Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos

Vascularisation is not necessary for gut colonisation by enteric neural crest cells

The vasculature and nervous system share striking similarities in their networked, tree-like architecture and in the way they are super-imposed in mature organs. It has previously been suggested that the intestinal microvasculature network directs the migration of enteric neural crest cells (ENCC) along the gut to promote the formation of the enteric nervous system (ENS). To investigate the inter-relationship of migrating ENCC, ENS formation and gut vascular development we combined fate-mapping of ENCC with immunolabelling and intravascular dye injection to visualise nascent blood vessel networks. We found that the enteric and vascular networks initially had very distinct patterns of development. In the foregut, ENCC migrated through areas devoid of established vascular networks. In vessel-rich areas, such as the midgut and hindgut, the distribution of migrating ENCC did not support the idea that these cells followed a pre-established vascular network. Moreover, when gut vascular development was impaired, either genetically in Vegfa120/120 or Tie2-Cre;Nrp1fl/- mice or using an in vitro Wnt1-Cre;Rosa26Yfp/+ mouse model of ENS development, ENCC still colonised the entire length of the gut, including the terminal hindgut. These results demonstrate that blood vessel networks are not necessary to guide migrating ENCC during ENS development. Conversely, in miRet51 mice, which lack ENS in the hindgut, the vascular network in this region appeared to be normal suggesting that in early development both networks form independently of each other

An essential function of the mitogen‐activated protein kinase Erk2 in mouse trophoblast development

The closely related mitogen-activated protein kinase isoforms extracellular signal-regulated kinase 1 (ERK1) and ERK2 have been implicated in the control of cell proliferation, differentiation and survival. However, the specific in vivo functions of the two ERK isoforms remain to be analysed. Here, we show that disruption of the Erk2 locus leads to embryonic lethality early in mouse development after the implantation stage. Erk2 mutant embryos fail to form the ectoplacental cone and extra-embryonic ectoderm, which give rise to mature trophoblast derivatives in the fetus. Analysis of chimeric embryos showed that Erk2 functions in a cell-autonomous manner during the development of extra-embryonic cell lineages. We also found that both Erk2 and Erk1 are widely expressed throughout early-stage embryos. The inability of Erk1 to compensate for Erk2 function suggests a specific function for Erk2 in normal trophoblast development in the mouse, probably in regulating the proliferation of polar trophectoderm cells

Mutations in pericentrin cause Seckel syndrome with defective ATR-dependent DNA damage signaling

Large brain size is one of the defining characteristics of modern humans. Seckel syndrome (MIM 210600), a disorder of markedly reduced brain and body size, is associated with defective ATR-dependent DNA damage signaling. Only a single hypomorphic mutation of ATR has been identified in this genetically heterogeneous condition. We now report that mutations in the gene encoding pericentrin (PCNT)--resulting in the loss of pericentrin from the centrosome, where it has key functions anchoring both structural and regulatory proteins--also cause Seckel syndrome. Furthermore, we find that cells of individuals with Seckel syndrome due to mutations in PCNT (PCNT-Seckel) have defects in ATR-dependent checkpoint signaling, providing the first evidence linking a structural centrosomal protein with DNA damage signaling. These findings also suggest that other known microcephaly genes implicated in either DNA repair responses or centrosomal function may act in common developmental pathways determining human brain and body size

3D human liver tissue from pluripotent stem cells displays stable phenotype in vitro and supports compromised liver function in vivo.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to the shortage of donor organs. Developing renewable sources of human liver tissue is therefore attractive. Pluripotent stem cell-derived liver tissue represents a potential alternative to cadaver derived hepatocytes and whole organ transplant. At present, two-dimensional differentiation procedures deliver tissue lacking certain functions and long-term stability. Efforts to overcome these limiting factors have led to the building of three-dimensional (3D) cellular aggregates. Although enabling for the field, their widespread application is limited due to their reliance on variable biological components. Our studies focused on the development of 3D liver tissue under defined conditions. In vitro generated 3D tissues exhibited stable phenotype for over 1 year in culture, providing an attractive resource for long-term in vitro studies. Moreover, 3D derived tissue provided critical liver support in two animal models, including immunocompetent recipients. Therefore, we believe that our study provides stable human tissue to better model liver biology 'in the dish', and in the future may permit the support of compromised liver function in humans

CoNIC Challenge: Pushing the Frontiers of Nuclear Detection, Segmentation, Classification and Counting

Nuclear detection, segmentation and morphometric profiling are essential in helping us further understand the relationship between histology and patient outcome. To drive innovation in this area, we setup a community-wide challenge using the largest available dataset of its kind to assess nuclear segmentation and cellular composition. Our challenge, named CoNIC, stimulated the development of reproducible algorithms for cellular recognition with real-time result inspection on public leaderboards. We conducted an extensive post-challenge analysis based on the top-performing models using 1,658 whole-slide images of colon tissue. With around 700 million detected nuclei per model, associated features were used for dysplasia grading and survival analysis, where we demonstrated that the challenge's improvement over the previous state-of-the-art led to significant boosts in downstream performance. Our findings also suggest that eosinophils and neutrophils play an important role in the tumour microevironment. We release challenge models and WSI-level results to foster the development of further methods for biomarker discovery

The kinetochore protein, CENPF, is mutated in human ciliopathy and microcephaly phenotypes

Background: Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly. Methods and results Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F. Conclusions: Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis

Rôle du facteur de transcription Otx2 lors de la maintenance de l'identité du mésencéphale et de la différenciation neuronale dans le mésencéphale murin

Le facteur de transcription Otx2 détermine l'identité mésencéphalique face à l'identité métencéphalique (rhombencéphale antérieur) notamment en positionant et maintenant le centre organisateur de l'isthme au niveau de la frontière mésencéphalique/métencéphalique. Cependant le rôle de Otx2 n'est pas restreint à cette étape du développement comme le suggère la maintenance de son expression dans le mésence phale bien après l'établissement du centre organisateur de l'isthme. Pour étudier, le rôle plus tardif de Otx2 dans le mésencéphale, j'ai utilisé une stratégie d'invalidation conditionnelle du gène Otx2 à l'aide du système CRE-LoxP et je me suis plus particulièrement intéressé à l'étude de l'identité neuronale dans le mésencéphale en l'absence de Otx2. Suite à la perte de Otx2 dans les progéniteurs neuronaux, une partie d'entre eux adoptent un programme de différenciation caractéristique du rhombencéphale antérieur ou métencéphale : cellules cérébelleuses dorsalement et neurones sérotoninergiques ventralement. Nous avons montré que ce phénotype est indépendant de tout déplacement du centre organisateur de l'isthme dans le mésencéphale mutant. L'analyse de l'expression de différents gènes nous a permis de montrer que les progéniteurs mésencéphalique adoptent un programme de différenciation métencéphalique (cellules cérébelleuses et neurones sérotoninergiques) au dépend des programmes mésencéphaliques qui déterminent les différentes classes de neurones mésencéphaliques (neurones dopaminergiques, noyau rouge). Otx2 en réprimant les gènes Math1 (cervelet) et Nkx2-2 (neurones sérotoninergiques) dans les progéniteurs mésencéphaliques empêche la mise en place des programmes de différenciation rhombencéphalique. En résumé ce travail démontre que Otx2 est requis dansle mésencéphale après 10,5 jpc pour maintenir l'identité mésencéphalique des progéniteurs et réprimer tout programme de différenciation rhombencéphalique.The transcription factor Otx2 is required to determine mesencephalic versus metencephalic (anterior rhombencephalon) territory during embryogenesis. So far, this function of Otx2 involves positioning and maintaining the mid-hindbrain organizer (MHO) at the border between midbrain and anterior hindbrain. Otx2 expression is maintained in midbrain progenitors long after this organizer is established suggesting that Otx2 could play a role during neurogenesis. I used a conditional knock-out strategy (system CRE-LoxP) to study Otx2 function during neurogenesis in the neuroectoderme and my analysis is focused on neuronal identity in mesencephalon in absence of Otx2. Following Otx2 deletion in mesencephalic neuronal progenitors, some progenitors adopt a rhombencephalic fate that is normally repressed by Otx2 in mesencephalon. Indeed, ectopic cerebellar cells and ectopic serotonergic neurons are observed in dorsal and ventral mesencephalon respectively. Gene expression analysis show that the mesencephalic neuronal progenitors adopt rhombencephalic fate at the expanse of mesencephalic fate (dopaminergic and red nuclei neurons) and we conclude that Otx2 prevents the formation of cerebellar neurons and serotonergic neurons in midbrain presumably by repressing the expression of genes such as Math1 (cerebellum) and Nkx2-2 (serotonergic neurons) in progenitors. In summary, this work demonstrates that Otx2 is required in midbrain progenitors to maintain mesencephalic fate and repress rhombencephalic differentiation program independently of its earlier patterning function at the mid-hindbrain border.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Propagating information from snow observations with CrocO ensemble data assimilation system: a 10-years case study over a snow depth observation network

International audienceThe mountainous snow cover is highly variable at all temporal and spatial scales. Snowpack models only imperfectly represent this variability, because of uncertain meteorological inputs, physical parameterizations, and unresolved terrain features. In situ observations of the height of snow (HS), despite their limited representativeness, could help constrain intermediate and large-scale modeling errors by means of data assimilation. In this work, we assimilate HS observations from an in situ network of 295 stations covering the French Alps, Pyrenees, and Andorra, over the period 2009–2019. In view of assimilating such observations into a spatialized snow cover modeling framework, we investigate whether such observations can be used to correct neighboring snowpack simulations. We use CrocO, an ensemble data assimilation framework of snow cover modeling, based on a particle filter suited to the propagation of information from observed to unobserved areas. This ensemble system already benefits from meteorological observations, assimilated within SAFRAN analysis scheme. CrocO also proposes various localization strategies to assimilate snow observations. These approaches are evaluated in a leave-one-out setup against the operational deterministic model and its ensemble open-loop counterpart, both running without HS assimilation. Results show that an intermediate localization radius of 35–50 km yields a slightly lower root mean square error (RMSE), and a better spread–skill than the strategy of assimilating all the observations from a whole mountain range. Significant continuous ranked probability score (CRPS) improvements of about 13 % are obtained in the areas where the open-loop modeling errors are the largest, e.g., the Haute-Ariège, Andorra, and the extreme southern Alps. Over these areas, weather station observations are generally sparser, resulting in more uncertain meteorological analyses and, therefore, snow simulations. In situ HS observations thus show an interesting complementarity with meteorological observations to better constrain snow cover simulations over large areas