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Bicuspid aortic valve (BAV), the most common cardiovascular malformation occurs in 0.5-1.2% of the population. Although highly heritable, few causal mutations have been identified in BAV patients. Here, we report the targeted sequencing of HOXA1 in a cohort of BAV patients and theidentification of rare indel variants in the homopolymeric histidine tract of HOXA1. In vitro analysis shows that disruption of this motif leads to a significant reduction in protein half-life and defective transcriptional activity of HOXA1. In zebrafish, targeting hoxa1a ortholog results in aortic valve defects. In vivo assays indicates that these variants behave as dominant negatives leading abnormal valve development. In mice, deletion of Hoxa1 leads to BAV with a very small, rudimentary non-coronary leaflet. We also show that 17% of homozygous Hoxa1-1His knock-in mice present similar phenotype. Genetic lineage tracing in Hoxa1-/- mutant mice reveals an abnormal reduction of neural crest-derived cells in the valve leaflet, which is caused by a failure of early migration of these cells.

International audience; Bicuspid aortic valve (BAV), the most common cardiovascular malformation occurs in 0.5–1.2% of the population. Although highly heritable, few causal mutations have been identified in BAV patients. Here, we report the targeted sequencing of HOXA1 in a cohort of BAV patients and the identification of rare indel variants in the homopolymeric histidine tract of HOXA1. In vitro analysis shows that disruption of this motif leads to a significant reduction in protein half-life and defective transcriptional activity of HOXA1. In zebrafish, targeting hoxa1a ortholog results in aortic valve defects. In vivo assays indicates that these variants behave as dominant negatives leading abnormal valve development. In mice, deletion of Hoxa1 leads to BAV with a very small, rudimentary non-coronary leaflet. We also show that 17% of homozygous Hoxa1 −1His knock-in mice present similar phenotype. Genetic lineage tracing in Hoxa1 −/− mutant mice reveals an abnormal reduction of neural crest-derived cells in the valve leaflet, which is caused by a failure of early migration of these cells.

The causes of chronic pancreatitis in children are partly different from those found in adults and remain incompletely understood. Thus, the presence of genetic mutations predisposing to pancreatitis is suspected to be a more frequent cause in children. In this context, among pediatric patients with precursor signs of pancreatitis, we identified a category of patients with symptoms typically associated with ciliopathies, a group of genetic disorders related to primary cilium dysfunction. In this work, we therefore aimed to identify mutations in ciliary genes in these patients, and to establish a link between these mutations and the development of pancreatitis. Via Whole Exome Sequencing of genomic DNA from 43 pediatric patients with idiopathic chronic pancreatitis, we first identified in three of them a mutation in the ciliary/ciliogenic genes PKHD1, HNF1β, and NPHP3. We then focused on the NPHP3 gene and generated by CRISPR/Cas9 technique two new transgenic mouse models; one replicates the NPHP3 gene mutations present in the corresponding patient (NPHP3mut1/mut2 model), while the other is a conditional inactivation of NPHP3 (NPHP3f/f model). Phenotypic analysis of these two models allowed us to conclude that loss of function of NPHP3 in pancreatic ductal cells led to inflammation and mild fibrosis associated with significant acinar atrophy and severe lipomatosis. This suggests a new form of pancreatitis in these murine models, characterized by a ciliopathic origin. In order to confirm the presence of pancreatic lipomatosis in patients with HNF1β and NPHP3 mutations, initial MRI analyses were performed and revealed a significant percentage of adipose tissue within the pancreas of these patients. Deeper analysis of mouse models, in particular to characterize the molecular mechanisms involved in the observed phenotypes, and the recruitment of a larger number of patients and control subjects, should allow us to confirm the existence of this new form of pancreatitis of ciliopathic origin. Lay abstract (English) The causes of chronic pancreatitis in children are partially different than in adults and are still incompletely understood. We found that a subset of pediatric patients with signs of early pancreatitis have clinical symptoms typically associated with ciliopathies. The objectives were to identify mutated ciliary genes in these patients and establish a link between mutations, ciliary phenotype, and pancreatitis development. Using a Whole Exome Sequencing strategy, we analyzed the genomic DNA of 43 children with idiopathic chronic pancreatitis. We generated with the CRISPR/Cas9 technique, two new transgenic mouse models to determine in vivo how ciliary gene mutations cause pancreatitis. We identified 3 patients, each with mutations in a ciliary gene: PKHD1, HNF1β and NPHP3. In both murine models, either the one reproducing the mutations found in one patient or the one with a specific deletion of NPHP3 in pancreatic ductal cells, an atypic pancreatitis characterized by the presence of lipomatosis was observed. In order to confirm the presence of pancreatic lipomatosis in patients suffering from NPHP3 or HNF1β mutations, MRI analysis was performed and revealed an important percentage of adipose tissue inside their pancreas. We validated some steps in our project with the discovery of three ciliary gene mutations in three patients. Analysis of mouse models reproducing these mutations confirms that the renal and pancreatic phenotypes found in a patient are indeed due to mutations of this gene. Lipomatosis development in the pancreas following a ciliary gene defect could be a sign of a new form of pancreatitis.

Les causes à l’origine de la pancréatite chronique chez l’enfant sont en partie différentes de celles trouvées chez l’adulte et restent mal connues. Ainsi, la présence de mutations génétiques prédisposant à la pancréatite est suspectée être une cause plus fréquente chez l’enfant. Dans ce contexte, parmi des patients pédiatriques présentant des signes précurseurs de pancréatite, nous avons identifié une catégorie de patients souffrant de symptômes typiquement associés aux ciliopathies, un groupe de désordres génétiques liés à un dysfonctionnement du cil primaire. Dans ce travail, nous avions donc pour objectif d’identifier des mutations de gènes ciliaires chez ces patients, et d’établir un lien entre ces mutations et le développement d’une pancréatite. Via le Whole Exome Sequencing de l’ADN génomique de 43 patients pédiatriques présentant une pancréatite chronique idiopathique, nous avons tout d’abord identifié chez trois d’entre eux une mutation dans les gènes ciliaires/ciliogéniques PKHD1, HNF1β et NPHP3. Nous nous sommes ensuite focalisés sur le gène NPHP3 et avons généré par la technique de CRISPR/Cas9 deux nouveaux modèles murins transgéniques ; l’un reproduit les mutations du gène NPHP3 présentes chez le patient correspondant (modèle NPHP3mut1/mut2), tandis que l’autre est une inactivation conditionnelle de NPHP3 (modèle NPHP3f/f). L’analyse phénotypique de ces deux modèles nous a permis de conclure qu’une perte de fonction de NPHP3 dans les cellules canalaires pancréatiques conduisait à une inflammation et une fibrose légère associées à une atrophie acinaire importante et une lipomatose sévère. Ce tableau fait penser à la présence, dans ces modèles de souris, d’une nouvelle forme de pancréatite caractérisée par une origine ciliopathique. Afin de confirmer la présence de lipomatose pancréatique chez des patients présentant des mutations de HNF1β et NPHP3, des premières analyses par IRM ont été réalisées et ont permis de révéler un pourcentage important de tissu adipeux au sein du pancréas de ces patients. Une analyse plus approfondie des modèles murins, notamment pour caractériser les mécanismes moléculaires impliqués dans les phénotypes observés, et le recrutement d’un plus grand nombre de patients et de sujets contrôles doit nous permettre de confirmer l’existence de cette nouvelle forme de pancréatite d’origine ciliopathique.

In the spinal cord, ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities including locomotion. Interneurons arise during embryonic development from distinct progenitor domains orderly distributed along the dorso-ventral axis of the neural tube. The p2 progenitor domain generates at least five V2 interneuron populations. However, identification and characterization of all V2 populations remain currently incomplete and the mechanisms that control their development remain only partly understood. In this study, we report the generation of a Vsx1-CreER BAC transgenic mouse line that drives CreER recombinase expression mimicking endogenous Vsx1 expression pattern in the developing spinal cord. We showed that the Vsx1-CreER transgene can mediate recombination in V2 precursors with a high efficacy and specificity. Lineage tracing demonstrated that all the V2 interneurons in the mouse developing spinal cord derive from cells expressing Vsx1. Finally, we confirmed that V2 precursors generate additional V2 populations that are not characterized yet. Thus, the Vsx1-CreER line described here is a useful genetic tool for lineage tracing and for functional studies of the mouse spinal V2 interneurons.

[no abstract available]

Kras mutations are not sufficient to induce precancerous lesions in the pancreas, unlike in other organs such as lungs. Pancreatic intraepithelial neoplasia (PanIN) appear when mutated Kras is associated with pancreatitis. Since acinar cells are thought to be the cell origin of PanIN and pancreatic ductal adenocarcinoma (PDAC), our aim is to understand how acinar cells initially resist to Kras mutations and how pancreatitis enables mutated Kras to promote pancreatic neoplasia. Pancreatitis and associated inflammation were induced by injections of cerulein in tamoxifen-treated Elastase-CreER/LSLKrasG12D mice, in which mutated Kras expression is induced exclusively in acinar cells. RNAseq analysis on FACS-sorted acinar cells revealed that Kras expression and several cancer-associated pathways were rapidly induced in the presence of inflammation. In situ hybridization, RTqPCR, and Western blot (WB) experiments confirmed that Kras and/or KrasG12D mRNA and protein were significantly increased in acinar cells aftercerulein treatment. To further explore the control of Kras expression, we generated a novel mouse model in which a citrine gene (a variant of GFP) is fused in frame with the endogenous Kras gene, allowing to detect Kras on tissue sections using a GFP antibody. We demonstrated by WB that the protein expression of Kras, KrasG12D and citrine-Kras fusion was similarly regulated in response to cerulein treatment, suggesting the involvement of the same regulatory elements on all three alleles. The ability of citrine-Kras fusion to bind GTP and translocate to the plasma membrane in acini was not altered compared to wild-type Kras protein. Little or no citrine-Kras was visualized in acinar cells of tamoxifen-treated Elastase-CreER/LSLKrasG12D/citrine-Kras mice. However, a 30-fold increase of citrine-Kras at the cell membrane of neoplastic lesions was detected after cerulein treatment. This indicates that inflammation is required for expression and plasma membrane targeting of Kras and KrasG12D in metaplastic acini. Importantly, protein expression of FT-α/β, Rce1 and ICMT, three enzymes necessary for Kras targeting to the membrane was greatly increased in murine acinar cells during pancreatitis. Basal FT-α/β, Rce1 and ICMT levels were found to be very low in normal mouse and human pancreata, compared to other organs, and their expression increased in primary mouse and human acini grown under conditions inducing the formation of preneoplastic lesions. In addition, computational analysis of a PDAC cohort (n=178) from The Cancer Genome Atlas (TCGA) revealed a positive correlation between expression of KrasG12D and its regulating enzymes. We conclude that Kras and KrasG12D are normally not present in acinar cells in vivo and that inflammation induces a coordinated program driving their expression and translocation to plasma membrane. Our findings provide a first clue to the long-standing observation that in several organs, Kras mutations, alone, are not sufficient to affect tissue homeostasis.

In a subset of patients suffering from myeloproliferative neoplasms (MPNs), calreticulin (CALR) exon 9 frameshift mutations are known to be responsible for the development of either essential thrombocythemia (ET) or primary myelofibrosis (PMF) (1, 2). The most prevalent mutations are a 52-bp deletion (del52, type-1 mutation) and a 5-bp TTGTC insertion (ins5, type-2 mutation). In these patients, the mutational status is almost always heterozygous. Our group and collaborators have recently shown that the pathogenic mutant CALR proteins require interaction with and activation of the thrombopoietin receptor (TpoR) for activation of the JAK-STAT pathway (3, 4). Until now, no knock-in mouse model of these diseases has been published. In this abstract, we show how we succeeded in creating such a model. We had shown that the murine CALR mutant proteins behave just like their human counterparts (5). Specifically, the del52, ins5 and del61 (61bp deletion, type-1) Calr mutations were able to transform Ba/F3 cells (murine pro-B lymphocytic cells normally dependent on IL-3 for growth) expressing the thrombopoietin receptor (TpoR) and render them cytokine-independent. Importantly, we also mutated the Ba/F3 genome using the widely adopted CRISPR/Cas9 system in order to create a 61-bp deletion of the exon 9 of Calr. This too successfully transformed the Ba/F3 cells, showing that endogenous levels of expression of a mutant CALR protein are sufficient to induce phenotype in vitro. Now, using the same approach, we injected C57BL/6J mouse zygotes with the same CRISPR/Cas9 constructs to create the same 61-bp deletion in the murine Calr gene. Out of 46 pups born from the procedure, one male pup was heterozygous for the 61-bp deletion. By in vitro fertilization, we subsequently obtained heterozygous Calr del61/WT pups. After inter-breeding the mice, we analyzed the blood of 12 Calr del61/WT males and 12 Calr WT/WT males (littermates) at three different timepoints (15, 18 and 22 weeks old) and found that the Calr del61/WT mice showed significantly higher levels of circulating platelets. Conversely, red blood and white blood cell numbers were the same between both groups at all time points. We further show that expression of a mutant CALR protein, in a heterozygous state, is sufficient to induce abnormal proliferation of megakaryocytes and develop an ET phenotype in vivo in mice. Follow-up in dynamics of the phenotype and bone marrow and spleen pathology (examination of myeloproliferation and fibrosis) allow comparison with the retroviral murine models of CALR-mutant MPNs and with the known features of the human disease. The only limitation of our model is the fact that the Calr del61 mutation is parentally acquired and widespread throughout the organism. With this new model, we aim to test the efficiency of various drugs to prevent or cure the MPN phenotype, such as ruxolitinib, a JAK2 type-1 inhibitor that is already used in clinics in patients suffering from CALR-mutated MPNs. We also now have a means to generate a high number of Calr del61/WT bone marrow cells to extensively study the oncogenic properties of the Calr mutations at different stages of the hematopoeisis. It will also be of great interest to study, if generated, a homozygous mutational status of Calr del61 in vivo. Thus, our system will shed light on the importance of the negatively charged tail of CALR and on the effects of the novel positively charged tail on myeloproliferation.

Cardiac development arises from two sources of mesoderm progenitors, the first heart field (FHF) and the second (SHF). Mesp1 has been proposed to mark the most primitive multipotent cardiac progenitors common for both heart fields. Here, using clonal analysis of the earliest prospective cardiovascular progenitors in a temporally controlled manner during early gastrulation, we found that Mesp1 progenitors consist of two temporally distinct pools of progenitors restricted to either the FHF or the SHF. FHF progenitors were unipotent, whereas SHF progenitors were either unipotent or bipotent. Microarray and single-cell PCR with reverse transcription analysis of Mesp1 progenitors revealed the existence of molecularly distinct populations of Mesp1 progenitors, consistent with their lineage and regional contribution. Together, these results provide evidence that heart development arises from distinct populations of unipotent and bipotent cardiac progenitors that independently express Mesp1 at different time points during their specification, revealing that the regional segregation and lineage restriction of cardiac progenitors occur very early during gastrulation.

We report the sequence of a human cDNA that encodes a 46 kDa transmembrane protein homologous to bacterial transporters for phosphate esters. This protein presents at its carboxy terminus the consensus motif for retention in the endoplasmic reticulum. Northern blots of rat tissues indicate that the corresponding mRNA is mostly expressed in liver and kidney. In two patients with glycogen storage disease type Ib, mutations were observed that either replaced a conserved Gly to Cys or introduced a premature stop codon. The encoded protein is therefore most likely the glucose 6-phosphate translocase that is functionally associated with glucose-6-phosphatase.