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Researchers who aim to globally analyze the gastrointestinal immune system via flow cytometry have many protocol options to choose from, with specifics generally tied to gut wall layers of interest. To get a clearer idea of the approach we should use on full-thickness colon samples from mice, we first undertook a systematic comparison of three tissue dissociation techniques: two based on enzymatic cocktails and the other one based on manual crushing. Using flow cytometry panels of general markers of lymphoid and myeloid cells, we found that the presence of cell-surface markers and relative cell population frequencies were more stable with the mechanical method. Both enzymatic approaches were associated with a marked decrease of several cell-surface markers. Using mechanical dissociation, we then developed two minimally overlapping panels, consisting of a total of 26 antibodies, for serial profiling of lymphoid and myeloid lineages from the mouse colon in greater detail. Here, we highlight how we accurately delineate these populations by manual gating, as well as the reproducibility of our panels on mouse spleen and whole blood. As a proof-of-principle of the usefulness of our general approach, we also report segment- and life stage-specific patterns of immune cell profiles in the colon. Overall, our data indicate that mechanical dissociation is more suitable and efficient than enzymatic methods for recovering immune cells from all colon layers at once. Additionally, our panels will provide researchers with a relatively simple tool for detailed immune cell profiling in the murine gastrointestinal tract, regardless of life stage or experimental conditions.

DNA replication and repair are basic yet essential molecular processes for all cells. RFC1 encodes the largest subunit of the Replication Factor C, an essential clamp-loader for DNA replication and repair. Intronic repeat expansion in RFC1 has recently been associated with so-called RFC1 -related disorders, which mainly encompass late-onset cerebellar ataxias. However, the mechanisms making certain tissues more susceptible to defects in these universal pathways remain mysterious. Here, we provide the first investigation of RFC1 gene function in vivo using zebrafish. We showed that RFC1 is expressed in neural progenitor cells within the developing cerebellum, where it maintains their genomic integrity during neurogenic maturation. Accordingly, RFC1 loss-of-function leads to a severe cerebellar phenotype due to impaired neurogenesis of both Purkinje and granule cells. Our data point to a specific role of RFC1 in the developing cerebellum, paving the way for a better understanding of the pathogenic mechanisms underlying RFC1 -related disorders. RFC1 is a replication and repair protein, and mutations to this gene are associated with rare movement disorders like late-onset cerebellar ataxias. Here they show that RFC1 is essential for zebrafish cerebellar development by preserving genome integrity in neural progenitors during neurogenesis.

Previously focused primarily on enteric neurons, studies of the enteric nervous system (ENS) in both health and disease are now broadening to recognize the equally significant role played by enteric glial cells (EGCs). Commensurate to the vast array of gastrointestinal functions they influence, EGCs exhibit considerable diversity in terms of location, morphology, molecular profiles, and functional attributes. However, the mechanisms underlying this diversification of EGCs remain largely unexplored. To begin unraveling the mechanistic complexities of EGC diversity, the current study aimed to examine its spatiotemporal aspects in greater detail, and to assess whether the various sources of enteric neural progenitors contribute differentially to this diversity. Based on established topo-morphological criteria for categorizing EGCs into four main subtypes, our detailed immunofluorescence analyses first revealed that these subtypes emerge sequentially during early postnatal development, in a coordinated manner with the structural changes that occur in the ENS. When combined with genetic cell lineage tracing experiments, our analyses then uncovered a strongly biased contribution by Schwann cell-derived enteric neural progenitors to particular topo-morphological subtypes of EGCs. Taken together, these findings provide a robust foundation for further investigations into the molecular and cellular mechanisms governing EGC diversity.

Excess of histamine in gut lumen generates a pronounced gastrointestinal discomfort, which may include diarrhea and peristalsis dysfunctions. Deleterious effects of histamine can be alleviated with antihistamine drugs targeting histamine receptors. However, many antihistamine agents come with various undesirable side effects. Vegetal diamine oxidase (vDAO) might be a relevant alternative owing to its histaminase activity. Mammalian intestinal mucosa contains an endogenous DAO, yet possessing lower activity compared to that of vDAO preparation. Moreover, in several pathological conditions such as inflammatory bowel disease and irritable bowel syndrome, this endogenous DAO enzyme can be lost or inactivated. Here, we tested the therapeutic potential of vDAO by focusing on the well-known effect of histamine on gut motility. Using ex vivo and in vitro assays, we found that vDAO is more potent than commercial anti-histamine drugs at inhibiting histamine-induced contraction of murine distal colon muscles. We also identified pyridoxal 5′-phosphate (the biologically active form of vitamin B6) as an effective enhancer of vDAO antispasmodic activity. Furthermore, we discovered that rectally administered vDAO can be retained on gut mucosa and remain active. These observations make administration of vDAO in the gut lumen a valid alternative treatment for histamine-induced intestinal dysfunctions.

The first volume of this Special Issue met its goal of covering several aspects regarding both the normal and abnormal development of neural crest cells, which form a truly unique multipotent and highly migratory cell population that only exists in vertebrates [...]

CHARGE syndrome—which stands for coloboma of the eye, heart defects, atresia of choanae, retardation of growth/development, genital abnormalities, and ear anomalies—is a severe developmental disorder with wide phenotypic variability, caused mainly by mutations in CHD7 (chromodomain helicase DNA-binding protein 7), known to encode a chromatin remodeler. The genetic lesions responsible for CHD7 mutation-negative cases are unknown, at least in part because the pathogenic mechanisms underlying CHARGE syndrome remain poorly defined. Here, we report the characterization of a mouse model for CHD7 mutation-negative cases of CHARGE syndrome generated by insertional mutagenesis of Fam172a (family with sequence similarity 172, member A). We show that Fam172a plays a key role in the regulation of cotranscriptional alternative splicing, notably by interacting with Ago2 (Argonaute-2) and Chd7. Validation studies in a human cohort allow us to propose that dysregulation of cotranscriptional alternative splicing is a unifying pathogenic mechanism for both CHD7 mutation-positive and CHD7 mutation-negative cases. We also present evidence that such splicing defects can be corrected in vitro by acute rapamycin treatment.

Hirschsprung disease (HSCR) is a complex genetic disorder of neural crest development resulting in incomplete formation of the enteric nervous system (ENS). This life-threatening neurocristopathy affects 1/5000 live births, with a currently unexplained male-biased ratio. To address this lack of knowledge, we took advantage of the TashT mutant mouse line, which is the only HSCR model to display a robust male bias. Our prior work revealed that the TashT insertional mutation perturbs a Chr.10 silencer-enriched non-coding region, leading to transcriptional dysregulation of hundreds of genes in neural crest-derived ENS progenitors of both sexes. Here, through sex-stratified transcriptome analyses and targeted overexpression in ENS progenitors, we show that male-biased ENS malformation in TashT embryos is not due to upregulation of Sry-the murine ortholog of a candidate gene for the HSCR male bias in humans-but instead involves upregulation of another Y-linked gene, Ddx3y. This discovery might be clinically relevant since we further found that the DDX3Y protein is also expressed in the ENS of a subset of male HSCR patients. Mechanistically, other data including chromosome conformation captured-based assays and CRISPR/Cas9-mediated deletions suggest that Ddx3y upregulation in male TashT ENS progenitors is due to increased transactivation by p53, which appears especially active in these cells yet without triggering apoptosis. Accordingly, in utero treatment of TashT embryos with the p53 inhibitor pifithrin-α decreased Ddx3y expression and abolished the otherwise more severe ENS defect in TashT males. Our data thus highlight novel pathogenic roles for p53 and DDX3Y during ENS formation in mice, a finding that might help to explain the intriguing male bias of HSCR in humans.

Neural crest cells (NCC) are a transient migratory cell population that generates diverse cell types such as neurons and glia of the enteric nervous system (ENS). Via an insertional mutation screen for loci affecting NCC development in mice, we identified one line-named TashT-that displays a partially penetrant aganglionic megacolon phenotype in a strong male-biased manner. Interestingly, this phenotype is highly reminiscent of human Hirschsprung's disease, a neurocristopathy with a still unexplained male sex bias. In contrast to the megacolon phenotype, colonic aganglionosis is almost fully penetrant in homozygous TashT animals. The sex bias in megacolon expressivity can be explained by the fact that the male ENS ends, on average, around a \"tipping point\" of minimal colonic ganglionosis while the female ENS ends, on average, just beyond it. Detailed analysis of embryonic intestines revealed that aganglionosis in homozygous TashT animals is due to slower migration of enteric NCC. The TashT insertional mutation is localized in a gene desert containing multiple highly conserved elements that exhibit repressive activity in reporter assays. RNAseq analyses and 3C assays revealed that the TashT insertion results, at least in part, in NCC-specific relief of repression of the uncharacterized gene Fam162b; an outcome independently confirmed via transient transgenesis. The transcriptional signature of enteric NCC from homozygous TashT embryos is also characterized by the deregulation of genes encoding members of the most important signaling pathways for ENS formation-Gdnf/Ret and Edn3/Ednrb-and, intriguingly, the downregulation of specific subsets of X-linked genes. In conclusion, this study not only allowed the identification of Fam162b coding and regulatory sequences as novel candidate loci for Hirschsprung's disease but also provides important new insights into its male sex bias.

Waardenburg syndrome is a neurocristopathy characterized by a combination of skin and hair depigmentation, and inner ear defects. In the type 4 form, these defects show comorbidity with Hirschsprung disease, a disorder marked by an absence of neural ganglia in the distal colon, triggering functional intestinal obstruction. Here, we report that the Spot mouse line – obtained through an insertional mutagenesis screen for genes involved in neural crest cell (NCC) development – is a model for Waardenburg syndrome type 4. We found that the Spot insertional mutation causes overexpression of an overlapping gene pair composed of the transcription-factor-encoding Nr2f1 and the antisense long non-coding RNA A830082K12Rik in NCCs through a mechanism involving relief of repression of these genes. Consistent with the previously described role of Nr2f1 in promoting gliogenesis in the central nervous system, we further found that NCC-derived progenitors of the enteric nervous system fail to fully colonize Spot embryonic guts owing to their premature differentiation in glial cells. Taken together, our data thus identify silencer elements of the Nr2f1-A830082K12Rik gene pair as new candidate loci for Waardenburg syndrome type 4.

GATA4 is an essential transcription factor required for the development and function of multiple tissues, including a major role in gonadogenesis. Despite its crucial role, the molecular mechanisms that regulate Gata4 expression in vivo remain poorly understood. We recently found that the Gata4 gene is expressed as multiple transcripts with distinct 5' origins. These co-expressed alternative transcripts are generated by different non-coding first exons with transcripts E1a and E1b being the most prominent. Moreover, we previously showed that an Ebox element, located in Gata4 5' flanking sequences upstream of exon 1a, is important for the promoter activity of these sequences in cell lines. To confirm the importance of this element in vivo, we generated and characterized Gata4 Ebox knockout mice. Quantitative PCR analyses realized on gonads, heart and liver at three developmental stages (embryonic, pre-pubertal and adult) revealed that the Ebox mutation leads to a robust and specific decrease (up to 89%) of Gata4 E1a transcript expression in all tissues and stages examined. However, a detailed characterization of the gonads revealed normal morphology and GATA4 protein levels in these mutants. Our qPCR data further indicate that this outcome is most likely due to the presence of Gata4 E1b mRNA, whose expression levels were not decreased by the Ebox mutation. In conclusion, our work clearly confirms the importance of the proximal Ebox element and suggests that adequate GATA4 protein expression is likely protected by a compensation mechanism between Gata4 E1a and E1b transcripts operating at the translational level.