The Interactive Fly

Genes involved in tissue and organ development

Salivary Gland

Genetic Control of Salivary Gland development in Drosophila

Joining Morphogenesis and Salivary Gland Development

Massive excretion of calcium oxalate from late prepupal salivary glands of Drosophila melanogaster demonstrates active nephridial-like anion transport

Tango7 regulates cortical activity of caspases during reaper-triggered changes in tissue elasticity

Molecular mechanisms of developmentally programmed crinophagy in Drosophila

Endosomal vacuoles of the prepupal salivary glands of Drosophila play an essential role in the metabolic reallocation of iron

Radially-patterned cell behaviours during tube budding from an epithelium

A molecular switch orchestrates enzyme specificity and secretory granule morphology

Drosophila HUWE1 ubiquitin ligase regulates endoreplication and antagonizes JNK signaling during salivary gland development

Role of tbc1 in Drosophila embryonic salivary glands

Role of inward rectifier potassium channels in salivary gland function and sugar feeding of the fruit fly, Drosophila melanogaster

Tango1 coordinates the formation of ER/Golgi docking sites to mediate secretory granule formation

Model to Link Cell Shape and Polarity with Organogenesis

Larval salivary gland chromosomes undergo endoreduplication and become polyploid. For information about the regulation of this process, see Polytene chromosomes, endoreduplication and puffing.

Genetic Control of Salivary Gland Development in Drosophila

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).

Joining Morphogenesis and Salivary Gland Development

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).

Massive excretion of calcium oxalate from late prepupal salivary glands of Drosophila melanogaster demonstrates active nephridial-like anion transport

The Drosophila salivary glands (SGs) were well known for the puffing patterns of their polytene chromosomes and so became a tissue of choice to study sequential gene activation by the steroid hormone ecdysone. One well-documented function of these glands is to produce a secretory glue, which is released during pupariation to fix the freshly formed puparia to the substrate. Over the past two decades SGs have been used to address specific aspects of developmentally-regulated programmed cell death (PCD), as it was thought that they are doomed for histolysis and after pupariation are just awaiting their fate. More recently, however, it has been shown that for the first 3-4 h after pupariation SGs undergo tremendous endocytosis and vacuolation followed by vacuole neutralization and membrane consolidation. Furthermore, from 8 to 10 h after puparium formation (APF) SGs display massive apocrine secretion of a diverse set of cellular proteins. This study shows that during the period from 11 to 12 h APF, the prepupal glands are very active in calcium oxalate (CaOx) extrusion that resembles renal or nephridial excretory activity. Genetic evidence that Prestin, a Drosophila homologue of the mammalian electrogenic anion exchange carrier SLC26A5, is responsible for the instantaneous production of CaOx by the late prepupal SGs. Its positive regulation by the protein kinases encoded by fray and wnk lead to increased production of CaOx. The formation of CaOx appears to be dependent on the cooperation between Prestin and the vATPase complex as treatment with bafilomycin A1 or concanamycin A abolishes the production of detectable CaOx. These data demonstrate that prepupal SGs remain fully viable, physiologically active and engaged in various cellular activities at least until early pupal period, that is, until moments prior to the execution of PCD (Farkas, 2016).

Radially-patterned cell behaviours during tube budding from an epithelium

The budding of tubular organs from flat epithelial sheets is a vital morphogenetic process. Cell behaviours that drive such processes are only starting to be unraveled. Using live-imaging and novel morphometric methods this study shows that in addition to apical constriction, radially-oriented directional intercalation of cells plays a major contribution to early stages of invagination of the salivary gland tube in the Drosophila embryo. Extending analyses in 3D, it was found that near the pit of invagination, isotropic apical constriction leads to strong cell-wedging. Further from the pit cells interleave circumferentially, suggesting apically-driven behaviours. Supporting this, junctional myosin is enriched in, and neighbour exchanges are biased towards the circumferential orientation. In a mutant failing pit specification, neither are biased due to an inactive pit. Thus, tube budding involves radially-patterned pools of apical myosin, medial as well as junctional, and radially-patterned 3D-cell behaviours, with a close mechanical interplay between invagination and intercalation (Sanchez-Corrales, 2018).

A molecular switch orchestrates enzyme specificity and secretory granule morphology
Regulated secretion is an essential process where molecules destined for export are directed to membranous secretory granules, where they undergo packaging and maturation. This study identified a gene (pgant9) that influences the structure and shape of secretory granules within the Drosophila salivary gland. Loss of pgant9, which encodes an O-glycosyltransferase, results in secretory granules with an irregular, shard-like morphology, and altered glycosylation of cargo. Interestingly, pgant9 undergoes a splicing event that acts as a molecular switch to alter the charge of a loop controlling access to the active site of the enzyme. The splice variant with the negatively charged loop glycosylates the positively charged secretory cargo and rescues secretory granule morphology. This study highlights a mechanism for dictating substrate specificity within the O-glycosyltransferase enzyme family. Moreover, these in vitro and in vivo studies suggest that the glycosylation status of secretory cargo influences the morphology of maturing secretory granules (Ji, 2018).

Drosophila HUWE1 ubiquitin ligase regulates endoreplication and antagonizes JNK signaling during salivary gland development

The HECT-type ubiquitin ligase HECT, UBA and WWE Domain Containing 1, (HUWE1) regulates key cancer-related pathways, including the Myc oncogene. It affects cell proliferation, stress and immune signaling, mitochondria homeostasis, and cell death. HUWE1 is evolutionarily conserved from Caenorhabditis elegance to Drosophila melanogaster and humans. This study reports that the Drosophila ortholog, dHUWE1 (CG8184), is an essential gene whose loss results in embryonic lethality and whose tissue-specific disruption establishes its regulatory role in larval salivary gland development. dHUWE1 is essential for endoreplication of salivary gland cells and its knockdown results in the inability of these cells to replicate DNA. Remarkably, dHUWE1 is a survival factor that prevents premature activation of JNK signaling, thus preventing the disintegration of the salivary gland, which occurs physiologically during pupal stages. This function of dHUWE1 is general, as its inhibitory effect is observed also during eye development and at the organismal level. Epistatic studies revealed that the loss of dHUWE1 is compensated by dMyc protein expression or the loss of dmP53. dHUWE1 is therefore a conserved survival factor that regulates organ formation during Drosophila development (Yanku, 2018).

Role of tbc1 in Drosophila embryonic salivary glands

CG4552/tbc1 was identified as a downstream target of Fork head (Fkh), the single Drosophila member of the FoxA family of transcription factors and a major player in salivary gland formation and homeostasis. Tbc1 and its orthologues have been implicated in phagocytosis, the innate immune response, border cell migration, cancer and an autosomal recessive form of non-degenerative Pontocerebellar hypoplasia. Recently, the mammalian Tbc1 orthologue, Tbc1d23, has been shown to bind both the conserved N-terminal domains of two Golgins (Golgin-97 and Golgin-245) and the WASH complex on endosome vesicles. Through this activity, Tbc1d23 has been proposed to link endosomally-derived vesicles to their appropriate target membrane in the trans Golgi (TGN). This paper provides an initial characterization of Drosophila orthologue. Like its mammalian orthologue, Tbc1 localizes to the trans Golgi. It also colocalizes with a subset of Rabs associated with both early and recycling endosomes. Animals completely missing tbc1 survive, but females have fertility defects. Consistent with the human disease, loss of tbc1 reduces optic lobe size and increases response time to mechanical perturbation. Loss and overexpression of tbc1 in the embryonic salivary glands leads to secretion defects and apical membrane irregularities. These findings support a role for tbc1 in endocytic/membrane trafficking, consistent with its activities in other systems (Johnson, 2019).

Role of inward rectifier potassium channels in salivary gland function and sugar feeding of the fruit fly, Drosophila melanogaster

The arthropod salivary gland is of critical importance for horizontal transmission of pathogens, yet a detailed understanding of the ion conductance pathways responsible for saliva production and excretion is lacking. A superfamily of potassium ion channels, known as inward rectifying potassium (Kir) channels, is overexpressed in the Drosophila salivary gland by 32-fold when compared to the whole body mRNA transcripts. Therefore, this study aimed to test the hypothesis that pharmacological and genetic depletion of salivary gland specific Kir channels alters the efficiency of the gland and reduced feeding capabilities using the fruit fly Drosophila melanogaster as a model organism that could predict similar effects in arthropod disease vectors. Exposure to VU041, a selective Kir channel blocker, reduced the volume of sucrose consumption by up to 3.2-fold and was found to be concentration-dependent with an EC50 of 68mµM. Importantly, the inactive analog, VU937, was shown to not influence feeding, suggesting the reduction in feeding observed with VU041 is due to Kir channel inhibition. Next, a salivary gland specific knockdown of Kir1 was performed to assess the role of these channels specifically in the salivary gland. The genetically depleted fruit flies had a reduction in total volume ingested and an increase in the time spent feeding, both suggestive of a reduction in salivary gland function. Furthermore, a compensatory mechanism appears to be present at day 1 of RNAi-treated fruit flies, and is likely to be the Na(+)-K(+)-2Cl(-) cotransporter and/or Na(+)-K(+)-ATPase pumps that serve to supplement the inward flow of K(+) ions, which highlights the functional redundancy in control of ion flux in the salivary glands. These findings suggest that Kir channels likely provide, at least in part, a principal potassium conductance pathway in the Drosophila salivary gland that is required for sucrose feeding (Swale, 2017).

Despite the critical role the arthropod salivary gland serves in horizontal transmission of pathogens, an understanding of the machinery required for proper gland function is limited. Although rather limited in scope, pharmacological studies against the isolated tick salivary gland have implicated several components involved in the process of salivary secretion: dopaminergic pathway, Na+-K+-ATPase, GABA, and the muscarinic acetylcholine receptor. Although these pathways are clearly important for saliva production and excretion, the complex nature of the gland suggests other pathways are likely critical for proper function of the salivary gland. Indeed, the results of the present study provide compelling data that a superfamily of potassium ion channels, known as inward rectifier potassium channels, is an essential conductance pathway in the salivary gland that mediates proper feeding in the model organism Drosophila melanogaster (Swale, 2017).

Recent work on insect Kir channels have yielded insightful data suggesting these channels serve a critical role in Malpighian tubule function and fluid secretion. The Malpighian tubules and salivary glands are physiologically related tissues as both are a polarized epithelial tissue, require water and ion transport for function, and are considered, at least in part, to be an exocrine tissue. Furthermore, the Kir1 channel has been shown to constitute the primary inward K+ conductance in the mosquito Malpighian tubule and the analogous gene that encodes Kir1 in Drosophila is highly upregulated in the salivary glands of larval and adult flies. Therefore, it was hypothesized Kir channels also serve a critical role in salivary gland function, and this study aimed to elucidate the role of these channels through pharmacological and genetic manipulations of the Kir1 channel measured through feeding efficiency (Swale, 2017).

The recent identification of selective and potent small-molecules designed to target insect Kir channels have enabled researchers to begin to characterize the physiological role of these channels in various tissue systems. This study used the recently discovered insect Kir channel modulator (VU041) and its inactive analog (VU937) to characterize the influence these molecules have in the feeding cascade. Exposure to VU041 during feeding significantly reduced the volume of sucrose ingested, whereas VU937 had no influence to feeding efficiency, suggesting the observed phenotype is through Kir inhibition. However, due to the capability that small-molecules can inhibit unintended target sites and the fact Kir channels are highly expressed in the Malpighian tubules, it was impossible to ensure the observed effect to feeding was directly due to salivary gland failure. Therefore, salivary gland specific RNAi-mediated knockdown of the Kir1 encoding gene was performed. Results from this genetic depletion of Kir1 show a significantly less efficient salivary gland and, when combined with the VU041-mediated reduction in sucrose consumption, strongly suggests the Drosophila salivary gland relies on the inward conductance of K+ ions through Kir channels (Swale, 2017).

The data presented in this study raises the question as to what the physiological role Kir channels have in salivary gland function at the cellular level. Consideration of knowledge and hypotheses based on the role of Kir channels in mammalian salivary gland function and saliva production can be applied to expand understanding of salivary gland physiology in arthropods. First, it has been shown that electrolyte secretion in the mammalian salivary glands is based on the secondary active transport of anions, principally Cl- (and/or HCO3-) ions. In this model, K+ channels in the basolateral membrane of acinar cells maintain the membrane potential of the apical cell membrane to be more negative than the Nernst potential for anions, thereby providing a driving force for the sustained electrogenic anion efflux across the apical membrane. The second model for a role of Kir channels in the mammalian salivary gland was described through cell-attached patch and whole-cell patch-clamp studies. Here, researchers demonstrated the presence of four primary K+ channels, two of which are the outward mediated Ca2 +-activated K+ channel and a Kir channel. The inwardly rectifying property of the Kir channel was hypothesized to perform fast uptake of accumulated K+ ions, in concert with Na+-K+-ATPase, into acinar cells with the K+ influx depending on the relation between the membrane potential and the concentration gradient of K+ across the basolateral membrane. This buffering action likely provides an ion gradient enabling the outward flow of K+ ions through Ca2 +-activated K+ channels. Such K+ buffering action of Kir channels has been proposed in brain astrocytes, in retinal Müller cells and also in retinal pigmented epithelial cells (Swale, 2017).

To begin elucidating the role of Kir channels in the insect salivary gland based on the mammalian hypotheses, the potassium ion concentration in the sucrose solution was augmented to increase the potassium equilibrium constant (Ek; based on the Nernst equation), which ultimately reduces the efficacy of intracellular K+ channel inhibitors, such as Kir channel blockers. The loss of VU041 potency supports the notion that Kir channels function as a means to provide a pathway for rapid influx of K+ ions after depolarization events, a phenomenon oftentimes referred to as K+-spatial buffering. Therefore, it was hypothesize that Kir channels in Drosophila salivary glands are responsible, at least in part, for maintaining the high intracellular K+ concentration through a buffering-like action, which provides the K+ ion gradient to enable the outward flow of potassium ions, presumably through Ca2 +-activated K+-channels as is seen in mammals. However, further studies rooted in cellular electrophysiology and membrane physiology are needed to provide additional support for this hypothesis (Swale, 2017).

Although the data of this study suggest Kir channels are likely the primary mechanism for K+ spatial buffering within insect salivary gland cells, it is also evident that it is not the only transport pathway facilitating inward flow of K+ ions. Genetic depletion of Kir1 channels yielded a reduction of feeding at days 2, 3, and 4, but not on day 1 and similarly, the time spent feeding was not statistically different to controls at day 1 when compared to subsequent days. These data suggest the presence of a compensatory mechanism that accounts for the reduced expression of Kir1 in the genetically depleted animals, but one that is lost after day 1. Compensatory mechanisms are commonly observed in animals with genetic depletions of Kir channels and most oftentimes arise through upregulation of a different Kir gene. For instance, Wu and colleagues (2015) showed individual knockdown of any of the three Kir channel genes in Drosophila Malpighian tubules had no effect on the organ function, yet simultaneous knockdown of irk1 and irk2 had significant effects on transpeithelial K+ transport, suggesting Kir1 and Kir2 play redundant roles in Malpighian tubule function. Due to the expression of Kir1 and Kir2 mRNA in the Drosophila salivary gland, albeit at dramatic differences in mRNA expression level, it is plausible that the Ir2 gene is upregulated after genetic depletion of Kir1, which may account for the absence of an effect to feeding at day 1. Furthermore, the Malpighian tubules partially rely on the Na+-K+-2Cl- cotransporter and Na+-K+-ATPase pump to establish a high intracellular K+ ion gradient. The compensatory systems of the Malpighian tubules and the expression of the same conductance pathways in the salivary glands highlights the possibility that the Drosophila salivary gland is capable of utilizing these same pathways for establishing the intracellular K+ ion concentration as well as providing redundancy into the system for salivary gland K+ excretion. However, additional studies are required to validate this notion (Swale, 2017).

This study provided the first insight into the role of K+ ion channels in arthropod salivary gland physiology. Such knowledge helped understand the machinery arthropods evolved in the salivary gland to facilitate food acquisition. A significant amount of research remains to be performed to elucidate all the mechanisms of K+ ion transport that is required for proper gland function, but this study provides clear evidence that inward rectifying potassium channels expressed in the insect salivary gland are critical for its proper function as evidenced by alterations in feeding ability after chemical- or genetic- depletion. This study serves as a proof-of-concept that VU041 could serve as a lead compound for the development of new/novel vector control agents aimed at disrupting blood feeding and pathogen transport. Therefore, future studies will aim to expound on these data to characterize the functional relationship between Kir channels, Na+-K+-2Cl- cotransporter and Na+-K+-ATPase pumps as well as identify the role of Kir channels in the salivary glands of arthropod disease vectors as a means for the development of new/novel vector control agents aimed at disrupting blood feeding and pathogen transport (Swale, 2017).

Tango1 coordinates the formation of ER/Golgi docking sites to mediate secretory granule formation

Regulated secretion is a conserved process occurring across diverse cells and tissues. Current models suggest that the conserved cargo receptor Tango1 mediates the packaging of collagen into large coat protein complex II (COPII) vesicles that move from the endoplasmic reticulum (ER) to the Golgi apparatus. However, how Tango1 regulates the formation of COPII carriers and influences the secretion of other cargo remains unknown. Through high-resolution imaging of Tango1, COPII, Golgi and secretory cargo (mucins) in Drosophila larval salivary glands, this study found that Tango1 forms ring-like structures that mediate the formation of COPII rings, rather than vesicles. These COPII rings act as docking sites for the cis-Golgi. Moreover, nascent secretory mucins were observed emerging from the Golgi side of these Tango1/COPII/Golgi complexes, suggesting that these structures represent functional docking sites/fusion points between the ER exit sites and the Golgi. Loss of Tango1 disrupted the formation of COPII rings, the association of COPII with the cis-Golgi, mucin O-glycosylation and secretory granule biosynthesis. Additionally, this study identified a Tango1 self-association domain that is essential for formation of this structure. These results provide evidence that Tango1 organizes an interaction site where secretory cargo is efficiently transferred from the ER to Golgi and then to secretory vesicles. These findings may explain how the loss of Tango1 can influence Golgi/ER morphology and affect the secretion of diverse proteins across many tissues (Reynolds, 2019).

Secretion of proteins is a highly conserved event occurring in all eukaryotic species and across many tissues. This process begins in the ER where proteins destined to be secreted are synthesized and transported to the cis-region of the Golgi apparatus in a COPII-dependent process. Appropriately modified and folded proteins are packaged into secretory vesicles emanating from the trans-Golgi network that then await appropriate signals before fusing with the plasma membrane to release their contents. However, the mechanisms whereby bulky cargo is efficiently packaged into small vesicular transport vehicles and moved between compartments of the secretory apparatus are largely unknown (Reynolds, 2019).

Recently studies have identified an essential cargo receptor (Tango1 or MIA Src homology 3 (SH3) domain ER export factor 3; MIA3) responsible for the efficient packaging and secretion of high-molecular-weight collagen. Tango1, a type I transmembrane protein located at the ER exit sites (ERES) was first identified in a screen for genes that affect general secretion and Golgi morphology in Drosophila cells (Bard, 2006). Subsequent studies demonstrated a role for Tango1 in collagen secretion, whereby it is thought to mediate the formation of large COPII megacarriers capable of transporting the large procollagen rods from the ER to the Golgi apparatus. Current models suggest that the luminal SH3 domain of Tango1 binds to the procollagen chaperone HSP47, directing procollagen to sites of COPII vesicle formation. Tango1 is thought to modulate the size of the COPII vesicles by recruiting factors that limit Sar1GTPase activity, thus allowing the vesicles to grow in size to accommodate this large cargo (Reynolds, 2019).

Many recent studies have suggested that Tango1 is important for the secretion of additional molecules other than collagen. In mammalian cells, Tango1 affects the export of bulky lipid particles such as pre-chylomicrons/very low-density lipoproteins. In Drosophila, loss of Tango1 results in defects in the secretion of mucins, laminins, perlecan, and other extracellular matrix proteins. Additional genetic studies suggest that Tango1 influences general secretion rather than the specific secretion of certain proteins. These studies also identified many additional Golgi proteins that interact with Tango1, either directly or indirectly, such as Grasp65 and GM130, and suggest that there may exist more direct contacts between the ER and Golgi that are mediated by Tango1. Other work suggests that although Tango1 is important for the secretion of bulky cargo, it has an additional role in ER-Golgi morphology. However, high-resolution visualization of Tango1 dynamics and COPII vesicle formation relative to endogenous cargo biosynthesis and packaging has been challenging given the small size of these structures and the resolution limits of light microscopy. Thus, the exact roles Tango1 plays in the packaging and secretion of diverse cargos, as well as ER-Golgi morphology, remain unclear (Reynolds, 2019).

This study used the Drosophila larval salivary gland (SG) to image the relationship between Tango1 and the synthesis and packaging of secretory cargo (mucins) in real time, taking advantage of the increased spatial resolution unique to this gland. The SG undergoes hormonally regulated secretory granule formation that results in secretory granules of 3-8 microns in diameter (~10-100x larger than those seen in mammalian systems) that are filled with highly O-glycosylated mucin proteins (19-23). Drosophila mucins are similar in structure to mammalian mucins (having serine/threonine-rich O-glycosylated regions) but are typically smaller in size. Fly lines expressing GFP-tagged versions of one secretory mucin (Sgs3-GFP) have allowed high-resolution, real-time imaging of secretory granule formation and secretion. The genetic tractability of Drosophila has also allowed the identification of factors that control secretory vesicle formation, morphology, and extrusion of bulky cargo, such as mucins. Through real-time imaging using this system, this study found that Tango1 undergoes regulated self-association and dynamic shape changes during hormonally induced secretion to form ring structures that mediate the formation of COPII rings rather than vesicles. These Tango1-COPII rings act as docking sites for the cis-Golgi. Moreover, nascent secretory mucins were imaged emerging from the Golgi side of these Tango1-COPII-Golgi complexes, suggesting that these structures represent functional docking sites/fusion points between the ER exit sites and the Golgi. Taken together, these data suggest that Tango1 acts as a scaffold for the formation of functional ER-Golgi junctions that allow the efficient synthesis, intraorganellar transport, and packaging of diverse secretory cargo (Reynolds, 2019).

Taking advantage of the high spatial resolution of secretory structures in the Drosophila larval SG, this study has demonstrated that Tango1 coordinates the formation of ER-Golgi interaction sites to mediate secretory vesicle formation. High resolution imaging of Tango1 relative to COPII, Golgi, and secretory cargo demonstrates that Tango1 self-associates to form rings that appear to orchestrate the formation of COPII rings. Moreover, cis-Golgi markers localize within these rings, forming a distinct Tango1-COPII-Golgi structure. In support of this Tango1-COPII-Golgi structure being a functional site of interaction between the ER and Golgi, secretory granules were found emanating from the trans-Golgi face of this structure. These results support a model where Tango1 mediates a functional interaction point between the ERES and Golgi to allow efficient transfer of cargo and the subsequent formation of secretory granules (Reynolds, 2019).

This unique structure and the formation of secretory vesicles are dependent on Tango1. This study found that loss of Tango1 resulted in the loss of the COPII rings, loss of the organized association of the cis-Golgi with COPII, and loss of secretory vesicles. Additionally, Tango1 overexpression in Drosophila cells was sufficient to drive COPII ring formation and Golgi association. Previous studies have demonstrated direct binding of Tango1 and COPII components, which likely orchestrates the overlapping ring formation. Likewise, COPII components are known to interact with various cis-Golgi proteins, which likely drives their association in this structure. The formation of this entire structure depends on Tango1 self-association via the CCD1 domain in the cytoplasmic region. This is similar to the domain responsible for Tango1 self-association in mammals, suggesting conserved aspects of Tango1 action between these species (Reynolds, 2019).

Interestingly, the COPII structures present in this system exist as rings rather than spherical vesicular structures. Whether the COPII ring represents a structure unique to Drosophila or whether these structures might also be present in mammals awaits further investigation. However, evidence exists for diverse COPII structures in different systems, including tubules and protruding saccules from the ER membrane. The flexibility of the COPII coat may allow unique adaptations, depending on the biological context. Indeed, one recent study in mammalian cells suggests that procollagen transport occurs via a 'short-loop pathway' from the ER to the Golgi in the absence of large COPII vesicular carriers. This study offers support for the possibility that similar COPII-dependent ER-Golgi interaction sites may exist in mammals (Reynolds, 2019).

Previous work in Drosophila also supports a model where Tango1 mediates a connection to the Golgi. In this study, the authors demonstrated that overexpression of the Tango1 cytoplasmic domain can increase the size and density of ERES and increase the number of Golgi units, strongly suggesting a role for Tango1 in organizing both ERES and the Golgi. Indeed, the authors propose that large COPII carriers may begin fusion with the Golgi before separating from the ERES. The current imaging clearly demonstrates that Tango1, COPII, and the Golgi lie in close proximity and that their spatial separation likely precludes the formation of a separate, large COPII vesicular carrier. Moreover, the finding that mucin cargo emerges from the trans-Golgi face of this structure strongly supports a model where Tango1 serves to organize ordered ERES/Golgi interactions sites through which cargo passes. The results and model would also explain previous Drosophila studies where loss of Tango1 affects Golgi structure as well as the secretion of diverse proteins. Tango1 was originally discovered in an RNAi screen in Drosophila cells for genes that affect both secretion and Golgi structure (Tango = transport and Golgi organization). Likewise, more recent studies have suggested that Tango1 plays a role in the interaction of the Golgi and ER that is independent of its role in trafficking bulky cargo proteins. Many of these studies also present evidence that loss of Tango1 affects constitutive secretion of all proteins, including small reporter proteins. If Tango1 functions to mediate docking sites between ERES and Golgi, then the loss of Tango1 would be expected to result in changes in Golgi structure. Indeed, evidence was seen of Golgi structural changes when Tango1 was deleted from this system. Likewise, if the rate of constitutive secretion also benefits from these contact sites, one would expect the loss of Tango1 to affect this as well. These results and model are therefore consistent with previous studies that suggest a role for Tango1 in Golgi structure and constitutive secretion (Reynolds, 2019).

Studies investigating the role of Tango1 in other systems have proposed diverse models for how Tango1 coordinates the secretion of specific cargo. In mammals, it is proposed that the SH3 region of Tango1 interacts with the HSP47 chaperone, which then binds collagen to mediate its entry into the nascent COPII vesicle. However, this model does not explain how diverse cargo and constitutive secretion can be affected by the loss of Tango1. Additionally, this model necessitates a second packaging event for bulky cargo on the trans-side of the Golgi that must take place. The data presented in this study suggest that tissues under a high secretory burden may use Tango1 to reduce the number of independent packaging steps required for bulky, highly glycosylated cargo (such as mucins) by forming direct connection points between the ER and Golgi. This may ensure the efficient production and packaging of large amounts of cargo into secretory vesicles over a short period of time (Reynolds, 2019).

The size of the secretory structures present in this genetically tractable system and its amenability to real-time imaging during the secretory process have led to key insights with regard to secretory granule biogenesis and secretion. Using this system, it was previously shown that clathrin and AP-1, which localize to the trans-Golgi network, are required for proper secretory granule formation. Subsequently, the activity of the phosphatidylinositol kinase PI4KII was shown to be essential for secretory granules to reach mature size, likely because of influences on homotypic fusion events. Previous work has identified a role for O-glycosylation in secretory granule morphology during granule maturation. Real-time imaging has outlined the steps involved in secretory granule fusion with the apical plasma membrane and identified factors required for proper secretion of the mucinous contents. The current results are consistent with these prior studies and shed light on how cargo moves efficiently from the ER to the Golgi through a unique secretory structure whose organization depends on Tango1. This structure may explain how Tango1 has diverse effects on both regulated and constitutive secretion across many cell and tissue types. Moving forward, this tractable genetic imaging system will be amenable to identifying additional factors responsible for the highly organized and incredibly robust secretory program of the SG. Moreover, understanding the mechanisms by which biological systems maximize secretory capacity and efficiency may provide insights into novel strategies to restore defective secretion in disease states (Reynolds, 2019).

Model to Link Cell Shape and Polarity with Organogenesis

How do flat sheets of cells form gut and neural tubes? Across systems, several mechanisms are at play: cells wedge, form actomyosin cables, or intercalate. As a result, the cell sheet bends, and the tube elongates. It is unclear to what extent each mechanism can drive tube formation on its own. To address this question, this study computationally probe if one mechanism, either cell wedging or intercalation, may suffice for the entire sheet-to-tube transition. Using a physical model with epithelial cells represented by polarized point particles, it was shown that either cell intercalation or wedging alone can be sufficient and that each can both bend the sheet and extend the tube. When working in parallel, the two mechanisms increase the robustness of the tube formation. The successful simulations of the key features in Drosophila salivary gland budding, sea urchin gastrulation, and mammalian neurulation support the generality of these results (Nielsen, 2020).

Tango7 regulates cortical activity of caspases during reaper-triggered changes in tissue elasticity

Caspases perform critical functions in both living and dying cells; however, how caspases perform physiological functions without killing the cell remains unclear. This study identified a novel physiological function of caspases at the cortex of Drosophila salivary glands. In living glands, activation of the initiator caspase Dronc triggers cortical F-actin dismantling, enabling the glands to stretch as they accumulate secreted products in the lumen. Tango7 (Eukaryotic translation initiation factor 3 subunit m), not the canonical Apaf-1-adaptor Dark, regulates Dronc activity at the cortex; in contrast, dark is required for cytoplasmic activity of dronc during salivary gland death. Therefore, tango7 and dark define distinct subcellular domains of caspase activity. Furthermore, Tango7-dependent cortical Dronc activity is initiated by a sublethal pulse of the inhibitor of apoptosis protein (IAP) antagonist Reaper. The results support a model in which biological outcomes of caspase activation are regulated by differential amplification of IAP antagonists, unique caspase adaptor proteins, and mutually exclusive subcellular domains of caspase activity. Caspases are known for their role in cell death, but they can also participate in other physiological functions without killing the cells. In this study the authors show that unique caspase adaptor proteins can regulate caspase activity within mutually-exclusive and independently regulated subcellular domains (Kang, 2017).

Principles that govern the activation and function of caspases have fallen short in providing an understanding for how these enzymes can be activated to perform both delicate intracellular remodeling in living cells and total destruction in dying cells. This paper provides new insights into the mechanisms that regulate caspase activation by comparing two completely different biological outcomes in the same tissue that both require caspase function. The Drosophila homolog of caspase-9, dronc, is required for dismantling of the cortical F-actin cytoskeleton during salivary gland development -- a role that is distinct from its known function in the salivary gland death response during metamorphosis. By systematically dissecting the regulation of dronc function at the cortex, this study showed that cortical functions of dronc are regulated independently from its cytoplasmic functions. The cytoplasmic functions of activated dronc require the canonical adaptor protein Dark, while the cortical roles of dronc require tango7. In this manner, tango7 and Dark restrict the function of dronc to distinct subcellular domains. Moreover, this study also showed that these two functions can be initiated independently through differential amplification of IAP antagonist expression, providing a model for how lethal and vital roles of caspases can be differentially activated in the same cell. Finally, a new non-apoptotic function was identified for caspases in the control of tissue elasticity to accommodate buildup of secreted products in the lumen of secretory tissues, facilitating their timely release (Kang, 2017).

The results demonstrate that caspases can be activated in distinct, mutually exclusive subcellular domains within a single cell, and that these subcellular domains are generated by use of unique caspase adaptor proteins. Local activation of caspases, as detected by staining with antibodies to activated caspases, has been reported before; however, this study demonstrates that local activation is achieved by targeting caspases to subcellular domains, and this targeting is necessary for subcellular functions of these caspases. Importantly, this study shows that caspases can be activated specifically in one domain without being activated in another, providing a mechanism that allows control of caspase activity with a previously unknown level of subcellular precision. However, the mechanisms that restrict caspase cascades to distinct subcellular compartments remain unclear. It is possible that caspase expression levels are intentionally kept low during non-lethal responses, and localized enrichment mediates subcellular domain-specific activation. For example, if most of the Dronc protein present in the cell localizes to the cortex, then this specific localization may restrict caspase functions to the cortical compartment. This model fits with the results at the end of larval development; however, in dying glands, caspases are independently activated in cortical and cytoplasmic compartments, suggesting that additional mechanisms are in play to restrict caspase activity to the appropriate subcellular compartment. For example, it is possible that caspase cascades occur within a physical complex consisting of initiator caspases, their adaptor proteins, effector caspases, and their substrates. In this model, only one of these proteins, likely the initiator caspase, would need to be subcellularly localized in order to generate a compartment-specific caspase cascade. However, resolution of this possible mechanism will require further studies. This subcellular domain-specific model for caspase activation contrasts with the commonly held belief that activated caspase cascades passively perpetuate themselves and spread throughout the cell, and also opens the possibility that caspases, through specific subcellular localization mediated by adaptor proteins, may play a role in many yet-to-be-identified biological processes (Kang, 2017).

This study demonstrates that differential amplification of IAP antagonists at specific developmental stages determines lethal vs. non-lethal outcomes of caspase activation. In the system used in this study, differential amplification is accomplished through the use of transcription factors that function downstream of a steroid hormone signal. However, caspases must have an ability to 'sense' the magnitude of the IAP antagonist pulse, ensuring that they initiate the appropriate lethal or non-lethal responses. One possible 'sensing' mechanism may involve the aforementioned selectivity of initiator caspase adaptor proteins, like was observed with tango7 and dark. In this model, some adaptor protein complexes would require a lower IAP antagonist threshold for initiator caspase activation than others. However, elucidation of the detailed molecular mechanisms mediating 'sensing' of IAP antagonist expression levels will require further study. Finally, the results indicate that small pulses of IAP antagonist expression are tissue specific, raising the possibility that many more of these pulses are generated in other tissues and developmental stages that have not yet been detected or characterized. The data suggests that non-lethal, physiological functions of caspases may be more widespread than previously thought (Kang, 2017).

These results show that caspases play a novel role during the secretion of glue proteins. Glue proteins are essential to allow a newly formed prepupa to adhere to a solid surface; however, when cortical F-actin dismantling fails, glue precociously 'leaks' onto the surface of the animal. Although precocious expulsion of glue does not appear to have a deleterious effect in the lab, in the wild, it may adversely affect fitness by inhibiting larval movement or reducing the ability of the animal to stick securely to a surface during metamorphosis. Additionally, the results raise the question of whether other exocrine tissues in different species, such as the mammary gland, may utilize caspases in a similar manner to accommodate large amounts of secreted luminal products prior to their release (Kang, 2017).

In conclusion, systematic analysis of vital and lethal responses to caspase activation in the same cells has revealed mechanisms that allow caspases to be activated without killing the cell. The results demonstrate that caspases can be activated in mutually exclusive subcellular domains, where activation of caspases in one domain does not trigger activation of caspases in another domain. These subcellular domains were shown to. e generated by different caspase adaptor proteins. It is likely that yet-to-be-identified adaptor proteins define other subcellular domains and, in so doing, help regulate the many physiological functions of caspases. Moreover, the results demonstrate that some of these subcellular domains have lower thresholds for activation of caspases, thereby allowing sublethal pulses of IAP antagonists to selectively initiate physiological functions of caspases. Together, these results outline a simple conceptual framework for controlling caspase activation during normal development and physiology (Kang, 2017).

Molecular mechanisms of developmentally programmed crinophagy in Drosophila

At the onset of metamorphosis, Drosophila salivary gland cells undergo a burst of glue granule secretion to attach the forming pupa to a solid surface. This study shows that excess granules evading exocytosis are degraded via direct fusion with lysosomes, a secretory granule-specific autophagic process known as crinophagy. This study found that the tethering complex HOPS (homotypic fusion and protein sorting); the small GTPases Rab2, Rab7, and its effector, PLEKHM1; and a SNAP receptor complex consisting of Syntaxin 13, Snap29, and Vamp7 are all required for the fusion of secretory granules with lysosomes. Proper glue degradation within lysosomes also requires the Uvrag-containing Vps34 lipid kinase complex and the v-ATPase proton pump, whereas Atg genes involved in macroautophagy are dispensable for crinophagy. This work establishes the molecular mechanism of developmentally programmed crinophagy in Drosophila and paves the way for analyzing this process in metazoans (Csizmadia, 2017).

Endosomal vacuoles of the prepupal salivary glands of Drosophila play an essential role in the metabolic reallocation of iron

After fruit flies release their glycoprotein-rich secretory glue during pupariation, early-to-mid prepupal salivary glands undergo extensive endocytosis with widespread vacuolation of the cytoplasm followed by massive apocrine secretion. This study describes additional novel properties of these endosomes. The use of vital pH-sensitive probes provided confirmatory evidence that these endosomes have acidic contents and that there are two types of endocytosis seen in the prepupal glands. The salivary glands simultaneously generate mildly acidic, small, basally-derived endosomes and strongly acidic, large and apical endosomes. Multiple lines of evidence were obtained that the small basally-derived endosomes are chiefly involved in the uptake of dietary Fe(3+) iron. The fusion of basal endosomes with the larger and strongly acidic apical endosomes appears to facilitate optimal conditions for ferrireductase activity inside the vacuoles to release metabolic Fe(2+) iron. This study found increasing fluorescence of a glutathione-sensitive probe in large vacuoles, which appeared to depend on the amount of iron released by ferrireductase. Moreover, labeled mammalian iron-bound transferrin is actively taken up, providing direct evidence for active iron uptake by basal endocytosis. In addition, it was serendipitously found that small (basal) endosomes were uniquely recognized by PNA lectin, whereas large (apical) vacuoles bound DBA lectin (Farkas, 2018).


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genes expressed in salivary gland

Genes involved in organ development

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