Dlx1/2 mice have abnormal enteric nervous system function

Wright, Christina M.; Garifallou, James; Schneider, Sabine; Mentch, Heather L; Kothakapa, Deepika R.; Maguire, Beth A.; Heuckeroth, Robert O.

doi:10.1172/jci.insight.131494

Cited by 18 publications

(15 citation statements)

References 63 publications

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“…We used ChAT-EGFP-L10A mice that reliably express EGFP at high levels in CHAT+ cells, as CHAT antibody staining is often weak. 29 Consistent with RNA-seq, SATB1, RBFOX1, and PBX3 are preferentially in Chat- EGFP+ neurons ( Figure 8 A , C–E , I–K ). TBX3 was primarily in NOS1+ neurons ( Figure 8 A , F , and L ).…”

Section: Resultssupporting

confidence: 70%

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scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3

Wright

Schneider

Smith‐Edwards

et al. 2021

Cellular and Molecular Gastroenterology and Hepatology

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113

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Background and Aims Bowel function requires coordinated activity of diverse enteric neuron subtypes. Our aim was to define gene expression in these neuron subtypes to facilitate development of novel therapeutic approaches to treat devastating enteric neuropathies, and to learn more about enteric nervous system function. Methods To identify subtype–specific genes, we performed single-nucleus RNA-seq on adult mouse and human colon myenteric plexus, and single-cell RNA-seq on E17.5 mouse ENS cells from whole bowel. We used immunohistochemistry, select mutant mice, and calcium imaging to validate and extend results. Results RNA-seq on 635 adult mouse colon myenteric neurons and 707 E17.5 neurons from whole bowel defined seven adult neuron subtypes, eight E17.5 neuron subtypes and hundreds of differentially expressed genes. Manually dissected human colon myenteric plexus yielded RNA-seq data from 48 neurons, 3798 glia, 5568 smooth muscle, 377 interstitial cells of Cajal, and 2153 macrophages. Immunohistochemistry demonstrated differential expression for BNC2, PBX3, SATB1, RBFOX1, TBX2, and TBX3 in enteric neuron subtypes. Conditional Tbx3 loss reduced NOS1-expressing myenteric neurons. Differential Gfra1 and Gfra2 expression coupled with calcium imaging revealed that GDNF and neurturin acutely and differentially regulate activity of ∼50% of myenteric neurons with distinct effects on smooth muscle contractions. Conclusion Single cell analyses defined genes differentially expressed in myenteric neuron subtypes and new roles for TBX3, GDNF and NRTN. These data facilitate molecular diagnostic studies and novel therapeutics for bowel motility disorders.

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Section: Resultssupporting

confidence: 70%

“…In these mice, EGFP marks CHAT-lineage and TDTOMATO identifies NOS1 -lineage neurons ( Figure 9 A–C ; see also Supplementary Figure 2). 29 These lineages were chosen to minimize the presence of less-differentiated precursors in the sample and were sequenced together. A total of 1005 cells were sequenced at 83% saturation.…”

Section: Resultsmentioning

confidence: 99%

scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3

Wright

Schneider

Smith‐Edwards

et al. 2021

Cellular and Molecular Gastroenterology and Hepatology

Self Cite

113

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“…For example, in mouse Phox2a mutants, early ENS development was not affected and later ENS neurogenesis was not analyzed 9 . In contrast mouse Dlx1 mutants have normal ENS neuronal density but slowed intestinal gut motility 10 .…”

Section: Discussionmentioning

confidence: 97%

A rapid F0 CRISPR screen in zebrafish to identify regulators of neuronal development in the enteric nervous system

Davidson

Straquadine

Cook

et al. 2021

Preprint

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The enteric nervous system (ENS) provides the intrinsic innervation of the gastrointestinal (GI) tract with millions of neurons and diverse neuronal subtypes and glial cells. The ENS regulates essential gut functions such as motility, nutrient uptake, and immune response, but basic information about the genes that control ENS neuronal specification and differentiation remains largely unknown. Deficits in ENS neuron numbers and composition cause gut dysfunction with debilitating GI symptoms, and are associated with e.g. Hirschsprung disease, inflammatory gut diseases, autism spectrum disorder, and neurodegenerative diseases such as Parkinson's disease. The genetic basis of most of these ENS disorders remains unknown. Recent transcriptomic analyses have identified many candidate genes for regulating ENS neurogenesis. However, functional evaluation of these candidate genes significantly lags because experimental testing of their role in ENS neurogenesis is time-consuming and expensive. Here, we have developed a rapid, scalable F0 CRISPR genome editing screen in zebrafish to functionally determine which candidate genes control neuronal development in the ENS. Proof-of-concept experiments targeting the known ENS regulators sox10 and ret phenocopy stable mutants with high efficiency and precision showing that our approach is reliable to identify regulators ENS neurogenesis using F0 guide RNA-injected larvae (F0 crispants). We then evaluate the role of 10 transcription factor genes for regulating ENS neurogenesis and function. Pools of guide RNAs targeting 2-3 candidate genes are co-injected with Cas9 protein into one-cell stage phox2bb:GFP transgenic zebrafish embryos to directly assess qualitative change in ENS neuron numbers compared to controls in 6-day old F0 crispants. Target genes from crispant pools exhibiting reduced ENS neuronal numbers were then tested individually to identify the responsible gene(s). We identify five transcription factors that show a reduction in ENS neurons indicating an influence on enteric progenitor cell differentiation into ENS neurons. Adding a simple and efficient test to further assess crispant gut motility, we find that loss-of-function of two of the transcription factor genes reduced intestinal transit of fluorescently labeled food through the gut. In summary, our novel, multistep, yet straight-forward CRISPR screening approach in zebrafish enables testing the genetic basis of ENS developmental and disease gene functions that will facilitate high-throughput evaluation of the manifold candidate genes emerging from transcriptomic, genome-wide association or other ENS-omics studies. Such in vivo ENS crispant screens will contribute to a better understanding of ENS neuronal development regulation in vertebrates and what goes awry in ENS disorders.

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“…Although the number of additional pathways or molecules revealed in recent years remains limited, some interesting advances have been made in relation to mitogen-activated protein kinase (MAPK) activity, and distal-less homeobox (Dlx)/vasoactive intestinal peptide (VIP) signaling in relation to ENS development. A recent in vitro study has shown that miR-4516, a cell migration suppressor The basic helix-loop-helix TF that is required for the development of subsets of autonomic neurons (Blaugrund et al, 1996); suppresses SOX10 (Kim et al, 2003 Wright et al, 2020).…”

Section: New Molecular Playersmentioning

confidence: 99%

“…Dysfunction of Mapk10, which is highly expressed in the mammalian ENS, also causes migration delay in zebrafish (Heanue et al, 2016). Another recent study links ENS development-related gene Dlx to the expression of the VIP neurotransmitter (Wright et al, 2020). The transcription factors Dlx1 and Dlx2 are expressed during ENS development and, although Dlx1/2 −/− mice show no defects on the topography of the ENS, they do show severe intestinal motility dysfunction at postnatal day (P) 0.…”

Section: N/amentioning

confidence: 99%

Gut innervation and enteric nervous system development: a spatial, temporal and molecular tour de force

2021

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During embryonic development, the gut is innervated by intrinsic (enteric) and extrinsic nerves. Focusing on mammalian ENS development, in this Review we highlight how important the different compartments of this innervation are to assure proper gut function. We specifically address the three-dimensional architecture of the innervation, paying special attention to the differences in development along the longitudinal and circumferential axes of the gut. We review recent information about the formation of both intrinsic innervation, which is fairly well-known, as well as the establishment of the extrinsic innervation, which, despite its importance in gut-brain signaling, has received much less attention. We further discuss how external microbial and nutritional cues or neuroimmune interactions may influence development of gut innervation. Finally, we provide summary tables, describing the location and function of several well-known molecules, along with some newer factors that have more recently been implicated in the development of gut innervation.

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Dlx1/2 mice have abnormal enteric nervous system function

Cited by 18 publications

References 63 publications

scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3

scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3

A rapid F0 CRISPR screen in zebrafish to identify regulators of neuronal development in the enteric nervous system

Gut innervation and enteric nervous system development: a spatial, temporal and molecular tour de force

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