Loss of Tbx3 in murine neural crest reduces enteric glia and causes cleft palate, but does not influence heart development or bowel transit

Abstract

Transcription factors that coordinate migration, differentiation or proliferation of enteric nervous system (ENS) precursors are not well defined. To identify novel transcriptional regulators of ENS development, we performed microarray analysis at embryonic day (E) 17.5 and identified many genes that were enriched in the ENS compared to other bowel cells. We decided to investigate the T-box transcription factor Tbx3, which is prominently expressed in developing and mature ENS. Haploinsufficiency for TBX3 causes ulnar-mammary syndrome (UMS) in humans, a multi-organ system disorder. TBX3 also regulates several genes known to be important for ENS development. To test the hypothesis that Tbx3 is important for ENS development or function, we inactivated Tbx3 in all neural crest derivatives, including ENS progenitors using Wnt1-Cre and a floxed Tbx3 allele. Tbx3 fl/fl; Wnt1-Cre conditional mutant mice die shortly after birth with cleft palate and difficulty feeding. The ENS of mutants was well-organized with a normal density of enteric neurons and nerve fiber bundles, but small bowel glial cell density was reduced. Despite this, bowel motility appeared normal. Furthermore, although Tbx3 is expressed cardiac neural crest, Tbx3 fl/fl; Wnt1-Cre mice had structurally normal hearts. Thus, loss of Tbx3 within neural crest has selective effects on Tbx3-expressing neural crest derivatives.

Dlx1/2 mice have abnormal enteric nervous system function

“…To test if Dlx1 -/mice had poor bowel motility, which can cause slow growth, we gavage-fed 37-day-old Dlx1 -/mice with fluorescein isothiocyanate-dextran (FITC-dextran) and evaluated transit through the bowel lumen. FITC-dextran is poorly absorbed across bowel epithelium, and transit of FITC-dextran through the bowel lumen is a well-established method to assess motility (20,24). We observed no delays in SI transit ( Figure 1, J and K).…”

“…backgrounds were reported to die by 1 month of age (14,15), a time frame similar to mouse models with defined enteric neuropathies (12,16). Consistent with the hypothesis that Dlx1 and Dlx2 mutations affect ENS function or development, both genes are expressed in developing ENS at ages when ENCDCs are migrating, proliferating, and differentiating into neurons and glia, including embryonic day 12.5 (E12.5) (10,17), E14.5 (18,19), E17.5 (20), and postnatal day 0 (P0) (18). Further supporting a role in ENS development, Dlx2 enhances expression of the transcription factor Zfhx1b (also called SIP1 and Zeb2) in the CNS (21), and ZFHX1B mutations can cause Hirschsprung disease (a problem where distal bowel lacks ENS) (22,23).…”

“…RNA-seq is possible in human ENS and might also be valuable in analysis of bowel from people with CIPO (34, 35). We chose to investigate bowel motility in Dlx1 and Dlx1/2 mutants because Dlx1 and Dlx2 are expressed in developing ENS at ages relevant for ENCDC migration, differentiation, and subtype specification, and because the original description of Dlx2 -/mice suggested they die from bowel dysmotility (10,(17)(18)(19)(20). We showed that neonatal Dlx1/2 -/mice have profound bowel function defects, including delayed gastric emptying and slow SI transit via an in vivo FITC-dextran assay.…”

Enteric Glia: S100, GFAP, and Beyond

“…Equally to enteric neurons, EGCs are derived from proliferating neural crest‐derived cells that colonize the embryonic gut at E9‐E9.5 in mice (Heanue and Pachnis, ; Rothman et al, ). A reduced density of EGCs was found in the small bowel after the conditional loss of T‐box transcription factor Tbx3 in the Wnt1‐Cre mutant mice (Lopez et al, ). Cell fate decision toward a glial phenotype is driven by the notch and sonic hedgehog signaling pathways (Liu and Ngan, ; Taylor et al, ).…”

Cadherin-11 is required for neural crest determination and survival

“…In avian embryos, NC cells begin as tightly adherent neuroepithelial cells in the dorsal neural tube but detach from each other and the basal lamina to migrate using the process of EMT, which is controlled by alterations in the expression of type I and II cadherin proteins (Rogers et al, 2013;Scarpa et al, 2015;Taneyhill et al, 2007). Abnormal NC development can cause congenital defects known as neurocristopathies, which include cleft palate, craniofacial abnormalities, albinism, and defects in the enteric and peripheral nervous systems among others (Lopez et al, 2018;Reissmann and Ludwig, 2013). Bi-allelic mutations in CDH11 have specifically been linked to Elsahy-Waters syndrome, which is a combination of abnormal craniofacial developmental morphologies including those likely induced by neurocristopathies (Harms et al, 2018).…”