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.     

The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels

Ecdysteroids regulate a wide variety of cellular processes during arthropod development, yet little is known about the genes involved in the biosynthesis of these hormones. Previous studies have suggested that production of 20-hydroxyecdysone in Drosophila and other arthropods involves a series of cytochrome P450 catalyzed hydroxylations of cholesterol. In this report, we show that the disembodied (dib) locus of Drosophila codes for a P450-like sequence. In addition, we find that dib mutant embryos have very low titers of ecdysone and 20-hydroxyecdysone (20E) and fail to express IMP-E1 and L1, two 20E-inducible genes, in certain tissues of the embryo. In situ hybridization studies reveal that dib is expressed in a complex pattern in the early embryo, which eventually gives way to restricted expression in the prothoracic portion of the ring gland. In larval and adult tissues, dib expression is observed in the prothoracic gland and follicle cells of the ovaries respectively, two tissues known to synthesize ecdysteroids. Phenotypic analysis reveals that dib mutant embryos produce little or no cuticle and exhibit severe defects in many late morphogenetic processes such as head involution, dorsal closure and gut development. In addition, we examined the phenotypes of several other mutants that produce defective embryonic cuticles. Like dib, mutations in the spook (spo) locus result in low embryonic ecdysteroid titers, severe late embryonic morphological defects, and a failure to induce IMP-E1. From these data, we conclude that dib and spo likely code for essential components in the ecdysone biosynthetic pathway and that ecdysteroids regulate many late embryonic morphogenetic processes such as cell movement and cuticle deposition.     

Regulation and function of Scr, exd, and hth in the Drosophila salivary gland

Salivary gland formation in the Drosophila embryo is dependent on the homeotic gene Sex combs reduced (Scr). When Scr function is missing, salivary glands do not form, and when SCR is expressed everywhere in the embryo, salivary glands form in new places. Scr is normally expressed in all the cells that form the salivary gland. However, as the salivary gland invaginates, Scr mRNA and protein disappear. Homeotic genes, such as Scr, specify tissue identity by regulating the expression of downstream target genes. For many homeotic proteins, target gene specificity is achieved by cooperatively binding DNA with cofactors. Therefore, it is likely that SCR also requires a cofactor(s) to specifically bind to DNA and regulate salivary gland target gene expression. Here, we show that two homeodomain-containing proteins encoded by the extradenticle (exd) and homothorax (hth) genes are also required for salivary gland formation. exd and hth function at two levels: (1) exd and hth are required to maintain the expression of Scr in the salivary gland primordia prior to invagination and (2) exd and hth are required in parallel with Scr to regulate the expression of downstream salivary gland genes. We also show that Scr regulates the nuclear localization of EXD in the salivary gland primordia through repression of homothorax (hth) expression, linking the regulation of Scr activity to the disappearance of Scr expression in invaginating salivary glands.     

Coordination of murine parotid secretory protein and salivary amylase expression.

PSP, parotid secretory protein, and salivary amylase are the major secretory proteins of mouse parotid gland where they appear in a constant ratio. Here we describe the isolation of the PSP gene and show through expression analysis on this and the salivary amylase gene that the two genes are transcribed in a coordinate fashion in adult animals, whereas the activation profiles are different during postnatal development. An explanation is put forward that involves activation of the genes at different stages of the acinar cell differentiation, leading in adults to the maximal and thus proportionate expression.     

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.     

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.