Efficient enzyme-free method to assess the development and maturation of the innate and adaptive immune systems in the mouse colon.

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.

CHD7 regulates craniofacial cartilage development via controlling HTR2B expression.

Mutations in the Chromodomain helicase DNA-binding protein 7 - coding gene (CHD7) cause CHARGE syndrome (CS). Although craniofacial and skeletal abnormalities are major features of CS patients, the role of CHD7 in bone and cartilage development remain largely unexplored. Here, using a zebrafish (Danio rerio) CS model, we show that chd7-/- larvae display abnormal craniofacial cartilage development and spinal deformities. The craniofacial and spine defects are accompanied by a marked reduction of bone mineralization. At the molecular level, we show that these phenotypes are associated with significant reduction in the expression levels of osteoblast differentiation markers. Additionally, we detected a marked depletion of collagen 2α1 in the cartilage of craniofacial regions and vertebrae, along with significantly reduced number of chondrocytes. Chondrogenesis defects are at least in part due to downregulation of htr2b, which we found to be also dysregulated in human cells derived from an individual with CHD7 mutation-positive CS. Overall, this study thus unveils an essential role for CHD7 in cartilage and bone development, with potential clinical relevance for the craniofacial defects associated with CS.

Patients with CHARGE syndrome exhibit skeletal defects. CHARGE syndrome is primarily caused by mutations in the chromatin remodeler-coding gene CHD7. To investigate the poorly characterized role of CHD7 in cartilage and bone development, here, we examine the craniofacial and bone anomalies in a zebrafish chd7-/- mutant model. We find that zebrafish mutant larvae exhibit striking dysmorphism of craniofacial structures and spinal deformities. Notably, we find a significant reduction in osteoblast, chondrocyte, and collagen matrix markers. This work provides important insights to improve our understanding of the role of chd7 in skeletal development.

Harnessing the Power of Enteric Glial Cells' Plasticity and Multipotency for Advancing Regenerative Medicine.

The enteric nervous system (ENS), known as the intrinsic nervous system of the gastrointestinal tract, is composed of a diverse array of neuronal and glial cell subtypes. Fascinating questions surrounding the generation of cellular diversity in the ENS have captivated ENS biologists for a considerable time, particularly with recent advancements in cell type-specific transcriptomics at both population and single-cell levels. However, the current focus of research in this field is predominantly restricted to the study of enteric neuron subtypes, while the investigation of enteric glia subtypes significantly lags behind. Despite this, enteric glial cells (EGCs) are increasingly recognized as equally important regulators of numerous bowel functions. Moreover, a subset of postnatal EGCs exhibits remarkable plasticity and multipotency, distinguishing them as critical entities in the context of advancing regenerative medicine. In this review, we aim to provide an updated overview of the current knowledge on this subject, while also identifying key questions that necessitate future exploration.

The CHARGE syndrome-associated protein FAM172A controls AGO2 nuclear import.

CHARGE syndrome is a neural crest-related disorder mainly caused by mutation of the chromatin remodeler-coding gene CHD7 Alternative causes include mutation of other chromatin and/or splicing factors. One of these additional players is the poorly characterized FAM172A, which we previously found in a complex with CHD7 and the small RNA-binding protein AGO2 at the chromatin-spliceosome interface. Focusing on the FAM172A-AGO2 interplay, we now report that FAM172A is a direct binding partner of AGO2 and, as such, one of the long sought-after regulators of AGO2 nuclear import. We show that this FAM172A function mainly relies on its classical bipartite nuclear localization signal and associated canonical importin-α/ß pathway, being enhanced by CK2-induced phosphorylation and abrogated by a CHARGE syndrome-associated missense mutation. Overall, this study thus strengthens the notion that noncanonical nuclear functions of AGO2 and associated regulatory mechanisms might be clinically relevant.

Neural Crest Development in Health and Disease.

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 [...].

NR2F1 regulates a Schwann cell precursor-vs-melanocyte cell fate switch in a mouse model of Waardenburg syndrome type IV.

Waardenburg syndrome type 4 (WS4) combines abnormal development of neural crest cell (NCC)-derived melanocytes (causing depigmentation and inner ear dysfunction) and enteric nervous system (causing aganglionic megacolon). The full spectrum of WS4 phenotype is present in Spot mice, in which an insertional mutation close to a silencer element leads to NCC-specific upregulation of the transcription factor-coding gene Nr2f1. These mice were previously found to develop aganglionic megacolon because of NR2F1-induced premature differentiation of enteric neural progenitors into enteric glia. Intriguingly, this prior work also showed that inner ear dysfunction in Spot mutants specifically affects balance but not hearing, consistent with the absence of melanocytes in the vestibule only. Here, we report an analysis of the effect of Nr2f1 upregulation on the development of both inner ear and skin melanocytes, also taking in consideration their origin relative to the dorsolateral and ventral NCC migration pathways. In the trunk, we found that NR2F1 overabundance in Spot NCCs forces dorso-laterally migrating melanoblasts to abnormally adopt a Schwann cell precursor (SCP) fate and conversely prevents ventrally migrating SCPs to normally adopt a melanoblast fate. In the head, Nr2f1 upregulation appears not to be uniform, which might explain why SCP-derived melanocytes do colonize the cochlea while non-SCP-derived melanocytes cannot reach the vestibule. Collectively, these data point to a key role for NR2F1 in the control of SCP-vs-melanocyte fate choice and unveil a new pathogenic mechanism for WS4. Moreover, our data argue against the proposed existence of a transit-amplifying compartment of melanocyte precursors in hair follicles.

CHARGE syndrome-associated proteins FAM172A and CHD7 influence male sex determination and differentiation through transcriptional and alternative splicing mechanisms.

To gain further insight into chromatin-mediated regulation of mammalian sex determination, we analyzed the role of the CHARGE syndrome-associated proteins FAM172A and CHD7. This study is based on our prior discoveries that a subset of corresponding mutant mice display complete male-to-female sex reversal, and that both of these proteins regulate co-transcriptional alternative splicing in neural crest cells. Here, we report that FAM172A and CHD7 are present in the developing gonads when sex determination normally occurs in mice. The interactome of FAM172A in pre-Sertoli cells again suggests a role at the chromatin-spliceosome interface, like in neural crest cells. Accordingly, analysis of Fam172a-mutant pre-Sertoli cells revealed transcriptional and splicing dysregulation of hundreds of genes. Many of these genes are similarly affected in Chd7-mutant pre-Sertoli cells, including several known key regulators of sex determination and subsequent formation of testis cords. Among them, we notably identified Sry as a direct transcriptional target and WNT pathway-associated Lef1 and Tcf7l2 as direct splicing targets. The identified molecular defects are also associated with the abnormal morphology of seminiferous tubules in mutant postnatal testes. Altogether, our results thus identify FAM172A and CHD7 as new players in the regulation of male sex determination and differentiation in mice, and further highlight the importance of chromatin-mediated regulatory mechanisms in these processes.

Genetic Background Influences Severity of Colonic Aganglionosis and Response to GDNF Enemas in the Holstein Mouse Model of Hirschsprung Disease.

Hirschsprung disease is a congenital malformation where ganglia of the neural crest-derived enteric nervous system are missing over varying lengths of the distal gastrointestinal tract. This complex genetic condition involves both rare and common variants in dozens of genes, many of which have been functionally validated in animal models. Modifier loci present in the genetic background are also believed to influence disease penetrance and severity, but this has not been frequently tested in animal models. Here, we addressed this question using Holstein mice in which aganglionosis is due to excessive deposition of collagen VI around the developing enteric nervous system, thereby allowing us to model trisomy 21-associated Hirschsprung disease. We also asked whether the genetic background might influence the response of Holstein mice to GDNF enemas, which we recently showed to have regenerative properties for the missing enteric nervous system. Compared to Holstein mice in their original FVB/N genetic background, Holstein mice maintained in a C57BL/6N background were found to have a less severe enteric nervous system defect and to be more responsive to GDNF enemas. This change of genetic background had a positive impact on the enteric nervous system only, leaving the neural crest-related pigmentation phenotype of Holstein mice unaffected. Taken together with other similar studies, these results are thus consistent with the notion that the enteric nervous system is more sensitive to genetic background changes than other neural crest derivatives.

Chromatin remodeller CHD7 is required for GABAergic neuron development by promoting PAQR3 expression.

Mutations in the chromatin remodeller-coding gene CHD7 cause CHARGE syndrome (CS). CS features include moderate to severe neurological and behavioural problems, clinically characterized by intellectual disability, attention-deficit/hyperactivity disorder and autism spectrum disorder. To investigate the poorly characterized neurobiological role of CHD7, we here generate a zebrafish chd7-/- model. chd7-/- mutants have less GABAergic neurons and exhibit a hyperactivity behavioural phenotype. The GABAergic neuron defect is at least in part due to downregulation of the CHD7 direct target gene paqr3b, and subsequent upregulation of MAPK/ERK signalling, which is also dysregulated in CHD7 mutant human cells. Through a phenotype-based screen in chd7-/- zebrafish and Caenorhabditis elegans, we show that the small molecule ephedrine restores normal levels of MAPK/ERK signalling and improves both GABAergic defects and behavioural anomalies. We conclude that chd7 promotes paqr3b expression and that this is required for normal GABAergic network development. This work provides insight into the neuropathogenesis associated with CHD7 deficiency and identifies a promising compound for further preclinical studies.

Treatment and Prevention of Neurocristopathies.

Neurocristopathies form a heterogeneous group of rare diseases caused by abnormal development of neural crest cells. Heterogeneity of neurocristopathies directly relates to the nature of these migratory and multipotent cells, which generate dozens of specialized cell types throughout the body. Neurocristopathies are thus characterized by congenital malformations of tissues/organs that otherwise appear to have very little in common, such as the craniofacial skeleton and enteric nervous system. Treatment options are currently very limited, mainly consisting of corrective surgeries. Yet, as reviewed here, analyses of normal and pathological neural crest development in model organisms have opened up the possibility for better treatment options involving cellular and molecular approaches. These approaches provide hope that some neurocristopathies might soon be curable or preventable.

Dhh-expressing Schwann cell precursors contribute to skin and cochlear melanocytes, but not to vestibular melanocytes.

For a long time, melanocytes were believed to be exclusively derived from neural crest cells migrating from the neural tube toward the developing skin. This notion was then challenged by studies suggesting that melanocytes could also be made from neural crest-derived Schwann cell precursors (SCPs) on peripheral nerves. A SCP origin was inferred from lineage tracing studies in mice using a Plp1 promoter-controlled Cre driver transgene (Plp1-CreERT2) and a fluorescent Rosa26 locus-controlled Cre reporter allele (Rosa26FloxSTOP-YFP ). However, doubts were raised in part because another SCP-directed Cre driver controlled by the Dhh promoter (Dhh-Cre) was apparently unable to label melanocytes when used with a non-fluorescent Rosa26 locus-controlled Cre reporter (Rosa26FloxSTOP-LacZ ). Here, we report that the same Dhh-Cre driver line can efficiently label melanocytes when used in a pure FVB/N background together with the fluorescent instead of the non-fluorescent Rosa26 locus-controlled Cre reporter. Our data further suggest that the vast majority of skin melanocytes are SCP-derived. Interestingly, we also discovered that SCPs contribute inner ear melanocytes in a region-specific manner, extensively contributing to the cochlea but not to the vestibule.

Vegetal diamine oxidase alleviates histamine-induced contraction of colonic muscles.

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.

Male-biased aganglionic megacolon in the TashT mouse model of Hirschsprung disease involves upregulation of p53 protein activity and Ddx3y gene expression.

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.

Glial Cell-Derived Neurotrophic Factor Induces Enteric Neurogenesis and Improves Colon Structure and Function in Mouse Models of Hirschsprung Disease.

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is a life-threatening birth defect in which the distal colon is devoid of enteric neural ganglia. HSCR is treated by surgical removal of aganglionic bowel, but many children continue to have severe problems after surgery. We studied whether administration of glial cell derived neurotrophic factor (GDNF) induces enteric nervous system regeneration in mouse models of HSCR. METHODS: We performed studies with four mouse models of HSCR: Holstein (HolTg/Tg, a model for trisomy 21-associated HSCR), TashT (TashTTg/Tg, a model for male-biased HSCR), Piebald-lethal (Ednrbs-l//s-l, a model for EDNRB mutation-associated HSCR), and Ret9/- (with aganglionosis induced by mycophenolate). Mice were given rectal enemas containing GDNF or saline (control) from postnatal days 4 through 8. We measured survival times of mice, and colon tissues were analyzed by histology, immunofluorescence, and immunoblots. Neural ganglia regeneration and structure, bowel motility, epithelial permeability, muscle thickness, and neutrophil infiltration were studied in colon tissues and in mice. Stool samples were collected, and microbiomes were analyzed by 16S rRNA gene sequencing. Time-lapse imaging and genetic cell-lineage tracing were used to identify a source of GDNF-targeted neural progenitors. Human aganglionic colon explants from children with HSCR were cultured with GDNF and evaluated for neurogenesis. RESULTS: GDNF significantly prolonged mean survival times of HolTg/Tg mice, Ednrbs-l//s-l mice, and male TashTTg/Tg mice, compared with control mice, but not Ret9/- mice (which had mycophenolate toxicity). Mice given GDNF developed neurons and glia in distal bowel tissues that were aganglionic in control mice, had a significant increase in colon motility, and had significant decreases in epithelial permeability, muscle thickness, and neutrophil density. We observed dysbiosis in fecal samples from HolTg/Tg mice compared with feces from wild-type mice; fecal microbiomes of mice given GDNF were similar to those of wild-type mice except for Bacteroides. Exogenous luminal GDNF penetrated aganglionic colon epithelium of HolTg/Tg mice, inducing production of endogenous GDNF, and new enteric neurons and glia appeared to arise from Schwann cells within extrinsic nerves. GDNF application to cultured explants of human aganglionic bowel induced proliferation of Schwann cells and formation of new neurons. CONCLUSIONS: GDNF prolonged survival, induced enteric neurogenesis, and improved colon structure and function in 3 mouse models of HSCR. Application of GDNF to cultured explants of aganglionic bowel from children with HSCR induced proliferation of Schwann cells and formation of new neurons. GDNF might be developed for treatment of HSCR.

Gut microbiota-mediated Gene-Environment interaction in the TashT mouse model of Hirschsprung disease.

Based on the bilateral relationship between the gut microbiota and formation/function of the enteric nervous system (ENS), we sought to determine whether antibiotics-induced dysbiosis might impact the expressivity of genetically-induced ENS abnormalities. To address this, we took advantage of the TashT mouse model of Hirschsprung disease, in which colonic aganglionosis and hypoganglionosis are both much more severe in males. These defects result into two male-biased colon motility phenotypes: either megacolon that is lethal around weaning age or chronic constipation in adults, the latter being also associated with an increased proportion of nitrergic neurons in the distal ENS. Induction of dysbiosis using a cocktail of broad-spectrum antibiotics specifically impacted the colonic ENS of TashTTg/Tg mice in a stage-dependent manner. It further decreased the neuronal density at post-weaning age and differentially modulated the otherwise increased proportion of nitrergic neurons, which appeared normalized around weaning age and further increased at post-weaning age. These changes delayed the development of megacolon around weaning age but led to premature onset of severe constipation later on. Finally, local inhibition of nitric oxide signaling improved motility and prevented death by megacolon. We thus conclude that exposure to antibiotics can negatively influence the expressivity of a genetically-induced enteric neuropathy.

Molecular dissection of CHARGE syndrome highlights the vulnerability of neural crest cells to problems with alternative splicing and other transcription-related processes.

CHARGE syndrome is characterized by co-occurrence of multiple malformations due to abnormal development of neural crest cells. Here, we review the phenotypic and molecular overlap between CHARGE syndrome and similar pathologies, and further discuss the observation that neural crest cells appear especially sensitive to malfunction of the chromatin-transcription-splicing molecular hub.

Dysregulation of cotranscriptional alternative splicing underlies CHARGE syndrome.

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.

HLX is a candidate gene for a pattern of anomalies associated with congenital diaphragmatic hernia, short bowel, and asplenia.

Isolated congenital diaphragmatic hernia is often a sporadic event with a low recurrence risk. However, underlying genetic etiologies, such as chromosome anomalies or single gene disorders, are identified in a small number of individuals. We describe two fetuses with a unique pattern of multiple congenital anomalies, including diaphragmatic hernia, short bowel and asplenia, born to first-cousin parents. Whole exome sequencing showed that both were homozygous for a missense variant, c.950A>C, predicting p.Asp317Ala, in the H.20-Like Homeobox 1 (HLX1) gene. HLX is a homeobox transcription factor gene which is relatively conserved across species. Hlx homozygous null mice have a short bowel and reduced muscle cells in the diaphragm, closely resembling the anomalies in the two fetuses and we therefore suggest that the HLX mutation in this family could explain the fetal findings.

Toward a better understanding of enteric gliogenesis.

Most of gastrointestinal functions are controlled by the enteric nervous system (ENS), which contains a vast diversity of neurons and glial cells. In accordance with its key role, defective ENS formation is the cause of several diseases that affect quality of life and can even be life-threatening. Treatment of these diseases would greatly benefit from a better understanding of the molecular mechanisms underlying ENS formation. In this regard, although several important discoveries have been made over the years, how the full spectrum of enteric neuronal and glial cell subtypes is generated from neural crest cells during development still remains enigmatic. Because they also have stem cell properties, such knowledge would be especially important for the enteric glial cell lineage. In a recent study, we identified the NR2F1 transcription factor as a new key regulator of enteric gliogenesis. Here we discuss our recent findings and briefly review what is already known about the mechanisms and signaling pathways involved in enteric gliogenesis, with an emphasis on Hedgehog and Notch signaling.

Upregulation of the Nr2f1-A830082K12Rik gene pair in murine neural crest cells results in a complex phenotype reminiscent of Waardenburg syndrome type 4.

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.