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The number of embryonic primordial germ cells in Drosophila is determined by the quantity of germ plasm, whose assembly starts in the posterior region of the oocyte during oogenesis. Here, we report that extending JAK-STAT activity in the posterior somatic follicular epithelium leads to an excess of primordial germ cells in the future embryo. We show that JAK-STAT signaling is necessary for the differentiation of approximately 20 specialized follicle cells maintaining tight contact with the oocyte. These cells define, in the underlying posterior oocyte cortex, the anchoring of the germ cell determinant oskar mRNA. We reveal that the apical surface of these posterior anchoring cells extends long filopodia penetrating the oocyte. We identify two JAK-STAT targets in these cells that are each sufficient to extend the zone of contact with the oocyte, thereby leading to production of extra primordial germ cells. JAK-STAT signaling thus determines a fixed number of posterior anchoring cells required for anterior-posterior oocyte polarity and for the development of the future germline., (© 2024. The Author(s).)

The JAK-STAT pathway is evolutionary conserved. The simplicity of this signaling in Drosophila , due to the limited redundancy between pathway components, makes it an ideal model for investigation. In the Drosophila follicular epithelium, highly stereotyped functions of JAK-STAT signaling have been well characterized, but how signaling activity is regulated precisely to allow the different outcomes is not well understood. In this tissue, the ligand is secreted by the polar cells positioned at each follicle extremity, thus generating a gradient of JAK-STAT activity in adjacent cells. One way to control the delivered quantity of ligand is by regulating the number of polar cells, which is reduced by apoptosis to exactly two at each pole by mid-oogenesis. Hence, JAK-STAT activity is described as symmetrical between follicle anterior and posterior regions. Here, we show that JAK-STAT signaling activity is actually highly dynamic, resulting in asymmetry between poles by mid-oogenesis. Interestingly, we found similar temporal dynamics at follicle poles in the accumulation of the adherens junction E-cadherin protein. Remarkably, E-cadherin and JAK-STAT signaling not only display patterning overlaps but also share functions during oogenesis. In particular, we show that E-cadherin, like JAK-STAT signaling, regulates polar cell apoptosis non-cell-autonomously from follicle cells. Finally, our work reveals that E-cadherin is required for optimal JAK-STAT activity throughout oogenesis and that E-cadherin and Stat92E, the transcription factor of the pathway, form part of a physical complex in follicle cells. Taken together, our study establishes E-cadherin as a new positive regulator of JAK-STAT signaling during oogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mallart, Chalvet, Netter, Torres, Poidevin, Montagne, Pret and Malartre.)

Many studies have focused on the mechanisms of stem cell maintenance via their interaction with a particular niche or microenvironment in adult tissues, but how formation of a functional niche is initiated, including how stem cells within a niche are established, is less well understood. Adult Drosophila melanogaster ovary Germline Stem Cell (GSC) niches are comprised of somatic cells forming a stack called a Terminal Filament (TF) and associated Cap and Escort Cells (CCs and ECs, respectively), which are in direct contact with GSCs. In the adult ovary, the transcription factor Engrailed is specifically expressed in niche cells where it directly controls expression of the decapentaplegic (dpp) gene encoding a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules, which are key factors for GSC maintenance. In larval ovaries, in response to BMP signaling from newly formed niches, adjacent primordial germ cells become GSCs. The bric-à-brac paralogs (bab1 and bab2) encode BTB/POZ domain-containing transcription factors that are expressed in developing niches of larval ovaries. We show here that their functions are necessary specifically within precursor cells for TF formation during these stages. We also identify a new function for Bab1 and Bab2 within developing niches for GSC establishment in the larval ovary and for robust GSC maintenance in the adult. Moreover, we show that the presence of Bab proteins in niche cells is necessary for activation of transgenes reporting dpp expression as of larval stages in otherwise correctly specified Cap Cells, independently of Engrailed and its paralog Invected (En/Inv). Moreover, strong reduction of engrailed/invected expression during larval stages does not impair TF formation and only partially reduces GSC numbers. In the adult ovary, Bab proteins are also required for dpp reporter expression in CCs. Finally, when bab2 was overexpressed at this stage in somatic cells outside of the niche, there were no detectable levels of ectopic En/Inv, but ectopic expression of a dpp transgene was found in these cells and BMP signaling activation was induced in adjacent germ cells, which produced GSC-like tumors. Together, these results indicate that Bab transcription factors are positive regulators of BMP signaling in niche cells for establishment and homeostasis of GSCs in the Drosophila ovary., Competing Interests: The authors have declared that no competing interests exist.

Objective: Sleep restriction has been associated with deteriorated insulin sensitivity. The effects of short sleep duration have been explored little in patients with type 1 diabetes. This study addresses the question of whether sleep curtailment affects HbA1c levels in patients with type 1 diabetes., Research Design and Methods: Seventy-nine adult patients with type 1 diabetes (median age 40 years [IQR 23-49]; 47% men) were recruited to wear a wrist actimetry sensor during 3 consecutive days to assess mean sleep duration during normal daily life. A subsample of 37 patients also performed 24-h ambulatory blood pressure monitoring (ABPM). Medical history, sleep questionnaires, and diabetes-related quality of life (DQOL) were assessed., Results: Patients having shorter sleep duration--less than 6.5 h (n=21)--had higher levels of HbA1c (P=0.01) than patients with longer sleep duration, above 6.5 h (n=58). In a multivariable regression model including shorter versus longer sleep duration, diabetes duration, DQOL score, and daily activity, sleep duration was the only variable independently associated with HbA1c (R2=10%). In patients who performed 24-h ABPM, patients with a nondipping pattern of blood pressure exhibited shorter sleep duration than patients with a dipping pattern of blood pressure., Conclusions: Shorter sleep duration is associated with higher HbA1c levels in patients with type 1 diabetes, as well as with a nondipping pattern of blood pressure, anticipating a long-term deleterious impact on the risk of microvascular complications. Further studies should test whether extending the duration of sleep may improve both HbA1c and blood pressure in type 1 diabetes.

The genome of the human immunodeficiency virus type 1 (HIV-1) encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu), in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin-proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated.We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs) which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1). Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/β-TrCP protein, as in mammals, both SLIMB/βTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK) pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway.

The potential to produce new cells during adult life depends on the number of stem cell niches and the capacity of stem cells to divide, and is therefore under the control of programs ensuring developmental homeostasis. However, it remains generally unknown how the number of stem cell niches is controlled. In the insect ovary, each germline stem cell (GSC) niche is embedded in a functional unit called an ovariole. The number of ovarioles, and thus the number of GSC niches, varies widely among species. In Drosophila, morphogenesis of ovarioles starts in larvae with the formation of terminal filaments (TFs), each made of 8-10 cells that pile up and sort in stacks. TFs constitute organizers of individual germline stem cell niches during larval and early pupal development. In the Drosophila melanogaster subgroup, the number of ovarioles varies interspecifically from 8 to 20. Here we show that pipsqueak, Trithorax-like, batman and the bric-à-brac (bab) locus, all encoding nuclear BTB/POZ factors of the Tramtrack Group, are involved in limiting the number of ovarioles in D. melanogaster. At least two different processes are differentially perturbed by reducing the function of these genes. We found that when the bab dose is reduced, sorting of TF cells into TFs was affected such that each TF contains fewer cells and more TFs are formed. In contrast, psq mutants exhibited a greater number of TF cells per ovary, with a normal number of cells per TF, thereby leading to formation of more TFs per ovary than in the wild type. Our results indicate that two parallel genetic pathways under the control of a network of nuclear BTB factors are combined in order to negatively control the number of germline stem cell niches.

Aim: To investigate the effects of (dietary) glycine against oxidant-induced injury caused by bile duct ligation (BDL)., Methods: Either a diet containing 5% glycine or a standard diet was fed to male Sprague-Dawley (SD) rats. Three days later, BDL or sham-operation was performed. Rats were sacrificed 1 to 3 d after BDL. The influence of deoxycholic acid (DCA) in the presence or absence of glycine on liver cells was determined by measurement of calcium and chloride influx in cultivated Kupffer cells and lactate dehydrogenase (LDH) activity was determined in the supernatant of cultivated hepatocytes., Results: Serum alanine transaminase levels increased to about 600 U/L 1 d after BDL. However, enzyme release was blunted by about two third in rats receiving glycine. Release of the alkaline phosphatase and aspartate aminotransferase was also blocked significantly in the group fed glycine. Focal necrosis was observed 2 d after BDL. Glycine partially blocked the histopathological changes. Incubation of Kupffer cells with DCA led to increased intracellular calcium that could be blocked by incubation with glycine. However, systemic blockage of Kupffer cells with gadolinium chloride had no effects on transaminase release. Incubation of isolated hepatocytes with DCA led to a significant release of LDH after 4 h. This release was largely blocked when incubation with glycine was performed., Conclusion: These data indicate that glycine significantly decreased liver injury, most likely by a direct effect on hepatocytes. Kupffer cells do not appear to play an important role in the pathological changes caused by cholestasis.

The Drosophila thorax exhibits 11 pairs of large sensory organs (macrochaetes) identified by their unique position. Remarkably precise, this pattern provides an excellent model system to study the genetic basis of pattern formation. In imaginal wing discs, the achaete-scute proneural genes are expressed in clusters of cells that prefigure the positions of each macrochaete. The activities of prepatterning genes provide positional cues controlling this expression pattern. The three homeobox genes clustered in the iroquois complex (araucan, caupolican and mirror) are such prepattern genes. mirror is generally characterized as performing functions predominantly different from the other iroquois genes. Conversely, araucan and caupolican are described in previous studies as performing redundant functions in most if not all processes in which they are involved. We have addressed the question of the specific role of each iroquois gene in the prepattern of the notum and we clearly demonstrate that they are intrinsically different in their contribution to this process: caupolican and mirror, but not araucan, are required for the neural patterning of the lateral notum. However, when caupolican and/or mirror expression is reduced, araucan loss of function has an effect on thoracic bristles development. Moreover, the overexpression of araucan is able to rescue caupolican loss of function. We conclude that, although retaining some common functionalities, the Drosophila iroquois genes are in the process of diversification. In addition, caupolican and mirror are required for stripe expression and, therefore, to specify the muscular attachment sites prepattern. Thus, caupolican and mirror may act as common prepattern genes for all structures in the lateral notum.

Aim: To investigate the effects of heme oxygenase-1 (HO-1) against oxidant-induced injury caused by bile duct ligation (BDL)., Methods: Either cobalt protoporphyrin (CoPP), a HO-1 inducer, or saline were injected intraperitoneally in male SD-rats. Three days later, BDL or sham-operations were performed. Rats were sacrificed 3 wk after BDL and livers were harvested for histology. Fibrosis was evaluated by sirius red staining and image analysis. Alpha-smooth muscular actin, which indicates activation of stellate cells, was detected by immunohistochemical staining, and cytokine and collagen-Ialpha (Col-Ialpha) mRNA expression was detected using RNase protection assays., Results: Serum alanine transaminase increased 8-fold above normal levels one day after BDL. Surprisingly, enzyme release was not reduced in rats receiving CoPP. Liver fibrosis was evaluated 3 wk after BDL and the sirius red-positive area was found to be increased to about 7.8%. However, in CoPP pretreated rats sirius red-positive areas were increased to about 11.7% after BDL. Collagen-Ialpha and TGF-beta mRNA increased significantly by BDL. Again, this effect was increased by HO-1 overexpression., Conclusion: Hepatic fibrosis due to BDL is not reduced by the HO-1 inducer CoPP. In contrast, HO-1 overexpression increases liver injury in rats under conditions of experimental chronic cholestasis.

Kupffer cells (KC) are the resident macrophages of the liver and represent about 80% of the total fixed macrophage population. They are involved in disease states such as endotoxin shock, alcoholic liver diseases and other toxic-induced liver injury. They release physiologically active substances such as eicosanoids and inflammatory cytokines (IL-1, IL-6, TNFalpha), and produce free radical species. Thus, KC are attractive targets for anti-inflammatory therapies and potential candidates responsible for differences in inflammation in liver disease seen between different individuals. However, to perform parallel in vitro experiments with KC from different donors a suitable method for conservation of KC would be necessary. Therefore, the present study evaluated, whether rat and human KC can be frozen, stored and recovered without losing their functional integrity. Rat and human KC were isolated and either cultured under standard conditions (fresh KC) or cryopreserved in special freezing medium (cryopreserved KC). At least 24 h later, cryopreserved KC were thawed, brought into suspension and seeded in the same density as fresh cells for subsequent experiments. Viability of cultured KC was analyzed by trypan blue exclusion. LPS (or PBS as control) stimulation was performed at different time points and cytokine release was analyzed with IL-6 and TNFalpha ELISAs, respectively. Phagocytic capacity was investigated by using a specific phagocytosis assay and FACS analysis. The recovery rate after thawing was around 57% for rat and around 65% for human cryopreserved KC. The results indicate, that KC can successfully be cryopreserved with an adequate recovery rate of viable cells. The properties of fresh and frozen KC can also be compared after thawing. Freshly isolated and cryopreserved cultured KC showed near-normal morphology and did not differ in the cultivation profiles over a period of 72 h. One to three days after seeding, frozen rat or human KC also retained inducible functions such as the production of TNFalpha or IL-6 after LPS challenge. Finally, regardless if they were cryopreserved or not, no differences in the phagocytic activities of the cells were obtained. Taken together, it is concluded that cryopreservation of KC does not change the physiological characteristics of the cells in vitro. Therefore, the method used here for cryopreservation of especially human KC allows the accumulation of KC from several donors for parallel in vitro experiments.

In Drosophila, relocation of a euchromatic gene near centromeric or telomeric heterochromatin often leads to its mosaic silencing. Nevertheless, modifiers of centromeric silencing do not affect telomeric silencing, suggesting that each location requires specific factors. Previous studies suggest that a subset of Polycomb-group (PcG) proteins could be responsible for telomeric silencing. Here, we present the effect on telomeric silencing of 50 mutant alleles of the PcG genes and of their counteracting trithorax-group genes. Several combinations of two mutated PcG genes impair telomeric silencing synergistically, revealing that some of these genes are required for telomeric silencing. In situ hybridization and immunostaining experiments on polytene chromosomes revealed a strict correlation between the presence of PcG proteins and that of heterochromatic telomeric associated sequences (TASs), suggesting that TASs and PcG complexes could be associated at telomeres. Furthermore, lines harboring a transgene containing an X-linked TAS subunit and the mini-white reporter gene can exhibit pairing-sensitive repression of the white gene in an orientation-dependent manner. Finally, an additional binding site for PcG proteins was detected at the insertion site of this type of transgene. Taken together, these results demonstrate that PcG proteins bind TASs in vivo and may be major players in Drosophila telomeric position effect (TPE).

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes.

Transplantation of encapsulated living cells is a promising approach for the treatment of a wide variety of diseases. Large-scale application of the technique, however, is hampered by insufficient biocompatibility of the capsules. In the present study, we have implemented new as well as previously reported technologies to test biocompatibility issues of immunoisolating microcapsules on the long term (i.e. 2 years) instead of usually reported short time periods. When transplanted empty, the capsules proved to be highly biocompatible not only for short periods (i.e. 1 month) but also on the long term as evidenced by the absence of any significant biological response up to 2 years after implantation in rats. The immunoprotective properties of the capsules were confirmed by prolonged survival of encapsulated islet allografts up to 200 days. The surface of the applied capsule was analyzed and provides new insight in the chemical structure of true biocompatible and immunoprotective capsules applicable for transplantation of encapsulated islets in type I diabetes.

Polyhomeotic is a member of the Polycomb group of genes. The products of this group are chromatin-associated proteins that act together as multimeric complexes. These proteins are required for the maintenance of target gene repression in a permanent and heritable manner during development. In order to better understand the dynamics of their action during development, we generated transgenic flies expressing a polyhomeotic protein tagged with the enhanced green fluorescent protein. Here we show that this fusion protein (PH-EGFP) retains both the functional properties of the endogenous protein and its target specificity on polytene chromosomes. The distribution of the PH-EGFP protein is partly dependent on the presence of wildtype Polycomb protein, indicating that PH-EGFP behaves as does the wildtype PH protein. Therefore, the PH-EGFP chimera appears to be an appropriate reporter of PH protein distribution and a suitable tool for the study of Polycomb-group complex assembly in vivo. The subnuclear distribution of PH-EGFP is dynamic throughout development. In the interphase nucleus at the cellular blastoderm, a diffuse granular pattern is observed. From the early gastrula stage onward, a few brighter dots appear. As development progressed from germ band retraction through hatching of the larva, numerous discrete dots accumulate in the nucleus of epidermal cells. The increasing number of dots observed during development may indicate that PH-EGFP is recruited at different stages on different target sites, a result that is in good agreement with functional data previously reported.

The Polycomb-group genes (PcG) encode a group of repressors well known for their function in stably maintaining the inactive expression patterns of key developmental regulators, including homeotic genes. PcG genes are structurally and functionally conserved in Drosophila and Mammalians, and some homologues have been found in worms, yeast and plants. Their products act through different complexes and at least one of these complexes seems to induce histone deacetylation. In Drosophila, building of PcG complexes depends on both protein-protein interactions and recognition near target genes of specific DNA sequences called Polycomb-group response element (PRE). Together with the counteracting trithorax-group proteins, PcG products establish a form of cellular memory by faithfully maintaining transcription states determined early in embryogenesis. Here, we discuss several aspects of PcG functions: the composition of the different complexes, the establishment and the transmission of silencing to subsequent cell generations as well as the subnuclear localisation of the PcG products.

The P[MTW] transposon carries a functional MtnA (metallothionein) gene and a miniwhite reporter gene. When P[MTW] was transformed into Drosophila, many lines were found to show position-dependent expression patterns of the miniwhite or the MtnA transgene. Identification of several of the target genes indicated that this construct behaves as an enhancer or silencer trap. For instance, expression of at least one reporter transgene was shown to correlate with that of the endogenous gene in the case of insertions in Ultrabithorax, four-jointed, and the iroquois complex. The frequency of patterns recovered with P[MTW] is higher than that reported for P[LacW], suggesting that P[MTW] has unusual properties. The possibility of biased insertion of P[MTW] was assayed by screening a sample of 66 MTW lines for modifiers of the extra sex comb phenotype caused by a hypomorphic allele of polyhomeotic. Seven modifiers were recovered, which could be ranked in two classes: genes involved in leg morphogenesis (including four-jointed and spitz), and genes of the Polycomb- or trithorax-Group, including trithorax and batman, a new gene which encodes a product with a BTB/POZ domain. Taken together, these results indicate that P[MTW] allows the tagging of patterning and cell determination genes, and thus provides a useful tool for identifying new developmental functions.

We used the white gene as an enhancer trap and reporter of chromatin structure. We collected white+ transgene insertions presenting a peculiar pigmentation pattern in the eye: white expression is restricted to the dorsal half of the eye, with a clear-cut dorsal/ventral (D/V) border. This D/V pattern is stable and heritable, indicating that phenotypic expression of the white reporter reflects positional information in the developing eye. Localization of these transgenes led us to identify a unique genomic region encompassing 140 kb in 69D1-3 subject to this D/V effect. This region contains at least three closely related homeobox-containing genes that are constituents of the iroquois complex (IRO-C). IRO-C genes are coordinately regulated and implicated in similar developmental processes. Expression of these genes in the eye is regulated by the products of the Polycomb-group (Pc-G) and trithorax-group (trx-G) genes but is not modified by classical modifiers of position-effect variegation. Our results, together with the report of a Pc-G binding site in 69D, suggest that we have identified a novel cluster of target genes for the Pc-G and trx-G products. We thus propose that ventral silencing of the whole IRO-C in the eye occurs at the level of chromatin structure in a manner similar to that of the homeotic gene complexes, perhaps by local compaction of the region into a heterochromatin-like structure involving the Pc-G products.