protein disappear in the salivary glands as the cells begin to invaginate. The normal disappearance of Scr expression in the salivary gland is controlled by a regulatory pathway that includes Exd and Hth. Initially, exd and hth are expressed almost everywhere in the embryo, including the cells of the salivary gland primordia. As the cells begin to invaginate, both hth expression and Exd nuclear localization disappear specifically in the salivary gland, coincident with the disappearance of Scr transcripts and protein. The loss of hth expression in the region of PS2 that normally forms the salivary gland is Scr-dependent, suggesting that hth is itself a downstream target gene whose expression is repressed by Scr in the salivary gland. Thus, Scr, Exd and Hth are necessary to specify, but not maintain, salivary gland cell fates. Many of the target genes activated by Scr, Exd and Hth encode transcription factors required to maintain their own expression and to regulate expression of other salivary gland genes (Andrew, 2000 and references therein).
Although Scr is expressed in the entire ectoderm of PS2, salivary gland formation is limited to the ventral cells by the molecules that establish overall dorsal-ventral polarity. For example, mutations in dorsal (dl), a gene required to specify ventral cell fates throughout the embryo, result in a complete absence of salivary glands. How does global dorsal-ventral patterning information controlled by Dl and its downstream effectors integrate with the anterior-posterior patterning information provided by Scr? To answer this question, it is necessary to identify the molecules within the dorsal-ventral patterning pathway that directly mediate this dorsal-ventral restriction (Andrew, 2000 and references therein).
Dl encodes a transcription factor whose activity is regulated by nuclear translocation. Dl is required throughout the embryo to differentially regulate gene expression along the dorsal-ventral axis. In the most ventral cells, where levels of nuclear Dl protein are highest, Dl activates expression of the transcription factors twist (twi) and snail (sn), which are required for the specification of mesodermal fates. In both ventral and ventrolateral regions of the embryo, Dl blocks expression of dpp. Dpp is a secreted signaling molecule that specifies dorsal cell fates. Salivary glands form from the cells that do not express twi, sn or dpp. Since the PS2 cells that express twi and sn do not normally express Scr, it is clear why salivary glands do not arise from the mesoderm. However, even if Scr is expressed everywhere using a heat-shock driven enhancer, salivary glands do not form from the most ventral (mesodermal) cells. These results indicate regulation at two levels: one level of control is to block Scr expression in the mesoderm; an additional level is to block Scr activation of salivary gland target genes. Twi or Sn could directly block activation of salivary gland target genes by Scr since Twi and Sn are expressed in the entire mesoderm when transcription of the earliest salivary gland genes begins. Mutations in twi or sn have not been tested directly for their effects on salivary gland formation; however, in embryos mutant for both dl and dpp, salivary glands form from all PS2 cells including cells that should be mesodermal, presumably because twi and sn are not expressed in dl mutants and the requirement for Dl to repress dpp is circumvented by removing dpp function (Andrew, 2000 and references therein).
The block to salivary gland formation by dpp has been more thoroughly studied. Loss of dpp function results in an expansion of salivary gland gene expression throughout the dorsal ectoderm of PS2. Correspondingly, the global expression of dpp, achieved either through the loss of dl function or by heat-shock-induced expression of a dpp cDNA, blocks salivary gland formation throughout PS2. Dpp is a secreted signaling molecule of the transforming growth factor-beta (TGF-beta) family and thus its effects on Scr-directed transcription must be indirect. Indeed, Dpp blocks salivary gland formation by binding to the receptors Thick veins (Tkv) and Punt (Put). This signal is transduced from the receptors to the nucleus by two related proteins, Mothers Against Dpp (Mad) and Medea (Med), and through a nuclear zinc-finger protein, Schnurri (Shn). The nuclear proteins downstream of Dpp (Mad, Med and Shn) could bind the enhancers of salivary gland genes, thereby blocking their activation; or, these proteins could bind Scr and redirect it to non-salivary gland target genes that normally function in the dorsal ectoderm of PS2 (Andrew, 2000 and references therein).
dpp transcription begins in dorsal cells shortly after cell cycle 11, about 1.5 h after egg laying (AEL), and continues in the entire dorsal ectoderm through germ band extension, about 7.5 h of development. Expression of the earliest salivary gland genes begins at about 4 h, and many additional salivary gland genes are activated by 7.5 h. Thus, dpp and its downstream effectors are expressed, and presumably active, when early salivary gland genes are being induced by Scr, Exd and Hth. By blocking salivary gland gene activation in dorsal cells, Dpp signaling limits the number of cells recruited to a salivary gland fate (Andrew, 2000 and references therein).
The salivary duct cell fate requires EGF signaling. Localized signaling controls the fates of cells within the salivary gland. EGF signaling is highest in the most ventral ectoderm of all segments, including PS2. In the salivary gland primordia, signaling by EGF is required to distinguish duct from secretory cell fates. In the absence of EGF signaling, all salivary gland cells become secretory cells. Mutations in EGF signaling pathway genes, including single-minded (sim), rhomboid (rho), spitz (spi) and pointed (pnt), result in the expression of all tested secretory cell markers in cells that normally form the duct. A corresponding loss of expression of duct-specific genes is also observed in these EGF pathway mutants. Thus, Scr in combination with EGF signaling specifies a salivary duct cell fate, whereas Scr in the absence of EGF signaling specifies a salivary secretory cell fate (Andrew, 2000 and references therein).
Studies of an enhancer element for fork head (fkh), a gene normally expressed in the secretory cells and not duct cells, suggest that EGF signaling blocks Scr-mediated activation of secretory cell-specific genes in duct cells. A 1 kb fragment of the fkh gene drives Scr-dependent expression of a reporter gene, lacZ, in only the secretory cells of the salivary gland in wild-type embryos. Like the endogenous gene, fkh-lacZ is expressed in all the salivary gland primordia in spi and pnt mutants. Deletion of a 145 base pair (bp) sequence within this enhancer allows lacZ expression in both secretory and duct cells in wild-type embryos. This result suggests that binding sites for the activators Scr, Exd and Hth are present and functioning both in the intact 1 kb fkh enhancer and in this fkh enhancer. However, when the 145 bp fragment is present, transcriptional activation of the reporter gene by Scr, Exd and Hth is blocked by EGF signaling in duct cells. The transcription factor downstream of EGF signaling that directly interacts with this 145 bp sequence in the fkh enhancer has not been identified, although Pnt is a candidate (Andrew, 2000 and references therein).
Another salivary gland gene, trachealess (trh), is expressed initially in both the secretory and duct cells, indicating that early trh expression in the salivary gland is not affected by EGF signaling. At later stages, trh expression becomes restricted to the duct cells through repression in the secretory cells by Fkh. fkh and trh encode transcription factors that regulate the expression of secretory and duct genes, respectively. It has been proposed that fkh and trh are critical for the distinction between the duct and secretory cell fates in the salivary gland. In this model, EGF signaling limits fkh expression to secretory cells. In turn, Fkh blocks duct cell fates in the secretory primordia by limiting trh expression to only the most ventral cells. Trh then confers duct identity on the most ventral cells by activating expression of the duct-specific genes. At least two findings suggest different roles for Fkh and Trh. (1) The model predicts that in fkh mutants, all duct-specific genes would be turned on in the secretory primordia. While this is true for trh and Serrate (Ser), a putative Trh downstream target gene, it is not true for breathless (btl) or dead ringer (dri), two other duct-specific genes. (2) The model predicts that trh is required for the expression of all duct-specific genes. At least one duct-specific gene, dri, is expressed in the duct cell primordia in both wild-type and trh mutant embryos. Therefore, neither trh nor fkh appear to function to specify the identities of cells within the salivary gland. Instead, both genes are likely to have critical roles in the behavior of salivary gland cells after the two specific cell types have been determined by differential EGF signaling in the salivary gland primordia (Andrew, 2000 and references therein).
In summary, three main decisions are controlled by localized expression of transcription factors and localized signaling: where salivary glands will form in the embryo; the number of cells committed to a salivary gland fate, and which cells will become secretory versus duct cells. Salivary gland formation requires the transcription factors Scr, Exd and Hth. Based on studies of related proteins in mammals and in Drosophila, these proteins are likely to directly bind and either activate or repress expression of downstream target genes in the salivary gland. In support of this idea, it has been demonstrated that Scr and Exd directly bind a functional fkh enhancer element in vitro. When Scr is expressed everywhere, the salivary gland fate is limited to a subset of Scr-, Exd- and Hth-expressing cells by the actions of Tsh and Abd-B. Dpp, through its downstream effectors Tkv, Pnt, Mad, Med and Shn, blocks salivary gland formation in the dorsal cells of PS2. Twi and/or Sn are likely to block salivary gland formation in the mesoderm, the most ventral cells of the early embryo. EGF signaling, which is required to specify a salivary duct fate, blocks the activation of secretory genes through either Pnt or an unidentified downstream transcription factor. There is now a fairly good picture of the molecular circuitry that regulates salivary gland formation. In the near future, this picture will be completed by characterizing the interactions of the upstream salivary gland regulators with the enhancers of downstream target genes. The next frontier is the functional characterization of the downstream target genes, which will reveal how each gene product contributes to the unique morphology and physiology of the salivary gland (Andrew, 2000 and references therein).
Salivary gland target genes are being successfully identified using three approaches. The first approach is careful inspection of the expression patterns of cloned genes; several genes with known or suspected roles in other developmental processes are expressed in developing salivary glands. The second approach is a systematic enhancer-trap screen for genes expressed in salivary glands, which has revealed both known genes and new genes. Finally, many cDNAs expressed to relatively high levels in the salivary gland have been identified through expression pattern studies of cDNAs from the Berkeley Drosophila Genome Project (Andrew, 2000 and references therein).
Several salivary gland genes are expressed only in the secretory cells; these genes include fkh, Toll (Tl), pipe (pip), modulo (mod), hückebein (hkb), Notch (N), DHR78 and WRS-85D. Other salivary gland genes are expressed in both secretory and duct cells, although in some cases expression in one cell type is transient; these genes include trh, dCREB-A and eye gone (eyg). Several genes that are known or thought to establish and/or maintain epithelial polarity are also expressed to high levels in both secretory and duct cells. These genes include betaH-spectrin, coracle (cora), crumbs (crb), discs large (dlg), neurexin IV (nrx IV) and Shark. Finally, there is a group of genes expressed exclusively in the duct cells of the salivary gland; these genes include dsc73, breathless (btl), Serrate (Ser) and dead ringer (dri). Although mutations exist for many of these genes, their roles in salivary gland development are only beginning to be understood (Andrew, 2000 and references therein).
fkh was among the first identified salivary gland target genes. FKH mRNA is first detected in the secretory cells of the salivary gland during embryonic stage 9, making it one of the earliest expressed salivary gland genes: recent studies suggest regulation of fkh by Scr and Exd is direct. fkh continues to be expressed in the secretory cells throughout larval life. fkh encodes a nuclear protein with a 'winged-helix' DNA-binding domain, similar to that of linker histone H5. However, unlike the linker histones, Fkh family members do not compact nucleosomal DNA; instead, these proteins open the chromatin to an active configuration (Andrew, 2000 and references therein).
Mutations in the fkh gene have a profound effect on salivary gland development; the salivary glands fail to internalize to form their characteristic tubes. Histological sections using antibodies to dCREB-A reveal a distinct salivary gland primordia in fkh mutants. In the mutants, secretory cell invagination initiates at the proper location but fails to continue, leaving all of the primordia at or near the embryo surface. The salivary duct cells also fail to internalize. Because fkh is not expressed in the duct cells, this defect could be indirect and due to stalled secretory cells physically blocking invagination of the duct cells. In other tissues, fkh mutants have phenotypes that suggest homeotic transformations, specifically transformations of the non-segmental terminal regions into segmental derivatives. Since several different salivary gland genes are expressed normally in the presumptive salivary gland secretory cells of fkh mutants, the salivary gland defects are unlikely to be due to changes in cell identity. Instead, the salivary gland defects in fkh mutants are probably due to a failure in morphogenesis. Since fkh encodes a transcription factor, it must mediate salivary gland invagination through the regulation of target genes involved in controlling the cell shape changes and coordinated movements necessary for internalization. At least five genes require fkh for expression in the salivary gland during embryogenesis, but their phenotypes have not yet been described (Andrew, 2000 and references therein).
fkh is required for salivary gland morphogenesis and for the expression of two salivary gland-specific structural proteins during late larval stages. Expression of the 'glue' protein genes, Salivary gland secretion protein 3 (Sgs3) and Salivary gland secretion protein 4 (Sgs4), is directly activated by Fkh. Glue proteins are made in the salivary gland at the end of larval life. When secreted, they form a sticky matrix to which the larva adheres to prepare for pupariation. Because fkh is required early for morphogenesis, and later for the expression of genes encoding cell-type specific structural proteins, Fkh appears to control multiple distinct activities of salivary gland cells.
trh, another gene expressed early in salivary gland formation, appears to do for duct cells what fkh does for secretory cells: trh is required for duct cells to invaginate and form their characteristic tubes. trh encodes a basic helix-loop-helix PAS protein that functions as a transcription factor. Trh is a Drosophila homolog of human hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor that activates target gene expression via heterodimer formation with the aryl hydrocarbon receptor nuclear translocator (ARNT). Similarly, Trh activates gene expression by forming a heterodimeric DNA-binding complex with Tango, the Drosophila ARNT homolog (Andrew, 2000 and references therein).
trh is initially expressed in the entire salivary gland primordia under the control of Scr, Exd, Hth, Tsh and Dpp signaling. trh mRNA and protein disappear in the secretory cells in a Fkh-dependent manner as these cells invaginate. trh expression persists in the duct cells, which are the only salivary gland cells affected by the loss of trh function. trh is also expressed in the trachea throughout tracheogenesis, and in cells that form the filzkörper, the tubular air filters for the trachea. Using different markers for these tissues, it appears that in trh mutants the precursor cells for the salivary duct, trachea and filzkörper are present, but fail to invaginate to form their characteristic tubes, supporting a role for Trh, like that for Fkh, in regulating tissue morphogenesis, and not tissue identity (Andrew, 2000 and references therein).
The organization of sheets of epithelial cells into tubes is an essential feature of organ development not only in the Drosophila salivary duct, trachea and filzkörper, but in all higher eukaryotes. Since Trh is a transcription factor, understanding its role in tube formation requires the identification and characterization of the genes it regulates. Among the known Trh target genes in the salivary duct are Ser and btl. Ser's role in the salivary duct is not known. btl, which encodes an FGF-receptor homolog, is regulated by Trh not only in the salivary duct, but also in the trachea. In btl mutants, the tracheal cells invaginate but stall at a relatively early stage in branch migration. So far, no obvious defects have been observed in the salivary ducts of embryos mutant for btl or for its ligand, which is encoded by the branchless gene. The absence of a btl mutant phenotype in the salivary duct suggests three possibilities. Either the phenotypes are too subtle to be discerned with available markers, btl function is redundant in this tissue, or btl is expressed in the salivary duct only as an indirect consequence of btl activation by Trh in other tissues that require btl function.
So far, only one target gene for Trh is known to be required for normal duct development. Trh regulates late expression of eye gone (eyg), which encodes a Pax family transcription factor. Eyg is required for the formation of the individual ducts, which connect the central common duct to the secretory portions of the gland. In eyg mutants, a large fraction of the individual duct cells appears to contribute to the central common duct instead. Also, the level of btl expression, which is normally higher in the individual duct cells relative to the common duct cells, is reduced. It has been proposed that Eye functions to distinguish the individual duct cells from common duct cells. However, ectopic expression of eyg in all duct cells does not transform common duct cells into individual duct cells, as predicted by this model (Andrew, 2000 and references therein).
What controls the size, shape and final position of the salivary gland cells? Although very little is known, at least one gene has been identified that affects secretory cell morphology. The mod gene functions as a modifier of position-effect variegation and directly binds DNA. Mutations in mod affect different tissues including the cuticle, fat body, gut mesoderm and salivary gland. Although embryonic phenotypes have not been described, three phenotypes are observed in late larval salivary glands from mod mutants: there are more secretory cells, the secretory cells are smaller and the cells do not adhere to one another as well as in wild-type salivary glands. The increase in cell number in mod mutants is striking since in wild-type embryos salivary gland cells stop dividing once the primordia are established. It would be interesting to know when the additional salivary gland cells arise, and if cell number increases in other tissues in mod mutants. As with all DNA-binding proteins that affect salivary gland differentiation, understanding the role of Mod will require the identification and characterization of its target genes, assuming that Mod regulates gene expression. Because of its effects on position-effect variegation (PEV), Mod is proposed to regulate downstream target genes through changes in chromatin structure. Alternatively, Mod may play a direct role in the transition from the mitotic cycle to polyteny, which occurs earliest in salivary gland cells. A role in the transition from normal mitotic divisions to polytenization could explain the increase in salivary gland cell number in the mod mutants (Andrew, 2000 and references therein).
Salivary glands are polarized epithelia. Therefore, it is not surprising that mutations in four genes required to establish and/or to maintain epithelial polarity also cause salivary gland defects. Mutations in cora and nrx IV, which encode components of the septate junction, result in salivary gland necrosis at the first larval instar; earlier embryonic phenotypes of these mutations have not been described. Mutations in dlt, which encodes a novel PDZ protein that binds NRX IV and CRB, cause a loss of salivary cell polarity; proteins normally limited to the apical or lateral plasma membranes are mislocalized throughout the membrane. Mutations in crb, which confers apical character to the plasma membrane, significantly reduces the number of cells comprising the salivary gland. Since the number of cells in the salivary primordia appears normal in crb mutants, the loss of salivary gland cells after germ band retraction is likely to be due to cell death. In histological sections, the small salivary glands in crb mutants are normal and appear to have secretory activity, suggesting that crb function in the salivary gland may be partially redundant (Andrew, 2000 and references therein).
scab encodes an alphaPS3 integrin that is expressed in the salivary gland as well as other tissues. Zygotic mutations in scab cause mild defects in salivary gland morphology. One salivary gland is often misshapen and smaller than the other gland. The salivary glands are also thought to reside closer to the midline than in wild-type embryos. Integrins reside in the basal plasma membrane where they mediate both cell attachment and cell signaling. Since salivary glands are normally found in close contact with the thoracic muscles, it is possible that the aberrant position of the salivary glands in the scab mutant is due to a requirement for integrins in establishing or maintaining this contact (Andrew, 2000 and references therein).
The salivary glands are the largest secretory organs in the Drosophila embryo and larva. In light of this secretory activity, it is interesting to note that several recently identified salivary gland cDNAs encode open reading frames with homology to proteins in the secretory pathway in other organisms. Drosophila homologs to proteins involved in sorting nascent polypeptide chains to the endoplasmic reticulum (ER), in vesicular transport from the ER to the Golgi, in the refolding of misfolded proteins and in regulated secretion are all expressed to elevated levels in the salivary gland under the control of Scr. These findings suggest that transcriptional up-regulation of secretory genes is an early step in the differentiation of a secretory cell. This up-regulation could be mediated directly by Scr or, more likely, through some of the early transcription factors expressed in the salivary gland, such as Fkh, dCREB-A and HKB (Andrew, 2000 and references therein).
In cells with high levels of secretory activity, components required for protein synthesis are also expected to be up-regulated. Thus, it was exciting to discover that one of the early-expressed salivary gland genes identified through enhancer-trapping is the gene encoding tryptophanyl tRNA synthetase, WRS-85D. tRNA synthetases, such as WRS-85D, attach amino acids to their cognate tRNAs, providing essential substrates for protein translation. When the expression profiles of the other 19 tRNA synthetase genes were examined, only a few were expressed at high levels in the salivary gland, and no other tRNA synthetase gene was expressed to the levels observed with WRS-85D. Since tryptophan has the lowest amino acid usage frequency among all 20 amino acids in the known salivary gland proteins, the up-regulation of WRS-85D expression is now proposed to reflect an additional non-canonical activity of this enzyme, as described for aminoacyl tRNA synthetases in other organisms. The loss of elevated WRS-85D expression in the salivary gland through loss of zygotic expression does not cause obvious defects in salivary gland morphology. Whether WRS-85D is required for salivary function is unknown (Andrew, 2000 and references therein).
The Drosophila homologs of three other well-characterized enzymes are also up-regulated in the secretory cells of the early salivary gland. These genes include paps synthetase, which encodes an enzyme involved in sulfation; columbus, which encodes an HMG-CoA reductase, and pipe, which encodes a heparan sulfate 2-O sulfotransferase. columbus is required in the mesoderm for the migration of germ cells and pipe is required maternally for dorsal-ventral patterning of the entire embryo. Given the function of these enzymes in other contexts, it is difficult to predict whether they will have roles in morphogenesis, patterning, secretion or some novel function in the developing salivary gland. Mutations in columbus do not affect salivary gland migration (Andrew, 2000 and references therein).
In the process of branching morphogenesis of trachea, the final tubular network is formed by repeated branching of large tubes to form finer and ever finer tubes. There is an opposite form of morphogenesis in which small tubes join together to form larger and less numerous tubes. This joining, rather than branching, morphogenesis has been little studied though it is not unusual. For example, during tracheal development, dorsal tracheal branches from each segment fuse with their counterparts from the other side of the embryo to connect the left and right sides of the tracheal system. Similarly, segmental branches fuse along the sides of the embryo to form the lateral tracheal trunks which connect the tracheae of different segments. Formation of the larval salivary glands in Drosophila provides a simple example of joining morphogenesis. During salivary invagination, ducts from the two sides of the embryo meet at the ventral midline and fuse so that continued invagination produces a single common duct that connects to the oral cavity (Jones, 1998 and references). Before describing the regulation of joining morphogenesis, the regulation of salivary gland morphogenesis will be reviewed.
Salivary development occurs rapidly, beginning at 4.5 hours of development and finishing by 10 hours of development. The initial specification of salivary cells occurs within a two-dimensional sheet of cells, the ectoderm, with no known induction from underlying layers. This initial specification, which is complete by embryonic stage 10 (about 5.5 hours of development), occurs only within a specific region of the anterioposterior axis: parasegment two. The salivary primordium is bilaterally symmetric and consists of approximately 100 cells on either side of the ventral midline. The homeotic gene responsible for patterning parasegment 2, Sex combs reduced (Scr), encodes the primary inducer of salivary glands. Embryos lacking Scr have no salivary glands while ectopic Scr results in ectopic salivary glands (Panzer, 1992; Andrew, 1994). The salivary primordium is continuous across the ventral midline but is limited dorsally by decapentaplegic (Panzer, 1992). Because the salivary cells do not divide after the initial patterning, further development is not complicated by cell proliferation (Jones, 1998).
The major subdivision of the salivary primordium distinguishes pregland from preduct tissue. The most dorsal 80-90 cells on each side of the ventral midline constitute the circular pregland domain, also known as the salivary placode, while the most ventral 20-30 cells become the precursors of the salivary ducts. After the salivary primordium has been established, gland and duct primordia can be distinguished at the molecular level since expression of several genes is restricted to only one of these domains. For example, forkhead (fkh: Weigel, 1989 and Panzer, 1992), Toll (Gerttula, 1988) and huckebein (Brönner, 1994) are expressed in pregland cells and are specifically excluded from preduct cells. In contrast, expression of Serrate (Ser: Fleming, 1990 and Thomas, 1991), breathless (btl: Klämbt, 1992) and dead ringer (dri: Gregory, 1996) is restricted to the preduct cells (Jones, 1998).
Two regulatory activities interact to define the border between pregland and preduct cells (Kuo, 1996). The first is the EGF receptor (EGFR) signaling pathway. In the ventral ectoderm, Spitz is responsible for activating the EGFR pathway in a graded fashion that limits the ventral extent of fkh expression to the pregland cells. fkh itself is the second regulator of the positioning of the pregland/preduct border. In fkh-mutant embryos, expression of the duct marker Ser extends dorsally into the gland primordium (Kuo, 1996). fkh is also responsible for excluding expression of trachealess (trh) from the gland primordium (Isaac, 1996). Thus, fkh is critical for the establishment of the dorsal limit of duct fate. Together, the opposing activities of Fkh and the EGFR pathway precisely determine the border between the gland and duct primordia. The subdivision of the salivary primordium into pregland and preduct defines the expression domains of two regulators that are required for the subsequent development of these tissues. In addition to its function in the establishment of the gland/duct border, fkh has another salivary role: fkh is necessary for the activation of all tested genes expressed in the gland primordium after its initial establishment. In the duct primordium, trachealess functions to activate duct fate (Kuo, 1996) in a way analogous to the role of fkh in pregland cells: all tested genes that are expressed in the duct primordium after its initial establishment require trachealess for expression. It is also interesting to note that both fkh and trh encode autoregulatory gene products, suggesting the possibility that continued expression of these genes is important for maintenance of gland and duct fate, respectively (Jones, 1998 and references).
Once the initial specification and the primary patterning events are complete, the cells begin characteristic morphogenetic movements that result in mature salivary glands and ducts. The morphogenesis of salivary tissues can be separated into three successive events: formation first of the salivary glands, then the individual ducts and finally the common duct. At the end of stage 11 (about 7 hours of development), the most posterodorsal pregland cells begin to invaginate. The site of invagination progresses anteriorly until all of the pregland cells have been internalized, forming a tubular salivary gland with a single layer of secretory cells surrounding a tubular lumen. fkh is required for this first type of invagination, during which the pregland cells leave the ventral surface of the embryo (Weigel, 1989 and Panzer, 1992). As the gland cells invaginate, the preduct cells rearrange to form two parallel rows of cells extending across the ventral midline (Kuo, 1996). These cells will form the two types of duct tissue: the posterior row becomes the individual ducts and the anterior row becomes the common duct. The lateral ends of the individual ducts remain in contact with the gland cells and when they invaginate they continue the tube started during gland invagination. The diameter of the tube, however, is much smaller in the ducts. The individual duct invagination continues to the ventral midline where the left and right sites of invagination fuse (Kuo, 1996). Finally, the common duct is formed as the anterior row of duct cells move to the anterior and begin to invaginate. The resulting structure connects the lumen of the individual ducts to the pharynx. trh plays a critical role in both duct invaginations as neither of the preduct tissues invaginate from the ventral surface in trh-mutant embryos (Kuo, 1996; Isaac, 1996).
In wild-type embryos, the salivary ducts arise as a result of two successive convergence and extension events. Convergence and extension is a common developmental process during which cells intercalate to narrow the tissue while at the same time lengthening it in a perpendicular axis. As the germ band is retracting in wild-type embryos, the duct primordium narrows from about 6-8 cells in the anterioposterior axis to 2 rows of cells. At the same time, the primordium extends laterally across the ventral midline. The result of this first convergence and extension is two parallel rows of cells separated by a cleft. The anterior row of cells is fated to become common duct while the posterior row is fated to become individual ducts. The Pax gene eyegone (eyg) plays a critical role in joining morphogenesis. It is required to distinguish individual from common duct domains and is necessary for the morphogenesis of the cells of the individual ducts. It is expressed specifically in individual duct cells and is critical for development of these cells as individual rather than common duct. In eyg-mutant embryos, presumptive individual duct cells are converted to common duct. In eyg-mutant embryos, individual duct cells are defective in convergence and extension. The posterior row of cells expresses eyg and will form the individual ducts. The anterior row of non-eyg-expressing cells, however, converges toward the ventral midline and extends anteriorly to form the common duct. In embryos mutant for eyg, the duct primordium never completes the first convergence and extension. Instead of forming the two parallel rows of cells extended across the ventral midline, the preduct cells remain as compact clumps, often slightly separated by the ventral midline. Later, however, the duct cells in eyg-mutant embryos move anteriorly in a process that resembles that of wild type, although they do not obviously converge toward the ventral midline. In summary, in eyg-mutant embryos, the duct primordia fail to converge and extend across the midline resulting in the absence of individual ducts. Invagination of the salivary gland cells begins normally but becomes temporarily stalled at the gland/duct boundary until the gland cells finally break loose from the duct cells. In these mutant embryos, many of the presumptive individual duct cells join with the presumptive common duct cells to form an unusually large common duct that does not connect to the glands (Jones, 1998).
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Genes involved in organ development
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