Valine aminoacyl-tRNA synthetase promotes therapy resistance in melanoma.

Transfer RNA dynamics contribute to cancer development through regulation of codon-specific messenger RNA translation. Specific aminoacyl-tRNA synthetases can either promote or suppress tumourigenesis. Here we show that valine aminoacyl-tRNA synthetase (VARS) is a key player in the codon-biased translation reprogramming induced by resistance to targeted (MAPK) therapy in melanoma. The proteome rewiring in patient-derived MAPK therapy-resistant melanoma is biased towards the usage of valine and coincides with the upregulation of valine cognate tRNAs and of VARS expression and activity. Strikingly, VARS knockdown re-sensitizes MAPK-therapy-resistant patient-derived melanoma in vitro and in vivo. Mechanistically, VARS regulates the messenger RNA translation of valine-enriched transcripts, among which hydroxyacyl-CoA dehydrogenase mRNA encodes for a key enzyme in fatty acid oxidation. Resistant melanoma cultures rely on fatty acid oxidation and hydroxyacyl-CoA dehydrogenase for their survival upon MAPK treatment. Together, our data demonstrate that VARS may represent an attractive therapeutic target for the treatment of therapy-resistant melanoma.

The Osteoblast Transcriptome in Developing Zebrafish Reveals Key Roles for Extracellular Matrix Proteins Col10a1a and Fbln1 in Skeletal Development and Homeostasis.

Zebrafish are now widely used to study skeletal development and bone-related diseases. To that end, understanding osteoblast differentiation and function, the expression of essential transcription factors, signaling molecules, and extracellular matrix proteins is crucial. We isolated Sp7-expressing osteoblasts from 4-day-old larvae using a fluorescent reporter. We identified two distinct subpopulations and characterized their specific transcriptome as well as their structural, regulatory, and signaling profile. Based on their differential expression in these subpopulations, we generated mutants for the extracellular matrix protein genes col10a1a and fbln1 to study their functions. The col10a1a-/- mutant larvae display reduced chondrocranium size and decreased bone mineralization, while in adults a reduced vertebral thickness and tissue mineral density, and fusion of the caudal fin vertebrae were observed. In contrast, fbln1-/- mutants showed an increased mineralization of cranial elements and a reduced ceratohyal angle in larvae, while in adults a significantly increased vertebral centra thickness, length, volume, surface area, and tissue mineral density was observed. In addition, absence of the opercle specifically on the right side was observed. Transcriptomic analysis reveals up-regulation of genes involved in collagen biosynthesis and down-regulation of Fgf8 signaling in fbln1-/- mutants. Taken together, our results highlight the importance of bone extracellular matrix protein genes col10a1a and fbln1 in skeletal development and homeostasis.

Perinatal exposure to the fungicide ketoconazole alters hypothalamic control of puberty in female rats.

Introduction: Estrogenic endocrine disrupting chemicals (EDCs) such as diethylstilbestrol (DES) are known to alter the timing of puberty onset and reproductive function in females. Accumulating evidence suggests that steroid synthesis inhibitors such as ketoconazole (KTZ) or phthalates may also affect female reproductive health, however their mode of action is poorly understood. Because hypothalamic activity is very sensitive to sex steroids, we aimed at determining whether and how EDCs with different mode of action can alter the hypothalamic transcriptome and GnRH release in female rats. Design: Female rats were exposed to KTZ or DES during perinatal (DES 3-6-12µg/kg.d; KTZ 3-6-12mg/kg.d), pubertal or adult periods (DES 3-12-48µg/kg.d; KTZ 3-12-48mg/kg.d). Results: Ex vivo study of GnRH pulsatility revealed that perinatal exposure to the highest doses of KTZ and DES delayed maturation of GnRH secretion before puberty, whereas pubertal or adult exposure had no effect on GnRH pulsatility. Hypothalamic transcriptome, studied by RNAsequencing in the preoptic area and in the mediobasal hypothalamus, was found to be very sensitive to perinatal exposure to all doses of KTZ before puberty with effects persisting until adulthood. Bioinformatic analysis with Ingenuity Pathway Analysis predicted "Creb signaling in Neurons" and "IGF-1 signaling" among the most downregulated pathways by all doses of KTZ and DES before puberty, and "PPARg" as a common upstream regulator driving gene expression changes. Deeper screening ofRNAseq datasets indicated that a high number of genes regulating the activity of the extrinsic GnRH pulse generator were consistently affected by all the doses of DES and KTZ before puberty. Several, including MKRN3, DNMT3 or Cbx7, showed similar alterations in expression at adulthood. Conclusion: nRH secretion and the hypothalamic transcriptome are highly sensitive to perinatal exposure to both DES and KTZ. The identified pathways should be exploredfurther to identify biomarkers for future testing strategies for EDC identification and when enhancing the current standard information requirements in regulation.

Comparing the transcriptome of developing native and iPSC-derived mouse retinae by single cell RNA sequencing.

We report the generation and analysis of single-cell RNA-Seq data (> 38,000 cells) from mouse native retinae and induced pluripotent stem cell (iPSC)-derived retinal organoids at four matched stages of development spanning the emergence of the major retinal cell types. We combine information from temporal sampling, visualization of 3D UMAP manifolds, pseudo-time and RNA velocity analyses, to show that iPSC-derived 3D retinal organoids broadly recapitulate the native developmental trajectories. However, we observe relaxation of spatial and temporal transcriptome control, premature emergence and dominance of photoreceptor precursor cells, and susceptibility of dynamically regulated pathways and transcription factors to culture conditions in retinal organoids. We demonstrate that genes causing human retinopathies are enriched in cell-type specifying genes and identify a subset of disease-causing genes with expression profiles that are highly conserved between human retinae and murine retinal organoids. This study provides a resource to the community that will be useful to assess and further improve protocols for ex vivo recapitulation and study of retinal development.

Identification of an evolutionarily conserved domain in Neurod1 favouring enteroendocrine versus goblet cell fate.

ARP/ASCL transcription factors are key determinants of cell fate specification in a wide variety of tissues, coordinating the acquisition of generic cell fates and of specific subtype identities. How these factors, recognizing highly similar DNA motifs, display specific activities, is not yet fully understood. To address this issue, we overexpressed different ARP/ASCL factors in zebrafish ascl1a-/- mutant embryos to determine which ones are able to rescue the intestinal secretory lineage. We found that Ascl1a/b, Atoh1a/b and Neurod1 factors are all able to trigger the first step of the secretory regulatory cascade but distinct secretory cells are induced by these factors. Indeed, Neurod1 rescues the enteroendocrine lineage while Ascl1a/b and Atoh1a/b rescue the goblet cells. Gain-of-function experiments with Ascl1a/Neurod1 chimeric proteins revealed that the functional divergence is encoded by a 19-aa ultra-conserved element (UCE), present in all Neurod members but absent in the other ARP/ASCL proteins. Importantly, inserting the UCE into the Ascl1a protein reverses the rescuing capacity of this Ascl1a chimeric protein that cannot rescue the goblet cells anymore but can efficiently rescue the enteroendocrine cells. This novel domain acts indeed as a goblet cell fate repressor that inhibits gfi1aa expression, known to be important for goblet cell differentiation. Deleting the UCE domain of the endogenous Neurod1 protein leads to an increase in the number of goblet cells concomitant with a reduction of the enteroendocrine cells, phenotype also observed in the neurod1 null mutant. This highlights the crucial function of the UCE domain for NeuroD1 activity in the intestine. As Gfi1 acts as a binary cell fate switch in several tissues where Neurod1 is also expressed, we can envision a similar role of the UCE in other tissues, allowing Neurod1 to repress Gfi1 to influence the balance between cell fates.

A δ-cell subpopulation with a pro-ß-cell identity contributes to efficient age-independent recovery in a zebrafish model of diabetes.

Restoring damaged ß-cells in diabetic patients by harnessing the plasticity of other pancreatic cells raises the questions of the efficiency of the process and of the functionality of the new Insulin-expressing cells. To overcome the weak regenerative capacity of mammals, we used regeneration-prone zebrafish to study ß-cells arising following destruction. We show that most new insulin cells differ from the original ß-cells as they coexpress Somatostatin and Insulin. These bihormonal cells are abundant, functional and able to normalize glycemia. Their formation in response to ß-cell destruction is fast, efficient, and age-independent. Bihormonal cells are transcriptionally close to a subset of δ-cells that we identified in control islets and that are characterized by the expression of somatostatin 1.1 (sst1.1) and by genes essential for glucose-induced Insulin secretion in ß-cells such as pdx1, slc2a2 and gck. We observed in vivo the conversion of monohormonal sst1.1-expressing cells to sst1.1+ ins + bihormonal cells following ß-cell destruction. Our findings support the conclusion that sst1.1 δ-cells possess a pro-ß identity enabling them to contribute to the neogenesis of Insulin-producing cells during regeneration. This work unveils that abundant and functional bihormonal cells benefit to diabetes recovery in zebrafish.

The E3 ligase COP1 promotes ERα signaling and suppresses EMT in breast cancer.

ERα signaling drives proliferation, survival and cancer initiation in the mammary gland. Therefore, it is critical to elucidate mechanisms by which ERα expression is regulated. We show that the tumor suppressor E3 ligase COP1 promotes the degradative polyubiquitination of the microtubule-associated protein HPIP. As such, COP1 negatively regulates estrogen-dependent AKT activation in breast cancer cells. However, COP1 also induces ERα expression and ERα-dependent gene transcription, at least through c-Jun degradation. COP1 and ERα levels are positively correlated in clinical cases of breast cancer. COP1 also supports the metabolic reprogramming by estrogens, including glycolysis. On the other hand, COP1 suppresses EMT in breast cancer cells. COP1 deficiency also contributes to Tamoxifen resistance, at least through protective autophagy. Therefore, COP1 acts as an oncogenic E3 ligase by promoting ERα signaling but also acts as a tumor suppressor candidate by preventing EMT, which reflects a dual role of COP1 in breast cancer.

Single-cell RNA sequencing of human liver reveals hepatic stellate cell heterogeneity.

BACKGROUND & AIMS: The multiple vital functions of the human liver are performed by highly specialised parenchymal and non-parenchymal cells organised in complex collaborative sinusoidal units. Although crucial for homeostasis, the cellular make-up of the human liver remains to be fully elucidated. Here, single-cell RNA-sequencing was used to unravel the heterogeneity of human liver cells, in particular of hepatocytes (HEPs) and hepatic stellate cells (HSCs). METHOD: The transcriptome of ~25,000 freshly isolated human liver cells was profiled using droplet-based RNA-sequencing. Recently published data sets and RNA in situ hybridisation were integrated to validate and locate newly identified cell populations. RESULTS: In total, 22 cell populations were annotated that reflected the heterogeneity of human parenchymal and non-parenchymal liver cells. More than 20,000 HEPs were ordered along the portocentral axis to confirm known, and reveal previously undescribed, zonated liver functions. The existence of 2 subpopulations of human HSCs with unique gene expression signatures and distinct intralobular localisation was revealed (i.e. portal and central vein-concentrated GPC3 + HSCs and perisinusoidally located DBH + HSCs). In particular, these data suggest that, although both subpopulations collaborate in the production and organisation of extracellular matrix, GPC3 + HSCs specifically express genes involved in the metabolism of glycosaminoglycans, whereas DBH + HSCs display a gene signature that is reminiscent of antigen-presenting cells. CONCLUSIONS: This study highlights metabolic zonation as a key determinant of HEP transcriptomic heterogeneity and, for the first time, outlines the existence of heterogeneous HSC subpopulations in the human liver. These findings call for further research on the functional implications of liver cell heterogeneity in health and disease. LAY SUMMARY: This study resolves the cellular landscape of the human liver in an unbiased manner and at high resolution to provide new insights into human liver cell biology. The results highlight the physiological heterogeneity of human hepatic stellate cells.

Dual-specificity phosphatase 3 deletion promotes obesity, non-alcoholic steatohepatitis and hepatocellular carcinoma.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic hepatic pathology in Western countries. It encompasses a spectrum of conditions ranging from simple steatosis to more severe and progressive non-alcoholic steatohepatitis (NASH) that can lead to hepatocellular carcinoma (HCC). Obesity and related metabolic syndrome are important risk factors for the development of NAFLD, NASH and HCC. DUSP3 is a small dual-specificity protein phosphatase with a poorly known physiological function. We investigated its role in metabolic syndrome manifestations and in HCC using a mouse knockout (KO) model. While aging, DUSP3-KO mice became obese, exhibited insulin resistance, NAFLD and associated liver damage. These phenotypes were exacerbated under high fat diet (HFD). In addition, DEN administration combined to HFD led to rapid HCC development in DUSP3-KO compared to wild type (WT) mice. DUSP3-KO mice had more serum triglycerides, cholesterol, AST and ALT compared to control WT mice under both regular chow diet (CD) and HFD. The level of fasting insulin was higher compared to WT mice, though, fasting glucose as well as glucose tolerance were normal. At the molecular level, HFD led to decreased expression of DUSP3 in WT mice. DUSP3 deletion was associated with increased and consistent phosphorylation of the insulin receptor (IR) and with higher activation of the downstream signaling pathway. In conclusion, our results support a new role for DUSP3 in obesity, insulin resistance, NAFLD and liver damage.

Pancreatic and intestinal endocrine cells in zebrafish share common transcriptomic signatures and regulatory programmes.

BACKGROUND: Endocrine cells of the zebrafish digestive system play an important role in regulating metabolism and include pancreatic endocrine cells (PECs) clustered in the islets of Langerhans and the enteroendocrine cells (EECs) scattered in the intestinal epithelium. Despite EECs and PECs are being located in distinct organs, their differentiation involves shared molecular mechanisms and transcription factors. However, their degree of relatedness remains unexplored. In this study, we investigated comprehensively the similarity of EECs and PECs by defining their transcriptomic landscape and comparing the regulatory programmes controlled by Pax6b, a key player in both EEC and PEC differentiations. RESULTS: RNA sequencing was performed on EECs and PECs isolated from wild-type and pax6b mutant zebrafish. Data mining of wild-type zebrafish EEC data confirmed the expression of orthologues for most known mammalian EEC hormones, but also revealed the expression of three additional neuropeptide hormones (Proenkephalin-a, Calcitonin-a and Adcyap1a) not previously reported to be expressed by EECs in any species. Comparison of transcriptomes from EECs, PECs and other zebrafish tissues highlights a very close similarity between EECs and PECs, with more than 70% of genes being expressed in both endocrine cell types. Comparison of Pax6b-regulated genes in EECs and PECs revealed a significant overlap. pax6b loss-of-function does not affect the total number of EECs and PECs but instead disrupts the balance between endocrine cell subtypes, leading to an increase of ghrelin- and motilin-like-expressing cells in both the intestine and pancreas at the expense of other endocrine cells such as beta and delta cells in the pancreas and pyyb-expressing cells in the intestine. Finally, we show that the homeodomain of Pax6b is dispensable for its action in both EECs and PECs. CONCLUSION: We have analysed the transcriptomic landscape of wild-type and pax6b mutant zebrafish EECs and PECs. Our study highlights the close relatedness of EECs and PECs at the transcriptomic and regulatory levels, supporting the hypothesis of a common phylogenetic origin and underscoring the potential implication of EECs in metabolic diseases such as type 2 diabetes.

Transcriptome analysis of pancreatic cells across distant species highlights novel important regulator genes.

BACKGROUND: Defining the transcriptome and the genetic pathways of pancreatic cells is of great interest for elucidating the molecular attributes of pancreas disorders such as diabetes and cancer. As the function of the different pancreatic cell types has been maintained during vertebrate evolution, the comparison of their transcriptomes across distant vertebrate species is a means to pinpoint genes under strong evolutionary constraints due to their crucial function, which have therefore preserved their selective expression in these pancreatic cell types. RESULTS: In this study, RNA-sequencing was performed on pancreatic alpha, beta, and delta endocrine cells as well as the acinar and ductal exocrine cells isolated from adult zebrafish transgenic lines. Comparison of these transcriptomes identified many novel markers, including transcription factors and signaling pathway components, specific for each cell type. By performing interspecies comparisons, we identified hundreds of genes with conserved enriched expression in endocrine and exocrine cells among human, mouse, and zebrafish. This list includes many genes known as crucial for pancreatic cell formation or function, but also pinpoints many factors whose pancreatic function is still unknown. A large set of endocrine-enriched genes can already be detected at early developmental stages as revealed by the transcriptomic profiling of embryonic endocrine cells, indicating a potential role in cell differentiation. The actual involvement of conserved endocrine genes in pancreatic cell differentiation was demonstrated in zebrafish for myt1b, whose invalidation leads to a reduction of alpha cells, and for cdx4, selectively expressed in endocrine delta cells and crucial for their specification. Intriguingly, comparison of the endocrine alpha and beta cell subtypes from human, mouse, and zebrafish reveals a much lower conservation of the transcriptomic signatures for these two endocrine cell subtypes compared to the signatures of pan-endocrine and exocrine cells. These data suggest that the identity of the alpha and beta cells relies on a few key factors, corroborating numerous examples of inter-conversion between these two endocrine cell subtypes. CONCLUSION: This study highlights both evolutionary conserved and species-specific features that will help to unveil universal and fundamental regulatory pathways as well as pathways specific to human and laboratory animal models such as mouse and zebrafish.