The enteric neural crest progressively loses capacity to form enteric nervous system

Cells of the vagal neural crest (NC) form most of the enteric nervous system (ENS) by a colonising wave in the embryonic gut, with high cell proliferation and differentiation. Enteric neuropathies have an ENS deficit and cell replacement has been suggested as therapy. This would be performed post-natally, which raises the question of whether the ENS cell population retains its initial ENS-forming potential with age. We tested this on the avian model in organ culture in vitro (3 days) using recipient aneural chick midgut/hindgut combined with ENS- donor quail midgut or hindgut of ages QE5 to QE10. ENS cells from young donor tissues (<= QE6) avidly colonised the aneural recipient, but this capacity dropped rapidly 2-3 days after the transit of the ENS cell wavefront. This loss in capability was autonomous to the ENS population since a similar decline was observed in ENS cells isolated by HNK1 FACS. Using QE5, 6, 8 and 10 midgut donors and extending the time of assay to 8 days in chorio-allantoic membrane grafts did not produce 'catch up' colonisation. NC-derived cells were counted in dissociated quail embryo gut and in transverse sections of chick embryo gut using NC, neuron and glial marker antibodies. This showed that the decline in ENS-forming ability correlated with a decrease in proportion of ENS cells lacking both neuronal and glial differentiation markers, but there were still large numbers of such cells even at stages with low colonisation ability. Moreover, ENS cells in small numbers from young donors were far superior in colonisation ability to larger numbers of apparently undifferentiated cells from older donors. This suggests that the decline of ENS-forming ability has both quantitative and qualitative aspects. In this case, ENS cells for cell therapies should aim to replicate the embryonic ENS stage rather than using post-natal ENS stem/progenitor cells

Enteric Neural Cells From Hirschsprung Disease Patients Form Ganglia in Autologous Aneuronal Colon

Background & Aims: Hirschsprung disease (HSCR) is caused by failure of cells derived from the neural crest (NC) to colonize the distal bowel in early embryogenesis, resulting in absence of the enteric nervous system (ENS) and failure of intestinal transit postnatally. Treatment is by distal bowel resection, but neural cell replacement may be an alternative. We tested whether aneuronal (aganglionic) colon tissue from patients may be colonized by autologous ENS-derived cells. Methods: Cells were obtained and cryopreserved from 31 HSCR patients from the proximal resection margin of colon, and ENS cells were isolated using flow cytometry for the NC marker p75 (nine patients). Aneuronal colon tissue was obtained from the distal resection margin (23 patients). ENS cells were assessed for NC markers immunohistologically and by quantitative reverse-transcription polymerase chain reaction, and mitosis was detected by ethynyl-2\u27-deoxyuridine labeling. The ability of human HSCR postnatal ENS-derived cells to colonize the embryonic intestine was demonstrated by organ coculture with avian embryo gut, and the ability of human postnatal HSCR aneuronal colon muscle to support ENS formation was tested by organ coculture with embryonic mouse ENS cells. Finally, the ability of HSCR patient ENS cells to colonize autologous aneuronal colon muscle tissue was assessed. Results: ENS-derived p75-sorted cells from patients expressed multiple NC progenitor and differentiation markers and proliferated in culture under conditions simulating Wnt signaling. In organ culture, patient ENS cells migrated appropriately in aneural quail embryo gut, and mouse embryo ENS cells rapidly spread, differentiated, and extended axons in patient aneuronal colon muscle tissue. Postnatal ENS cells derived from HSCR patients colonized autologous aneuronal colon tissue in cocultures, proliferating and differentiating as neurons and glia. Conclusions: NC-lineage cells can be obtained from HSCR patient colon and can form ENS-like structures in aneuronal colonic muscle from the same patient

Loss of gastrokine-2 drives premalignant gastric inflammation and tumor progression

Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, therefore, requires tight control by suppressive counter mechanisms. Gastrokine-2 (GKN2) belongs to a family of secreted proteins expressed within normal gastric mucosal cells. GKN2 expression is frequently lost during GC progression, suggesting an inhibitory role; however, a causal link remains unsubstantiated. Here, we developed Gkn2 knockout and transgenic overexpressing mice to investigate the functional impact of GKN2 loss in GC pathogenesis. In mouse models of GC, decreased GKN2 expression correlated with gastric pathology that paralleled human GC progression. At baseline, Gkn2 knockout mice exhibited defective gastric epithelial differentiation but not malignant progression. Conversely, Gkn2 knockout in the IL-11/STAT3-dependent gp130[superscript F/F] GC model caused tumorigenesis of the proximal stomach. Additionally, gastric immunopathology was accelerated in Helicobacter pylori–infected Gkn2 knockout mice and was associated with augmented T helper cell type 1 (Th1) but not Th17 immunity. Heightened Th1 responses in Gkn2 knockout mice were linked to deregulated mucosal innate immunity and impaired myeloid-derived suppressor cell activation. Finally, transgenic overexpression of human gastrokines (GKNs) attenuated gastric tumor growth in gp130[superscript F/F] mice. Together, these results reveal an antiinflammatory role for GKN2, provide in vivo evidence that links GKN2 loss to GC pathogenesis, and suggest GKN restoration as a strategy to restrain GC progression

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Retinoic acid upregulates ret and induces chain migration and population expansion in vagal neural crest cells to colonise the embryonic gut.

Vagal neural crest cells (VNCCs) arise in the hindbrain, and at (avian) embryonic day (E) 1.5 commence migration through paraxial tissues to reach the foregut as chains of cells 1-2 days later. They then colonise the rest of the gut in a rostrocaudal wave. The chains of migrating cells later resolve into the ganglia of the enteric nervous system. In organ culture, E4.5 VNCCs resident in the gut (termed enteric or ENCC) which have previously encountered vagal paraxial tissues, rapidly colonised aneural gut tissue in large numbers as chains of cells. Within the same timeframe, E1.5 VNCCs not previously exposed to paraxial tissues provided very few cells that entered the gut mesenchyme, and these never formed chains, despite their ability to migrate in paraxial tissue and in conventional cell culture. Exposing VNCCs in vitro to paraxial tissue normally encountered en route to the foregut conferred enteric migratory ability. VNCC after passage through paraxial tissue developed elements of retinoic acid signalling such as Retinoic Acid Binding Protein 1 expression. The paraxial tissue's ability to promote gut colonisation was reproduced by the addition of retinoic acid, or the synthetic retinoid Am80, to VNCCs (but not to trunk NCCs) in organ culture. The retinoic acid receptor antagonist CD 2665 strongly reduced enteric colonisation by E1.5 VNCC and E4.5 ENCCs, at a concentration suggesting RARα signalling. By FACS analysis, retinoic acid application to vagal neural tube and NCCs in vitro upregulated Ret; a Glial-derived-neurotrophic-factor receptor expressed by ENCCs which is necessary for normal enteric colonisation. This shows that early VNCC, although migratory, are incapable of migrating in appropriate chains in gut mesenchyme, but can be primed for this by retinoic acid. This is the first instance of the characteristic form of NCC migration, chain migration, being attributed to the application of a morphogen

Why are enteric ganglia so small? Role of differential adhesion of enteric neurons and enteric neural crest cells. [v1; ref status: indexed, http://f1000r.es/59q]

The avian enteric nervous system (ENS) consists of a vast number of unusually small ganglia compared to other peripheral ganglia. Each ENS ganglion at mid-gestation has a core of neurons and a shell of mesenchymal precursor/glia-like enteric neural crest (ENC) cells. To study ENS cell ganglionation we isolated midgut ENS cells by HNK-1 fluorescence-activated cell sorting (FACS) from E5 and E8 quail embryos, and from E9 chick embryos. We performed cell-cell aggregation assays which revealed a developmentally regulated functional increase in ENS cell adhesive function, requiring both Ca2+ -dependent and independent adhesion. This was consistent with N-cadherin and NCAM labelling. Neurons sorted to the core of aggregates, surrounded by outer ENC cells, showing that neurons had higher adhesion than ENC cells. The outer surface of aggregates became relatively non-adhesive, correlating with low levels of NCAM and N-cadherin on this surface of the outer non-neuronal ENC cells. Aggregation assays showed that ENS cells FACS selected for NCAM-high and enriched for enteric neurons formed larger and more coherent aggregates than unsorted ENS cells. In contrast, ENS cells of the NCAM-low FACS fraction formed small, disorganised aggregates.  This suggests a novel mechanism for control of ENS ganglion morphogenesis where i) differential adhesion of ENS neurons and ENC cells controls the core/shell ganglionic structure and ii) the ratio of neurons to ENC cells dictates the equilibrium ganglion size by generation of an outer non-adhesive surface

Vagal paraxial tissues improve colonisation of aneural gut by E1.5 VNCCs.

<p>Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064077#pone-0064077-g003" target="_blank">Figure 3</a>, plus vPA = E1.5 vagal neural and paraxial tissues.</p

E1.5 VNCC donors are poor at colonising aneural gut in catenary cultures, relative to E4.5 ENCC donors.

<p>Colonisation performance (cell number and chain formation) of QCPN+ cells was quantified on a five point scale (0 = nil to 4 = dense chains and aggregates, in 5 gut regions (MG1, MG2, Ceca, HG1, HG2). Averages are shown. The following shading system was applied: 0.0, white; 0.1–1.4, light gray; 1.5–2.4, medium gray; 2.5–3.4, dark gray; 3.5–4.0, black. * denotes cells in chains or aggregates. E = quail embryonic day, vNT = vagal neural tube, MG = midgut, wg = with gut.</p

RA signalling agonist and antagonist alter the percentages of BrdU positive and caspase-3 positive VNCCs in neural tube/NC cultures.

<p>Percent (±s.e.m.) of positive labelling is listed on the Y-axis, and culture and labeling conditions are listed on the X-axis. Number of explants in: TCM N = 7, 10 µM retinoic acid (RA) N = 21, 1 µM RAR inhibitor CD 2665 (RARi) N = 9. s = significant differences (p≤0.05); ns = not significant difference.</p

VNCCs form chains on arrival in foregut mesenchyme.

<p><b>A.</b> Sagittally sliced E2.5 quail embryo viewed from the medial aspect, with rostral to the right. Oto = otocyst; I = branchial arch I (mandibular division); II = branchial arch II; etc. (Light field microscopy) <b>B.</b> Pharyngeal endoderm of same specimen revealed by E-cadherin antibody labelling. The endoderm of the narrowed foregut caudal to the pharynx is not present in this half-specimen. Axons detected by E/C8 antibody converge ventrally from vagal somite levels (somite 1, 2 etc.), lateral and ventral to the foregut, in the boxed area. (Confocal microscopy) <b>C.</b> Boxed region in B showing chains of SoxE+ve nuclei of ENCC in the mesenchyme of the foregut. In the central part of this image, ENCC and axons are not associated; axons are more laterally placed and excluded from this confocal optical section. Ventrally (this image) and more laterally, NCC and axons are associated.</p