Salivary gland development in Drosophila melanogaster

The Drosophila salivary gland is proving to be an excellent experimental system for understanding how cells commit to specific developmental programs and, once committed, how cells implement such decisions. Through genetic studies, the factors that determine where salivary glands will form, the number of cells committed to a salivary gland fate, and the distinction between the two major cell types (secretory cells and duct cells) have been discovered. Within the next few years, we will learn the molecular details of the interactions among the salivary gland regulators and salivary gland target genes. We will also learn how the early-expressed salivary gland genes coordinate their activities to mediate the morphogenetic movements required to form the salivary gland and the changes in cell physiology required for high secretory activity.     

Rac function in epithelial tube morphogenesis

Epithelial cell migration and morphogenesis require dynamic remodeling of the actin cytoskeleton and cell-cell adhesion complexes. Numerous studies in cell culture and in model organisms have demonstrated the small GTPase Rac to be a critical regulator of these processes; however, little is known about Rac function in the morphogenic movements that drive epithelial tube formation. Here, we use the embryonic salivary glands of Drosophila to understand the role of Rac in epithelial tube morphogenesis. We show that inhibition of Rac function, either through loss of function mutations or dominant-negative mutations, disrupts salivary gland invagination and posterior migration. In contrast, constitutive activation of Rac induces motile behavior and subsequent cell death. We further show that Rac regulation of salivary gland morphogenesis occurs through modulation of cell-cell adhesion mediated by the E-cadherin/beta-catenin complex and that shibire, the Drosophila homolog of dynamin, functions downstream of Rac in regulating beta-catenin localization during gland morphogenesis. Our results demonstrate that regulation of cadherin-based adherens junctions by Rac is critical for salivary gland morphogenesis and that this regulation occurs through dynamin-mediated endocytosis.     

Fork head and Sage maintain a uniform and patent salivary gland lumen through regulation of two downstream target genes, PH4alphaSG1 and PH4alphaSG2

(Fkh) is required to block salivary gland apoptosis, internalize salivary gland precursors, prevent expression of duct genes in secretory cells and maintain expression of CrebA, which is required for elevated secretory function. Here, we characterize two new Fkh-dependent genes: PH4alphaSG1 and PH4alphaSG2. We show through in vitro DNA-binding studies and in vivo expression assays that Fkh cooperates with the salivary gland-specific bHLH protein Sage to directly regulate expression of PH4alphaSG2, as well as sage itself, and to indirectly regulate expression of PH4alphaSG1. PH4alphaSG1 and PH4alphaSG2 encode alpha-subunits of resident ER enzymes that hydroxylate prolines in collagen and other secreted proteins. We demonstrate that salivary gland secretions are altered in embryos missing function of PH4alphaSG1 and PH4alphaSG2; secretory content is reduced and shows increased electron density by TEM. Interestingly, the altered secretory content results in regions of tube dilation and constriction, with intermittent tube closure. The regulation studies and phenotypic characterization of PH4alphaSG1 and PH4alphaSG2 link Fkh, which initiates tube formation, to the maintenance of an open and uniformly sized secretory tube.     

Salivary gland determination in Drosophila: a salivary-specific, fork head enhancer integrates spatial pattern and allows fork head autoregulation

In the early Drosophila embryo, a system of coordinates is laid down by segmentation genes and dorsoventral patterning genes. Subsequently, these coordinates must be interpreted to define particular tissues and organs. To begin understanding this process for a single organ, we have studied how one of the first salivary gland genes, fork head (fkh), is turned on in the primordium of this organ, the salivary placode. A placode-specific fkh enhancer was identified 10 kb from the coding sequence. Dissection of this enhancer showed that the apparently homogeneous placode is actually composed of at least four overlapping domains. These domains appear to be developmentally important because they predict the order of salivary invagination, are evolutionarily conserved, and are regulated by patterning genes that are important for salivary development. Three dorsoventral domains are defined by EGF receptor (EGFR) signaling, while stripes located at the anterior and posterior edges of the placode depend on wingless signaling. Further analysis identified sites in the enhancer that respond either positively to the primary activator of salivary gland genes, SEX COMBS REDUCED (SCR), or negatively to EGFR signaling. These results show that fkh integrates spatial pattern directly, without reference to other early salivary gland genes. In addition, we identified a binding site for FKH protein that appears to act in fkh autoregulation, keeping the gene active after SCR has disappeared from the placode. This autoregulation may explain how the salivary gland maintains its identity after the organ is established. Although the fkh enhancer integrates information needed to define the salivary placode, and although fkh mutants have the most extreme effects on salivary gland development thus far described, we argue that fkh is not a selector gene for salivary gland development and that there is no master, salivary gland selector gene. Instead, several genes independently sense spatial information and cooperate to define the salivary placode.     

Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila

BACKGROUND: Cell growth arrest and autophagy are required for autophagic cell death in Drosophila. Maintenance of growth by expression of either activated Ras, Dp110, or Akt is sufficient to inhibit autophagy and cell death in Drosophila salivary glands, but the mechanism that controls growth arrest is unknown. Although the Warts (Wts) tumor suppressor is a critical regulator of tissue growth in animals, it is not clear how this signaling pathway controls cell growth. RESULTS: Here, we show that genes in the Wts pathway are required for salivary gland degradation and that wts mutants have defects in cell growth arrest, caspase activity, and autophagy. Expression of Atg1, a regulator of autophagy, in salivary glands is sufficient to rescue wts mutant salivary gland destruction. Surprisingly, expression of Yorkie (Yki) and Scalloped (Sd) in salivary glands fails to phenocopy wts mutants. By contrast, misexpression of the Yki target bantam was able to inhibit salivary gland cell death, even though mutations in bantam fail to suppress the wts mutant salivary gland-persistence phenotype. Significantly, wts mutant salivary glands possess altered phosphoinositide signaling, and decreased function of the class I PI3K-pathway genes chico and TOR suppressed wts defects in cell death. CONCLUSIONS: Although we have previously shown that salivary gland degradation requires genes in the Wts pathway, this study provides the first evidence that Wts influences autophagy. Our data indicate that the Wts-pathway components Yki, Sd, and bantam fail to function in salivary glands and that Wts regulates salivary gland cell death in a PI3K-dependent manner.     

Invasion of mosquito salivary glands by malaria parasites: Prerequisites and defense strategies

The interplay between vector and pathogen is essential for vector-borne disease transmission. Dissecting the molecular basis of refractoriness of some vectors may pave the way to novel disease control mechanisms. A pathogen often needs to overcome several physical barriers, such as the peritrophic matrix, midgut epithelium and salivary glands. Additionally, the arthropod vector elicites immune responses that can severely limit transmission success. One important step in the transmission of most vector-borne diseases is the entry of the disease agent into the salivary glands of its arthropod vector. The salivary glands of blood-feeding arthropods produce a complex mixture of molecules that facilitate blood feeding by inhibition of the host haemostasis, inflammation and immune reactions. Pathogen entry into salivary glands is a receptor-mediated process, which requires molecules on the surface of the pathogen and salivary gland. In most cases, the nature of these molecules remains unknown. Recent advances in our understanding of malaria parasite entry into mosquito salivary glands strongly suggests that specific carbohydrate molecules on the salivary gland surface function as docking receptors for malaria parasites.     

From fate to function: the Drosophila trachea and salivary gland as models for tubulogenesis

Tube formation is a ubiquitous process required to sustain life in multicellular organisms. The tubular organs of adult mammals include the lungs, vasculature, digestive and excretory systems, as well as secretory organs such as the pancreas, salivary, prostate, and mammary glands. Other tissues, including the embryonic heart and neural tube, have requisite stages of tubular organization early in development. To learn the molecular and cellular basis of how epithelial cells are organized into tubular organs of various shapes and sizes, investigators have focused on the Drosophila trachea and salivary gland as model genetic systems for branched and unbranched tubes, respectively. Both organs begin as polarized epithelial placodes, which through coordinated cell shape changes, cell rearrangement, and cell migration form elongated tubes. Here, we discuss what has been discovered regarding the details of cell fate specification and tube formation in the two organs; these discoveries reveal significant conservation in the cellular and molecular events of tubulogenesis.     

Salivary glands and salivary pumps in adult Nymphalidae (Lepidoptera)

The salivary glands and salivary pumps were investigated by means of dissection and serial semithin sections in order to expose the anatomy and histology of Nymphalidae in relation to feeding ecology. The paired salivary glands are tubular, they begin in the head, and extend through the thorax into the abdomen. The epithelium is a unicellular layer consisting of a single cell type. Despite the uniform composition, each salivary gland can be divided into five anatomically and histologically distinct regions. The bulbous end region of the gland lies within the abdomen and is composed of highly prismatic glandular cells with large vacuoles in their cell bodies. The tubular secretion region extends into the thorax where it forms large loops running backward and forward. It is composed of glandular cells that lack large vacuoles. The salivary duct lies in the thorax and also shows a looped formation but is composed of flat epithelial cells. The salivary reservoir begins in the prothorax and reaches the head. Its cells are hemispherical and bulge out into the large lumen of the tube. In the head the outlet tube connects the left and right halves of the salivary gland, and its epithelial cells are flat. The salivary pump lies in the head ventral to the sucking pump and leads directly into the food canal of the proboscis. It is not part of the salivary gland but is derived from the salivarium. Both the thin cuticle of the roof of the salivary pump and the thick bottom are ventrally arched. Paired muscles extend from the hypopharyngeal ridges and obviously serve as dilators for the pump. A functional interpretation of the salivary pump suggests that when not in use, the dilators are not contracted and the pump is tightly closed due to its own elasticity. When the dilator muscles repeatedly contract, the saliva is forced forward into the food canal of the proboscis. The salivary gland anatomy was found to be similar to other Lepidoptera. Furthermore, the histology of the salivary glands is identical in all examined butterflies, even in the species which exhibit specialized pollen-feeding behavior.     

Distinct localization of MUC5B glycoforms in the human salivary glands

Salivary mucins, encoded by the MUC5B gene, make up a heterogeneous family of molecules, which are secreted by several glands, including the submandibular, sublingual, and palatine glands. Previous studies have shown that heterogeneity in the salivary mucin population is related to its multiglandular origin. In the present study we address the question to what extent the mucin (MUC5B) population from a single human salivary gland is made up of different glycoforms. Using monoclonal antibodies to defined protein and sulfated carbohydrate epitopes specific to MUC5B, we conduct an immunohistochemical study of different salivary gland types, including submandibular, sublingual, and labial glands. In all tissues studied we found a mosaic expression pattern of sulfo-Lewis a antigen, recognized by mAb F2, which in salivary glands is exclusively present on MUC5B. On the other hand, mucous acini were uniformly labeled by mAb EU-MUC5Bb, evoked against a peptide-stretch of the tandem repeat region of MUC5B. Double staining with both antibodies confirmed the presence of MUC5B-positive/sulfo-Lewis a-positive cells, as well as MUC5B-positive/sulfo-Lewis a-negative cells within one glandular unit. These results indicate that one and the same salivary gland synthesizes different MUC5B glycoforms.     

Identification of major soluble salivary gland proteins in teneral Glossina morsitans morsitans

Salivary glands of tsetse flies (Diptera: Glossinidiae) contain molecules that are involved in preventing blood clotting during feeding as well as molecules thought to be intimately associated with trypanosome development and maturation. Here we present a protein microchemical analysis of the major soluble proteins of the salivary glands of Glossina morsitans morsitans, an important vector of African trypanosomes. Differential solubilization of salivary proteins was followed by reverse-phase, high-performance liquid chromatography (HPLC) and analysis of fractions by 1-D gel electrophoresis to reveal four major proteins. Each protein was subjected to amino acid microanalysis and N-terminal microsequencing. A protein chemical approach using high-resolution 2-D gel electrophoresis and mass spectrometry was also used to identify the salivary proteins. Matrix-assisted, laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry methods were used for peptide mass mapping and sequencing, respectively. Sequence information and peptide mass maps queried against the NCBI non-redundant database confirmed the identity of the first protein as tsetse salivary gland growth factor-1 (TSGF-1). Two proteins with no known function were identified as tsetse salivary gland protein 1 (Tsal 1) and tsetse salivary gland protein 2 (Tsal 2). The fourth protein was identified as Tsetse antigen-5 (TAg-5), which is a member of a large family of anti-haemostatic proteins. The results show that these four proteins are the most abundant soluble gene products present in salivary glands of teneral G. m. morsitans. We discuss the possible functions of these major proteins in cyclical transmission of African trypanosomes.     

Pro-apoptotic gene knockdown mediated by nanocomplexed siRNA reduces radiation damage in primary salivary gland cultures

A critical issue in the management of head and neck tumors is radioprotection of the salivary glands. We have investigated whether siRNA-mediated gene knock down of pro-apoptotic mediators can reduce radiation-induced cellular apoptosis in salivary gland cells in vitro. We used novel, pH-responsive nanoparticles to deliver functionally active siRNAs into cultures of salivary gland cells. The nanoparticle molecules are comprised of cationic micelles that electrostatically interact with the siRNA, protecting it from nuclease attack, and also include pH-responsive endosomolytic constituents that promote release of the siRNA into the target cell cytoplasm. Transfection controls with Cy3-tagged siRNA/nanoparticle complexes showed efficiently internalized siRNAs in more than 70% of the submandibular gland cells. We found that introduction of siRNAs specifically targeting the Pkcδ or Bax genes significantly blocked the induction of these pro-apoptotic proteins that normally occurs after radiation in cultured salivary gland cells. Furthermore, the level of cell death from subsequent radiation, as measured by caspase-3, TUNEL, and mitochondrial disruption assays, was significantly decreased. Thus, we have successfully demonstrated that the siRNA/nanoparticle-mediated knock down of pro-apoptotic genes can prevent radiation-induced damage in submandibular gland primary cell cultures.     

WRS-85D: A tryptophanyl-tRNA synthetase expressed to high levels in the developing Drosophila salivary gland

In a screen for genes expressed in the Drosophila embryonic salivary gland, we identified a tryptophanyl-tRNA synthetase gene that maps to cytological position 85D (WRS-85D). WRS-85D expression is dependent on the homeotic gene Sex combs reduced (Scr). In the absence of Scr function, WRS-85D expression is lost in the salivary gland primordia; conversely, ectopic expression of Scr results in expression of WRS-85D in new locations. Despite the fact that WRS-85D is a housekeeping gene essential for protein synthesis, we detected both WRS-85D mRNA and protein at elevated levels in the developing salivary gland. WRS-85D is required for embryonic survival; embryos lacking the maternal contribution were unrecoverable, whereas larvae lacking the zygotic component died during the third instar larval stage. We showed that recombinant WRS-85D protein specifically charges tRNATrp, and WRS-85D is likely to be the only tryptophanyl-tRNA synthetase gene in Drosophila. We characterized the expression patterns of all 20 aminoacyl-tRNA synthetases and found that of the four aminoacyl-tRNA synthetase genes expressed at elevated levels in the salivary gland primordia, WRS-85D is expressed at the highest level throughout embryogenesis. We also discuss the potential noncanonical activities of tryptophanyl-tRNA synthetase in immune response and regulation of cell growth.     

Xylose-linked proteoglycan synthesis does not have a primary role in the control of secretory cell differentiation in salivary glands

Xylose-linked proteoglycans, particularly chondroitin sulfate proteoglycan, have been shown to play a significant role in the regulation of salivary gland morphogenesis. The purpose of this study was to determine if xylose-linked proteoglycans are involved in the regulation of differentiation of salivary gland secretory cells. Embryonic rat submandibular salivary gland rudiments were cultured for 120 hr in the presence or absence of 0.75 to 1.0 mM p-nitrophenyl-beta-D-xylopyranoside (beta-D-xyloside), an inhibitor of xylose-linked proteoglycan assembly. beta-D-Xyloside has been shown to block submandibular gland morphogenesis (Thompson and Spooner, 1982). In the present study glandular morphogenesis was blocked in 93.3% of the rudiments cultured in the presence of beta-D-xyloside. However, secretory cell differentiation was observed in 71.4% of those rudiments in which morphogenesis had been inhibited. Biochemical evaluation confirmed that xylose-linked proteoglycan assembly had been inhibited by xyloside. These results indicate that while xylose-linked proteoglycans play a significant role in the control of salivary gland morphogenesis these molecules are not primary regulators for secretory cell differentiation within developing salivary glands.