Ultrasonic vocalizations (USVs) are used as a phenotypic marker in mouse models of neuropsychiatric disorders. Nevertheless, current methodologies still require time-consuming manual input or sound recordings clean of any background noise. We developed a method to overcome these two restraints to boost knowledge on mouse USVs. The methods are freely available and the USV analysis runs online at https://usv.pasteur.cloud. As little is currently known about usage and structure of ultrasonic vocalizations during social interactions over the long-term and in unconstrained context, we investigated mouse spontaneous communication by coupling the analysis of USVs with automatic labeling of behaviors. We continuously recorded during 3 days undisturbed interactions of same-sex pairs of C57BL/6J sexually naive males and females at 5 weeks and 3 and 7 months of age. In same-sex interactions, we observed robust differences between males and females in the amount of USVs produced, in the acoustic structure and in the contexts of emission. The context-specific acoustic variations emerged with increasing age. The emission of USVs also reflected a high level of excitement during social interactions. We finally highlighted the importance of studying long-term spontaneous communication by investigating female mice lacking Shank3, a synaptic protein associated with autism. While the previous short-time constrained investigations could not detect USV emission abnormalities, our analysis revealed robust differences in the usage and structure of the USVs emitted by mutant mice compared to wild-type female pairs.

International audienceRibosome biogenesis is an essential cellular process. Its impairment is associated with developmental defects and increased risk of cancer. The in vivo cellular responses to defective ribosome biogenesis and the underlying molecular mechanisms are still incompletely understood. In particular, the consequences of impaired ribosome biogenesis within the intestinal epithelium in mammals have not been investigated so far. Here we adopted a genetic approach to investigate the role of Notchless (NLE), an essential actor of ribosome biogenesis, in the adult mouse intestinal lineage. Nle deficiency led to defects in the synthesis of large ribosomal subunit in crypts cells and resulted in the rapid elimination of intestinal stem cells and progenitors through distinct types of cellular responses, including apoptosis, cell cycle arrest and biased differentiation toward the goblet cell lineage. Similar observations were made using the rRNA transcription inhibitor CX-5461 on intestinal organoids culture. Importantly, we found that p53 activation was responsible for most of the cellular responses observed, including differentiation toward the goblet cell lineage. Moreover, we identify the goblet cell-specific marker Muc2 as a direct transcriptional target of p53. Nle-deficient ISCs and progenitors disappearance persisted in the absence of p53, underlying the existence of p53-independent cellular responses following defective ribosome biogenesis. Our data indicate that NLE is a crucial factor for intestinal homeostasis and provide new insights into how perturbations of ribosome biogenesis impact on cell fate decisions within the intestinal epithelium

The contribution of basal cellular processes to the regulation of tissue homeostasis has just started to be appreciated. However, our knowledge of the modulation of ribosome biogenesis activity in situ within specific lineages remains very limited. This is largely due to the lack of assays that enable quantitation of ribosome biogenesis in small numbers of cells in vivo. We used a technique, named Flow-FISH, combining cell surface antibody staining and flow cytometry with intracellular ribosomal RNA (rRNA) FISH, to measure the levels of pre-rRNAs of hematopoietic cells in vivo. Here, we show that Flow-FISH reports and quantifies ribosome biogenesis activity in hematopoietic cell populations, thereby providing original data on this fundamental process notably in rare populations such as hematopoietic stem and progenitor cells. We unravel variations in pre-rRNA levels between different hematopoietic progenitor compartments and during erythroid differentiation. In particular, our data indicate that, contrary to what may be anticipated from their quiescent state, hematopoietic stem cells have significant ribosome biogenesis activity. Moreover, variations in pre-rRNA levels do not correlate with proliferation rates, suggesting that cell type-specific mechanisms might regulate ribosome biogenesis in hematopoietic stem cells and progenitors. Our study contributes to a better understanding of the cellular physiology of the hematopoietic system in vivo in unperturbed situations.

Inner cell Mass (ICM) specification into epiblast (Epi) and primitive endoderm (PrE) is an asynchronous and progressive process taking place between E3.0 to E3.75 under the control of the Fibroblast Growth Factor (FGF)/Extracellular signal-Regulated Kinase (ERK) signaling pathway. Here, we have analyzed in details the kinetics of specification and found that ICM cell responsiveness to the up and down regulation of FGF signaling activity are temporally distinct. We also showed that PrE progenitors are generated later than Epi progenitors. We further demonstrated that, during this late phase of specification, a 4 hours period of FGF/ERK inhibition prior E3.75 is sufficient to convert ICM cells into Epi. Finally, we showed that ICM conversion into Epi in response to inhibition during this short time window requires both transcription and proteasome degradation. Collectively, our data give new insights into the timing and mechanisms involved in the process of ICM specification.