Retinoic acid temporally orchestrates colonization of the gut by vagal neural crest cells

Abstract

Summary The enteric nervous system arises from neural crest cells that migrate as chains into and along the primitive gut, subsequently differentiating into enteric neurons and glia. Little is known about the mechanisms governing neural crest migration en route to and along the gut in vivo. Here, we report that Retinoic Acid (RA) temporally controls zebrafish enteric neural crest cell chain migration. In vivo imaging reveals that RA loss severely compromises the integrity and migration of the chain of neural crest cells during the window of time window when they are moving along the foregut. After loss of RA, enteric progenitors accumulate in the foregut and differentiate into enteric neurons, but subsequently undergo apoptosis resulting in a striking neuronal deficit. Moreover, ectopic expression of the transcription factor meis3 and/or the receptor ret, partially rescues enteric neuron colonization after RA attenuation. Collectively, our findings suggest that retinoic acid plays a critical temporal role in promoting enteric neural crest chain migration and neuronal survival upstream of Meis3 and RET in vivo.

Tracking neural crest cell cycle progression in vivo

“…Using the Gateway cloning system via LR Clonase II Plus enzyme (Invitrogen), mAG‐zGem(1/100) was cloned upstream of mCherry‐zCdt1(1/190), separated by the viral T2A peptide, and placed downstream of the −4.9kb Sox10 promoter in a plasmid containing Tol2 elements, creating ‐4.9sox10:mAG‐gmnn(1/100)‐2A‐mCherry‐cdt1(1/190) . The following entry and destination vector plasmids were used: p5E‐sox10 (Uribe, Hong, & Bronner, ), pME‐mAG‐zGem(1/100) (Sugiyama et al, ), p3E‐T2A‐mCherry‐zCdt1(1/190)pA and pDest‐Tol2pA2 (Kwan et al, ). One‐cell stage embryos were injected with pDEST ‐ 4.9sox10:mAG‐gmnn(1/100)‐2A‐mCherry‐cdt1(1/190)pA2 to generate a stable line as previously described in Kawakami, Shima, and Kawakami ().…”

“…All embryos to be used at time points older than 24 hpf were treated with 1phenyl-2-thiourea to prevent pigmentation from interfering with imaging. Transgenic lines used and their abbreviations here are: Tg (-4.9sox10:eGFP) (Wada et al, 2005) The following entry and destination vector plasmids were used: p5E-sox10 (Uribe, Hong, & Bronner, 2018), pME-mAG-zGem(1/100) (Sugiyama et al, 2009), p3E-T2A-mCherry-zCdt1(1/190)pA and pDest-Tol2pA2 (Kwan et al, 2007). One-cell stage embryos were injected with pDEST-4.9sox10:mAG-gmnn(1/100)-2A-mCherry-cdt1(1/190)pA2 to generate a stable line as previously described in Kawakami, Shima, and Kawakami (2000).…”

Specifying neural crest cells: From chromatin to morphogens and factors in between

“…First, RA is required for the induction and patterning of mesodermal and NP tissues (Sive, Draper, Harland, & Weintraub, 1990; Villanueva, Glavic, Ruiz, & Mayor, 2002), which have been established as necessary elements to allow NC formation. RA has long been recognized as a modulator of NC migration, and when it accumulates in migratory NC, it functions as a teratogen and causes defects in craniofacial morphogenesis (Table 1) (Jimenez et al, 2016; Laue et al, 2011; Martinez-Morales et al, 2011; Uribe, Hong, & Bronner, 2017; Watanabe, Goulding, & Pratt, 1988; Watanabe & Pratt, 1991). The early role of RA in patterning regulates NC specification and development, possibly due to the importance of RA in mesoderm specification.…”

Retinoic acid receptor beta variant‐related colonic hypoganglionosis

“…Hypoganglionosis and enteric nervous system migration have been linked to Ret , Gdnf, and Hlx variants in numerous animal models (Bates, Dunagan, Welch, Kaul, & Harvey, ; Natarajan, Marcos‐Gutierrez, Pachnis, & de Graaff, ). Notably, RET mutations are the most common cause of Hirschsprung disease in humans and multiple sources have demonstrated a key regulatory link between RA signaling and activation of RET gene expression (Chatterjee et al, ; Fu et al, ; Simkin, Zhang, Rollo, & Newgreen, ; Uribe, Hong, & Bronner, ). Ret is a Glial‐derived‐neurotrophic‐factor and tyrosine kinase receptor expressed on enteric neural crest cells.…”