JAK/STAT signaling coordinates stem cell proliferation and multilineage differentiation in the Drosophila intestinal stem cell lineage

Adult stem cells are the most primitive cells of a lineage and are distinguished by the properties of self-renewal and multipotency. Coordinated control of stem cell proliferation and multilineage differentiation is essential to ensure a steady output of differentiated daughter cells necessary to maintain tissue homeostasis. However, little is known about the signals that coordinate stem cell proliferation and daughter cell differentiation. Here we investigate the role of the conserved JAK/STAT signaling pathway in the Drosophila intestinal stem cell (ISC) lineage. We show first, that JAK/STAT signaling is normally active in both ISCs and their newly formed daughters, but not in terminally differentiated enteroendocrine (ee) cells or enterocyte (EC) cells. Second, analysis of ISC lineages shows that JAK/STAT signaling is necessary but not sufficient for daughter cell differentiation, indicating that competence to undergo multilineage differentiation depends upon JAK/STAT. Finally, our analysis reveals JAK/STAT signaling to be a potent regulator of ISC proliferation, but not ISC self-renewal. On the basis of these findings, we suggest a model in which JAK/STAT signaling coordinates the processes of stem cell proliferation with the competence of daughter cells to undergo multilineage differentiation, ensuring a robust cellular output in the lineage.     

Steroid signaling promotes stem cell maintenance in the Drosophila testis

Stem cell regulation by local signals is intensely studied, but less is known about the effects of hormonal signals on stem cells. In Drosophila, the primary steroid twenty-hydroxyecdysone (20E) regulates ovarian germline stem cells (GSCs) but was considered dispensable for testis GSC maintenance. Male GSCs reside in a microenvironment (niche) generated by somatic hub cells and adjacent cyst stem cells (CySCs). Here, we show that depletion of 20E from adult males by overexpressing a dominant negative form of the Ecdysone receptor (EcR) or its heterodimeric partner ultraspiracle (usp) causes GSC and CySC loss that is rescued by 20E feeding, uncovering a requirement for 20E in stem cell maintenance. EcR and USP are expressed, activated and autonomously required in the CySC lineage to promote CySC maintenance, as are downstream genes ftz-f1 and E75. In contrast, GSCs non-autonomously require ecdysone signaling. Global inactivation of EcR increases cell death in the testis that is rescued by expression of EcR-B2 in the CySC lineage, indicating that ecdysone signaling supports stem cell viability primarily through a specific receptor isoform. Finally, EcR genetically interacts with the NURF chromatin-remodeling complex, which we previously showed maintains CySCs. Thus, although 20E levels are lower in males than females, ecdysone signaling acts through distinct cell types and effectors to ensure both ovarian and testis stem cell maintenance.     

Myoblast fusion in Drosophila

The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process [1], [2], [3] and [4]. With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.     

Smurf-mediated differential proteolysis generates dynamic BMP signaling in germline stem cells during Drosophila testis development

Germline stem cells (GSCs) produce gametes throughout the reproductive life of many animals, and intensive studies have revealed critical roles of BMP signaling to maintain GSC self-renewal in Drospophila adult gonads. Here, we show that BMP signaling is downregulated as testes develop and this regulation controls testis growth, stem cell number, and the number of spermatogonia divisions. Phosphorylated Mad (pMad), the activated Drosophila Smad in germ cells, was restricted from anterior germ cells to GSCs and hub-proximal cells during early larval development. pMad levels in GSCs were then dramatically downregulated from early third larval instar (L3) to late L3, and maintained at low levels in pupal and adult GSCs. The spatial restriction and temporal down-regulation of pMad, reflecting the germ cell response to BMP signaling activity, required action in germ cells of E3 ligase activity of HECT domain protein Smurf. Analyses of Smurf mutant testes and dosage-dependent genetic interaction between Smurf and mad indicated that pMad downregulation was required for both the normal decrease in stem cell number during testis maturation in the pupal stage, and for normal limit of four rounds of spermatogonia cell division for control of germ cell numbers and testis size. Smurf protein was expressed at a constant low level in GSCs and spermatogonia during development. Rescue experiments showed that expression of exogenous Smurf protein in early germ cells promoted pMad downregulation in GSCs in a stage-dependent but concentration-independent manner, suggesting that the competence of Smurf to attenuate response to BMP signaling may be regulated during development. Taken together, our work reveals a critical role for differential attenuation of the response to BMP signaling in GSCs and early germ cells for control of germ cell number and gonad growth during development.     

Regulation of apoptosis of rbf mutant cells during Drosophila development

Inactivation of the retinoblastoma gene Rb leads to defects in cell proliferation, differentiation, or apoptosis, depending on specific cell or tissue types. To gain insights into the genes that can modulate the consequences of Rb inactivation, we carried out a genetic screen in Drosophila to identify mutations that affected apoptosis induced by inactivation of the Retinoblastoma-family protein (rbf) and identified a mutation that blocked apoptosis induced by rbf. We found this mutation to be a new allele of head involution defective (hid) and showed that hid expression is deregulated in rbf mutant cells in larval imaginal discs. We identified an enhancer that regulates hid expression in response to developmental cues as well as to radiation and demonstrated that this hid enhancer is directly repressed by RBF through an E2F binding site. These observations indicate that apoptosis of rbf mutant cells is mediated by an upregulation of hid. Finally, we showed that bantam, a miRNA that regulates hid translation, is expressed in the interommatidial cells in the larval eye discs and modulates the survival of rbf mutant cells.     

Hedgehog does not guide migrating Drosophila germ cells

In many species, the germ cells, precursors of sperm and egg, migrate during embryogenesis. The signals that regulate this migration are thus essential for fertility. In flies, lipid signals have been shown to affect germ cell guidance. In particular, the synthesis of geranylgeranyl pyrophosphate through the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Hmgcr) pathway is critical for attracting germ cells to their target tissue. In a genetic analysis of signaling pathways known to affect cell migration of other migratory cells, we failed to find a role for the Hedgehog (Hh) pathway in germ cell migration. However, previous reports had implicated Hh as a germ cell attractant in flies and suggested that Hh signaling is enhanced through the action of the Hmgcr pathway. We therefore repeated several critical experiments and carried out further experiments to test specifically whether Hh is a germ cell attractant in flies. In contrast to previously reported findings and consistent with findings in zebrafish our data do not support the notion that Hh has a direct role in the guidance of migrating germ cells in flies.     

Left-right asymmetric morphogenesis of the anterior midgut depends on the activation of a non-muscle myosin II in Drosophila

Many animals exhibit stereotypical left-right (LR) asymmetry in their internal organs. The mechanisms of LR axis formation required for the subsequent LR asymmetric development are well understood, especially in some vertebrates. However, the molecular mechanisms underlying LR asymmetric morphogenesis, particularly how mechanical force is integrated into the LR asymmetric morphogenesis of organs, are poorly understood. Here, we identified zipper (zip), encoding a Drosophila non-muscle myosin II (myosin II) heavy chain, as a gene required for LR asymmetric development of the embryonic anterior midgut (AMG). Myosin II is known to directly generate mechanical force in various types of cells during morphogenesis and cell migration. We found that myosin II was involved in two events in the LR asymmetric development of the AMG. First, it introduced an LR bias to the directional position of circular visceral muscle (CVMU) cells, which externally cover the midgut epithelium. Second, it was required for the LR-biased rotation of the AMG. Our results suggest that myosin II in CVMU cells plays a crucial role in generating the force leading to LR asymmetric morphogenesis. Taken together with previous studies in vertebrates, the involvement of myosin II in LR asymmetric morphogenesis might be conserved evolutionarily.     

Regulation of EGFR and Notch signaling by distinct isoforms of D-cbl during Drosophila development

Cells receive and interpret extracellular signals to regulate cellular responses such as proliferation, cell survival and differentiation. However, proper inactivation of these signals is critical for appropriate homeostasis. Cbl proteins are E3-ubiquitin ligases that restrict receptor tyrosine kinase (RTK) signaling, most notably EGFR (Epidermal Growth Factor Receptor), via the endocytic pathway. Consistently, many mutant phenotypes of Drosophila cbl (D-cbl) are due to inappropriate activation of EGFR signaling. However, not all D-cbl phenotypes can be explained by increased EGFR activity. Here, we report that D-Cbl also negatively regulates Notch activity during eye and wing development. D-cbl produces two isoforms by alternative splicing. The long isoform, D-CblL, regulates the EGFR. We found that the short isoform, D-CblS, preferentially restricts Notch signaling. Specifically, our data imply that D-CblS controls the activity of the Notch ligand Delta. Taken together, these data suggest that D-Cbl controls the EGFR and Notch/Delta signaling pathways through production of two alternatively spliced isoforms during development in Drosophila.     

Ectopic Repo suppresses expression of castor gene in Drosophila central nervous system

The ventral nerve cord (VNC) of the Drosophila embryo is derived from neuroblasts (NBs). NBs divide in a stem cell lineage to generate a series of ganglion mother cells (GMCs), each of which divides once to produce a pair of neurons or glial cells. One of the NB genes, castor (cas), is expressed in a subset of NBs and has never been identified in neurons and the peripheral nervous system; cas plays a role in axonogenesis. But its limited expression along the dorsal-ventral axis within the central nervous system has not been investigated yet. In the present study, we examined the expression patterns of both genes using confocal microscopy to determine the effects of repo mutation on cas expression. Cas was mainly expressed in layers different from repo-expressed layers during early embryogenesis: repo was expressed mostly from deep to mid layers, while cas, from mid to superficial layers. Loss-of-function of repo did not result in an ectopic expression of cas, but rather, a scattering of cas-expressing cells. However, repo gain-of-function mutation caused repression of cas. In addition, repo-expressing cells seemed to block the migration of cas-expressing cells.     

Identifying calpain substrates in intact S2 cells of Drosophila

Calpains are cysteine proteases involved in a number of physiological and pathological processes, yet our knowledge of substrates cleaved in vivo, in intact cells, is scarce. In this work we made an attempt to develop a technique for finding calpain substrates in intact Drosophila Schneider S2 cells. The procedure consists in comparative 2D gelelectrophoresis: three identical samples were treated in different ways: A (control, no addition), B, activated (Ca²⁺ and ionomycin added), C, inactivated (additions as in B + specific calpain inhibitor). 2D gel pattern were analyzed by densitometry. Spots showing density relation A > B << C were identified by mass spectroscopy. In a typical run, 11 candidate substrates were recognized; out of these, four were randomly selected: all four were verified to be calpain substrates, by digestion of the recombinant protein with recombinant calpain.     

The deubiquitinase emperor's thumb is a regulator of apoptosis in Drosophila

We have characterized the gene emperor's thumb (et) and showed that it is required for the regulation of apoptosis in Drosophila. Loss-of-function mutations in et result in apoptosis associated with a decrease in the concentration of DIAP1. Overexpression of one form of et inhibits apoptosis, consistent with et having an anti-apoptotic function; however, overexpression of a second form of et induces apoptosis, indicating that the two forms of et may have competing functions. et encodes a protein deubiquitinase, suggesting it regulates apoptosis by controlling the stability of apoptotic regulatory proteins.     

Thermal preference in Drosophila

Environmental temperature strongly affects physiology of ectotherms. Small ectotherms, like Drosophila, cannot endogenously regulate body temperature so must rely on behavior to maintain body temperature within a physiologically permissive range. Here we review what is known about Drosophila thermal preference. Work on thermal behavior in this group is particularly exciting because it provides the opportunity to connect genes to neuromolecular mechanisms to behavior to fitness in the wild.     

On the roles of Notch, Delta, kuzbanian, and inscuteable during the development of Drosophila embryonic neuroblast lineages

The generation of cellular diversity in the nervous system involves the mechanism of asymmetric cell division. Besides an array of molecules, including the Par protein cassette, a heterotrimeric G protein signalling complex, Inscuteable plays a major role in controlling asymmetric cell division, which ultimately leads to differential activation of the Notch signalling pathway and correct specification of the two daughter cells. In this context, Notch is required to be active in one sibling and inactive in the other. Here, we investigated the requirement of genes previously known to play key roles in sibling cell fate specification such as members of the Notch signalling pathway, e.g., Notch (N), Delta (Dl), and kuzbanian (kuz) and a crucial regulator of asymmetric cell division, inscuteable (insc) throughout lineage progression of 4 neuroblasts (NB1-1, MP2, NB4-2, and NB7-1). Notch-mediated cell fate specification defects were cell-autonomous and were observed in all neuroblast lineages even in cells born from late ganglion mother cells (GMC) within the lineages. We also show that Dl functions non-autonomously during NB lineage progression and clonal cells do not require Dl from within the clone. This suggests that within a NB lineage Dl is dispensable for sibling cell fate specification. Furthermore, we provide evidence that kuz is involved in sibling cell fate specification in the central nervous system. It is cell-autonomously required in the same postmitotic cells which also depend on Notch function. This indicates that KUZ is required to facilitate a functional Notch signal in the Notch-dependent cell for correct cell fate specification. Finally, we show that three neuroblast lineages (NB1-1, NB4-2, and NB7-1) require insc function for sibling cell fate specification in cells born from early GMCs whereas insc is not required in cells born from later GMCs of the same lineages. Thus, there is differential requirement for insc for cell fate specification depending on the stage of lineage progression of NBs.     

Intestinal stem cells in the adult Drosophila midgut

Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury.     

The role of stem cells in midgut growth and regeneration

Summary TheManduca sexta (L.) [Lepidoptera: Sphingidae] andHeliothis virescens (F.) [Lepidoptera: Noctuidae] midguts consist of a pseudostratified epithelium surrounded by striated muscle and tracheae. This epithelium contains goblet, columnar, and basal stem cells. The stem cells are critically important in that they are capable of massive proliferation and differentiation. This growth results in a fourfold enlargement of the midgut at each larval molt. The stem cells are also responsible for limited cell replacement during repair. While the characteristics of the stem cell population vary over the course of an instar, stem cells collected early in an instar and those collected late can start in vitro cultures. Cultures of larval stem, goblet, and columnar cells survive in vitro for several mo through proliferation and differentiation of the stem cells. One of the two polypeptide differentiation factors which have been identified and characterized from the culture medium has now been shown to be present in midgut in vivo. Thus the ability to examine lepidopteran midgut stem cell growth in vitro and in vivo is proving to be effective in determining the basic features of stem cell action and regulation. Mention of any product in this publication does not imply endorsement by the USDA.     

Sex-specific repression of dachshund is required for Drosophila sex comb development

The origin of new morphological structures requires the establishment of new genetic regulatory circuits to control their development, from initial specification to terminal differentiation. The upstream regulatory genes are usually the first to be identified, while the mechanisms that translate novel regulatory information into phenotypic diversity often remain obscure. In particular, elaborate sex-specific structures that have evolved in many animal lineages are inevitably controlled by sex-determining genes, but the genetic basis of sexually dimorphic cell differentiation is rarely understood. In this report, we examine the role of dachshund (dac), a gene with a deeply conserved function in sensory organ and appendage development, in the sex comb, a recently evolved male-specific structure found in some Drosophila species. We show that dac acts during metamorphosis to restrict sex comb development to the appropriate leg region. Localized repression of dac by the sex determination pathway is necessary for male-specific morphogenesis of sex comb bristles. This pupal function of dac is separate from its earlier role in leg patterning, and Dac at this stage is not dependent on the pupal expression of Distalless (Dll), the main regulator of dac during the larval period. Dll acts in the epithelial cells surrounding the sex comb during pupal development to promote sex comb rotation, a complex cellular process driven by coordinated cell rearrangement. Our results show that genes with well-conserved developmental functions can be re-used at later stages in development to regulate more recently evolved traits. This mode of gene co-option may be an important driver of evolutionary innovations.     

Drosophila Fragile X Protein controls cellular proliferation by regulating cbl levels in the ovary

FMRP is an RNA binding protein linked to the most common form of inherited mental retardation, Fragile X syndrome (FraX). In addition to severe cognitive deficits, FraX etiology includes postpubescent macroorchidism, which is thought to result from overproliferation. Using a Drosophila FraX model, we show that FMRP controls germline proliferation during oogenesis. dFmr1 null ovaries contain egg chambers with both fewer and supranumerary germ cells. The mutant germaria contain a significantly increased number of cyclin E and PhosphoHistone H3 positive cells, suggesting that loss of FMRP leads to defects in cell cycle progression. BrdU incorporation and flow cytometry data suggest that, in addition to proliferation, germline endoreplication and ploidy are also affected by the loss of FMRP during ovary development. Here we report that FMRP controls the levels of cbl mRNA in the ovary and that reducing cbl gene dosage by half rescues the dFmr1 oogenesis phenotypes. These data support a model whereby FMRP controls germline proliferation by regulating the expression of cbl in the developing ovary.