A Drosophila model to screen Alport syndrome COL4A5 variants for their functional pathogenicity.

Alport syndrome is a hereditary chronic kidney disease, attributed to rare pathogenic variants in either of three collagen genes (COL4A3/4/5) with most localized in COL4A5. Trimeric type IV Collagen α3α4α5 is essential for the glomerular basement membrane that forms the kidney filtration barrier. A means to functionally assess the many candidate variants and determine pathogenicity is urgently needed. We used Drosophila, an established model for kidney disease, and identify Col4a1 as the functional homolog of human COL4A5 in the fly nephrocyte (equivalent of human podocyte). Fly nephrocytes deficient for Col4a1 showed an irregular and thickened basement membrane and significantly reduced nephrocyte filtration function. This phenotype was restored by expressing human reference (wildtype) COL4A5, but not by COL4A5 carrying any of three established pathogenic patient-derived variants. We then screened seven additional patient COL4A5 variants; their ClinVar classification was either likely pathogenic or of uncertain significance. The findings support pathogenicity for four of these variants; the three others were found benign. Thus, demonstrating the effectiveness of this Drosophila in vivo kidney platform in providing the urgently needed variant-level functional validation.

Fat- and sugar-induced signals regulate sweet and fat taste perception in Drosophila.

In this study, we investigate the interplay between taste perception and macronutrients. While sugar's and protein's self-regulation of taste perception is known, the role of fat remains unclear. We reveal that in Drosophila, fat overconsumption reduces fatty acid taste in favor of sweet perception. Conversely, sugar intake increases fatty acid perception and suppresses sweet taste. Genetic investigations show that the sugar signal, gut-secreted Hedgehog, suppresses sugar taste and enhances fatty acid perception. Fat overconsumption induces unpaired 2 (Upd2) secretion from adipose tissue to the hemolymph. We reveal taste neurons take up Upd2, which triggers Domeless suppression of fatty acid perception. We further show that the downstream JAK/STAT signaling enhances sweet perception and, via Socs36E, fine-tunes Domeless activity and the fatty acid taste perception. Together, our results show that sugar regulates Hedgehog signaling and fat induces Upd2 signaling to balance nutrient intake and to regulate sweet and fat taste perception.

Single-cell profiling of the developing embryonic heart in Drosophila.

Drosophila is an important model for studying heart development and disease. Yet, single-cell transcriptomic data of its developing heart have not been performed. Here, we report single-cell profiling of the entire fly heart using ∼3000 Hand-GFP embryos collected at five consecutive developmental stages, ranging from bilateral migrating rows of cardiac progenitors to a fused heart tube. The data revealed six distinct cardiac cell types in the embryonic fly heart: cardioblasts, both Svp+ and Tin+ subtypes; and five types of pericardial cell (PC) that can be distinguished by four key transcription factors (Eve, Odd, Ct and Tin) and include the newly described end of the line PC. Notably, the embryonic fly heart combines transcriptional signatures of the mammalian first and second heart fields. Using unique markers for each heart cell type, we defined their number and location during heart development to build a comprehensive 3D cell map. These data provide a resource to track the expression of any gene in the developing fly heart, which can serve as a reference to study genetic perturbations and cardiac diseases.

The opportunities and challenges of using Drosophila to model human cardiac diseases.

The Drosophila heart tube seems simple, yet it has notable anatomic complexity and contains highly specialized structures. In fact, the development of the fly heart tube much resembles that of the earliest stages of mammalian heart development, and the molecular-genetic mechanisms driving these processes are highly conserved between flies and humans. Combined with the fly's unmatched genetic tools and a wide variety of techniques to assay both structure and function in the living fly heart, these attributes have made Drosophila a valuable model system for studying human heart development and disease. This perspective focuses on the functional and physiological similarities between fly and human hearts. Further, it discusses current limitations in using the fly, as well as promising prospects to expand the capabilities of Drosophila as a research model for studying human cardiac diseases.

Hedgehog-mediated gut-taste neuron axis controls sweet perception in Drosophila.

Dietary composition affects food preference in animals. High sugar intake suppresses sweet sensation from insects to humans, but the molecular basis of this suppression is largely unknown. Here, we reveal that sugar intake in Drosophila induces the gut to express and secrete Hedgehog (Hh) into the circulation. We show that the midgut secreted Hh localize to taste sensilla and suppresses sweet sensation, perception, and preference. We further find that the midgut Hh inhibits Hh signalling in the sweet taste neurons. Our electrophysiology studies demonstrate that the midgut Hh signal also suppresses bitter taste and some odour responses, affecting overall food perception and preference. We further show that the level of sugar intake during a critical window early in life, sets the adult gut Hh expression and sugar perception. Our results together reveal a bottom-up feedback mechanism involving a "gut-taste neuron axis" that regulates food sensation and preference.

Identification of a GABAergic neuroblast lineage modulating sweet and bitter taste sensitivity.

In Drosophila melanogaster, processing of gustatory information and controlling feeding behavior are executed by neural circuits located in the subesophageal zone (SEZ) of the brain.1 Gustatory receptor neurons (GRNs) project their axons in the primary gustatory center (PGC), which is located in the SEZ.1,2,3,4 To address the function of the PGC, we need detailed information about the different classes of gustatory interneurons that frame the PGC. In this work, we screened large collections of driver lines for SEZ interneuron-specific labeling and subsequently used candidate lines to access the SEZ neuroblast lineages. We converted 130 Gal4 lines to LexA drivers and carried out functional screening using calcium imaging. We found one neuroblast lineage, TRdm, whose neurons responded to both sweet and bitter tastants, and formed green fluorescent protein (GFP) reconstitution across synaptic partners (GRASP)-positive synapses with sweet sensory neurons. TRdm neurons express the inhibitory transmitter GABA, and silencing these neurons increases appetitive feeding behavior. These results demonstrate that TRdm generates a class of inhibitory local neurons that control taste sensitivity in Drosophila.

Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development.

BACKGROUND: A number of cellular processes have evolved in metazoans that increase the proteome repertoire in relation to the genome, such as alternative splicing and translation recoding. Another such process, translational stop codon readthrough (SCR), generates C-terminally extended protein isoforms in many eukaryotes, including yeast, plants, insects, and humans. While comparative genome analyses have predicted the existence of programmed SCR in many species including humans, experimental proof of its functional consequences are scarce. RESULTS: We show that SCR of the Drosophila POU/Oct transcription factor Ventral veins lacking/Drifter (Vvl/Dfr) mRNA is prevalent in certain tissues in vivo, reaching a rate of 50% in the larval prothoracic gland. Phylogenetically, the C-terminal extension is conserved and harbors intrinsically disordered regions and amino acid stretches implied in transcriptional activation. Elimination of Vvl/Dfr translational readthrough by CRISPR/Cas9 mutagenesis changed the expression of a large number of downstream genes involved in processes such as chromatin regulation, neurogenesis, development, and immune response. As a proof-of-principle, we demonstrate that the C-terminal extension of Vvl/Dfr is necessary for correct timing of pupariation, by increasing the capacity to regulate its target genes. The extended Vvl/Dfr isoform acts in synergy with the transcription factor Molting defective (Mld) to increase the expression and biosynthesis of the steroid hormone ecdysone, thereby advancing pupariation. Consequently, late-stage larval development was prolonged and metamorphosis delayed in vvl/dfr readthrough mutants. CONCLUSIONS: We demonstrate that translational recoding of a POU/Oct transcription factor takes place in a highly tissue-specific and temporally controlled manner. This dynamic and regulated recoding is necessary for normal expression of a large number of genes involved in many cellular and developmental processes. Loss of Vvl/Dfr translational readthrough negatively affects steroid hormone biosynthesis and delays larval development and progression into metamorphosis. Thus, this study demonstrates how SCR of a transcription factor can act as a developmental switch in a spatiotemporal manner, feeding into the timing of developmental transitions between different life-cycle stages.

Bab2 Functions as an Ecdysone-Responsive Transcriptional Repressor during Drosophila Development.

Drosophila development is governed by distinct ecdysone steroid pulses that initiate spatially and temporally defined gene expression programs. The translation of these signals into tissue-specific responses is crucial for metamorphosis, but the mechanisms that confer specificity to systemic ecdysone pulses are far from understood. Here, we identify Bric-à-brac 2 (Bab2) as an ecdysone-responsive transcriptional repressor that controls temporal gene expression during larval to pupal transition. Bab2 is necessary to terminate Salivary gland secretion (Sgs) gene expression, while premature Bab2 expression blocks Sgs genes and causes precocious salivary gland histolysis. The timely expression of bab2 is controlled by the ecdysone-responsive transcription factor Broad, and manipulation of EcR/USP/Broad signaling induces inappropriate Bab2 expression and termination of Sgs gene expression. Bab2 directly binds to Sgs loci in vitro and represses all Sgs genes in vivo. Our work characterizes Bab2 as a temporal regulator of somatic gene expression in response to systemic ecdysone signaling.

The POU/Oct Transcription Factor Nubbin Controls the Balance of Intestinal Stem Cell Maintenance and Differentiation by Isoform-Specific Regulation.

Drosophila POU/Oct transcription factors are required for many developmental processes, but their putative regulation of adult stem cell activity has not been investigated. Here, we show that Nubbin (Nub)/Pdm1, homologous to mammalian OCT1/POU2F1 and related to OCT4/POU5F1, is expressed in gut epithelium progenitor cells. We demonstrate that the nub-encoded protein isoforms, Nub-PB and Nub-PD, play opposite roles in the regulation of intestinal stem cell (ISC) maintenance and differentiation. Depletion of Nub-PB in progenitor cells increased ISC proliferation by derepression of escargot expression. Conversely, loss of Nub-PD reduced ISC proliferation, suggesting that this isoform is necessary for ISC maintenance, analogous to mammalian OCT4/POU5F1 functions. Furthermore, Nub-PB is required in enteroblasts to promote differentiation, and it acts as a tumor suppressor of Notch RNAi-driven hyperplasia. We suggest that a dynamic and well-tuned expression of Nub isoforms in progenitor cells is required for maintaining gut epithelium homeostasis.

Proteomic Analysis of Silkworm Antennae.

The silkworm Bombyx mori is an oligophagous insect that feeds mainly on mulberry leaves. The olfactory system of silkworm is a good model to study olfaction in Lepidoptera. Here, we carried out shotgun proteomic analysis and MS sequencing of the silkmoth antennae. A total of 364 proteins were detected, 77 were female specific, 143 were male specific, and 144 were expressed in both male and female antennae. Five odorant-binding proteins, two chemosensory proteins, and one olfactory receptor were identified. They may play a major role in the perception of odorants. An esterase and an aldehyde dehydrogenase were found only in male antennae. Glutathione S-transferases (GSTs) and cytochrome P450s, also found in silkworm antennae, may be involved in the degradation of xenobiotics. Additionally, antioxidation proteins and immunity proteins were identified. Juvenile hormone binding proteins (JHBP), juvenile hormone resistance protein II, and juvenile hormone episode hydrolase (JHEH) were found in the proteomic analysis, which suggests that the antennae are a target for juvenile hormone in the silkworm. Our results provide insight into the expression of proteins in the antennae of silkworm and will facilitate the future functional analysis of silkworm antennae.

The IMD innate immunity pathway of Drosophila influences somatic sex determination via regulation of the Doa locus.

The IMD pathway induces the innate immune response to infection by gram-negative bacteria. We demonstrate strong female-to-male sex transformations in double mutants of the IMD pathway in combination with Doa alleles. Doa encodes a protein kinase playing a central role in somatic sex determination through its regulation of alternative splicing of dsx transcripts. Transcripts encoding two specific Doa isoforms are reduced in Rel null mutant females, supporting our genetic observations. A role for the IMD pathway in somatic sex determination is further supported by the induction of female-to-male sex transformations by Dredd mutations in sensitized genetic backgrounds. In contrast, mutations in either dorsal or Dif, the two other NF-κB paralogues of Drosophila, display no effects on sex determination, demonstrating the specificity of IMD signaling. Our results reveal a novel role for the innate immune IMD signaling pathway in the regulation of somatic sex determination in addition to its role in response to microbial infection, demonstrating its effects on alternative splicing through induction of a crucial protein kinase.

A major facilitator superfamily protein participates in the reddish brown pigmentation in Bombyx mori.

MucK is a member of the major facilitator super family (MFS) and is within a subgroup involved in the uptake of organic acids. Here, we provide the first evidence that mucK is required for normal body color pattern in insect larvae. In the cts mutant strain of Bombyx mori (Linnaeus), the larval anal plates, prothoracic tergum, and the head are reddish brown, as opposed to the white color found in the wild type. Positional cloning of the cts gene was carried out and it was mapped within 90kb on the 16th chromosome. Two molecular markers, 182A4 and 242GA, were found co-segregated with the cts phenotype. Only one candidate gene in the region, BGIBMGA013242, which codes for a major facilitator super family protein named BmMucK, located between the two loci. Sequence analysis revealed that a ∼3kb deletion in the genome resulting in a 126bp deletion in the open reading frame (ORF) of BmmucK in the cts mutant. Fluorescence in situ hybridization (FISH) and quantitative reverse transcription PCR showed that BmmucK expression coincided with reddish brown pigmented regions in the cts mutant strain. BmmucK expression in the head and anal plates were markedly elevated at the molting stages. We performed RNA interference of the mucK homolog in Tribolium castaneum and observed brown coloration accompanied by lethality in ∼1/3 of the insects. The results suggest that MucK plays an important role in the pigmentation process of insect larvae.

[Mapping of non-lepis wing gene nlw in silkworm (Bombyx mori) using SSR and STS markers].

The non-lepis wing of silkworm (Bombyx mori) is controlled by the recessive gene, nlw. Owning to lack of crossing over in females, the reciprocal backcrossed F(1) (BC(1)) progenies were used for linkage analysis and mapping of nlw based on the SSR linkage map and STS markers using the wild type (+(nlw)/+(nlw)) silkworm strain P50 and U06 with scaleless wing (nlw/nlw). The nlw gene was linked to eight SSR markers and one STS marker. All the individuals with the wild type in the BC1F (Using F(1) as female to backcross to the recessive parent, that is (U06xP50)xU06) showed heterozygous profile of (U06xP50) F(1), and the ones with non-lepis wing in BC1F exhibited the homozygous profile of the strain U06. Using a reciprocal BC1M (Using F1 as male to backcross to the recessive parent, that is U06x(U06xP50))cross, we constructed a linkage map of 125.6 cM, and the distance between nlw and the nearest marker cash2p was 11.4 cM.

An integrated genetic linkage map for silkworms with three parental combinations and its application to the mapping of single genes and QTL.

BACKGROUND: Bombyx mori, the domesticated silkworm, is a well-studied model insect with great economic and scientific significance. Although more than 400 mutations have been described in silkworms, most have not been identified, especially those affecting economically-important traits. Simple sequence repeats (SSRs) are effective and economical tools for mapping traits and genetic improvement. The current SSR linkage map is of low density and contains few polymorphisms. The purpose of this work was to develop a dense and informative linkage map that would assist in the preliminary mapping and dissection of quantitative trait loci (QTL) in a variety of silkworm strains. RESULTS: Through an analysis of > 50,000 genotypes across new mapping populations, we constructed two new linkage maps covering 27 assigned chromosomes and merged the data with previously reported data sets. The integrated consensus map contains 692 unique SSR sites, improving the density from 6.3 cM in the previous map to 4.8 cM. We also developed 497 confirmed neighboring markers for corresponding low-polymorphism sites, with 244 having polymorphisms. Large-scale statistics on the SSR type were suggestive of highly efficient markers, based upon which we searched 16,462 available genomic scaffolds for SSR loci. With the newly constructed map, we mapped single-gene traits, the QTL of filaments, and a number of ribosomal protein genes. CONCLUSION: The integrated map produced in this study is a highly efficient genetic tool for the high-throughput mapping of single genes and QTL. Compared to previous maps, the current map offers a greater number of markers and polymorphisms; thus, it may be used as a resource for marker-assisted breeding.

Differentially expressed genes in resistant and susceptible Bombyx mori strains infected with a densonucleosis virus.

We investigated variations in the gene expression of Bombyx mori following infection with a densonucleosis virus (BmDNV-Z). Two B. mori near-isogenic lines, Jingsong and Jingsong.nsd-Z.NIL, which are highly susceptible and completely resistant to BmDNV-Z, respectively, were used in this study. The infection profiles of BmDNV-Z in the midguts of the B. mori Jingsong and Jingsong.nsd-Z.NIL larvae revealed that the virus invaded the midguts of both of these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, three cDNA libraries were constructed to compare BmDNV-Z responsive gene expression between the two silkworm lines. In total, 151 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that 11 genes were significantly up-regulated in the midgut of the Jingsong.nsd-Z.NIL strain following BmDNV-Z infection. Our results imply that these up-regulated genes might be involved in B. mori immune responses against BmDNV infection.

Inheritance and linkage analysis of co-dominant SSR markers on the Z chromosome of the silkworm (Bombyx mori L.).

Microsatellites or simple sequence repeats (SSRs) are co-dominant molecular markers. When we used fluorescent SSR markers to construct a linkage map for the female heterogametic silkworm (Bombyx mori, ZW), we found that some loci did not segregate in a Mendelian ratio of 1:1 in a backcross population. These loci segregated in a 3:1 ratio of single bands compared with double bands. Further examination of band patterns indicated that three types of SSR bands were present: two homozygotes and one heterozygote. In the beginning, we considered to discard these markers. By scoring male and female F1 individuals, we confirmed that these loci were located on the Z chromosome. Using the sex-linked visible mutation sch (K05) and its wild-type (C108), we constructed an F1 male backcross (BC1M) mapping population. The combination of sch backcross and SSR data enabled us to map the SSR markers to the Z chromosome. By adjusting input parameters based on these data, we were able to use Mapmaker software to construct a linkage map. This strategy takes advantage of co-dominant markers for positional cloning of genes on the Z chromosome. We localized sch to the Z chromosome relative to six SSR markers and one PCR marker, covering a total of 76.1 cM. The sch mutation is an important sex-linked visible mutation widely used in breeding of commercial silkworms (e.g. male silkworm selection rearing). Localization of the sch gene may prove helpful in cloning the gene and developing strains for marker-assisted selection in silkworm breeding.

[Cloning of silkworm 3-hydroxyisobutyrate dehydrogenase gene and its expression patterns analysis in simulated weightless environment].

The full length cDNA of silkworm hibadh gene was cloned by RT-PCR and RACE (Rapid amplification of cDNA ends) technique. The hibadh gene and its deduced amino acid sequences were analyzed. The tissue distribution of hibadh gene in 5th instar silkworm larvae was tested by RT-PCR. The expression patterns of hibadh gene in simulated weightless environment were analyzed by real time RT-PCR. The results showed that the full length hibadh cDNA sequence was 1074 bp in lenth, including an open read frame of 969 bp encoding the entire coding region of Hibadh (GenBank accession No. EU719652). The deduced amino acid sequence similarities of hibadh between silkworm and Burkholderia ambifaria, Drosophila melanogaster, Apis mellifera, Xenopus tropicalis, Mus musculus, Homo sapiens were 46%, 43%, 48%, 44%, 45%, 45%, respectively. Signal peptide analysis showed that Hibadh was a secretory protein. There wasn't glycosyl-phosphatidyl inositol anchor site in Hibadh amino acid sequence. Molecular weight and isoelectric point of Hibadh were 34.1 kD and 9.14 respectively. The RT-PCR tests indicated that the hibadh gene expressed in head, silk gland, midgut, cuticle, blood, fat body, tuba malpighii of the 5th instar silkworm larvae. There were different expression patterns of hibadh gene during different silkworm embryo period in simulated weightless environment. Simulated weightlessness resulted in the expression of silkworm hibadh gene up regulated 2.3-fold (P < 0.05), up regulated 4.6-fold (P<0.01), down regulated 7.6-fold (P < 0.01), down regulated 2.6-fold (P < 0.05) during apophysis formation period, inverse period, trachea formation period, and whole embryo period, respectively. There was no significant change of hibadh gene expression during other period of silkworm embryo between simulated weightless and control groups. There were different response patterns to simulated weightless environment between hibadh gene and whole body of silkworm. Gene showed much higher sensitivity compared to whole body in response to environment. This study is useful for the further research on the gravity biological mechanism of hibadh gene.