Retinoid biology

Retinoids and the ENS:

For more than 60 years vitamin A metabolites (retinoids) have been known to be essential for normal development. Retinoids also have many important roles after birth including in adults. Until recently essentially nothing was known about retinoids and the ENS.
Work by other investigators provided valuable proof that retinoids influence ENS development:
1. Excess vitamin A during pregnancy delays ENS precursor migration into distal mouse bowel and causes other defects in gut development. (Pitera RA teratogenicity paper). This is an important reminder that too much vitamin A is teratogenic (i.e., it can cause birth defects).
2. Mutations in retinaldehyde dehydrogenase 2 (RALDH2), an enzyme that makes the active metabolite retinoic acid (RA) in tissues from retinaldehyde, cause severe intestinal aganglionosis. (Niederreither RALDH2 KO paper) This paper provided the first evidence that RA was likely to be needed for ENS development.
Work by the Heuckeroth lab on retinoids:
1. Studies using ENS precursor cells isolated from the fetal mouse bowel and grown in culture demonstrate many roles for retinoids in ENS development (Sato RA culture paper). Retinoids influence ENS precursor proliferation and enhance neuronal differentiation. Remarkably however, unlike every other type of neuronal precursor studied where RA increases neurite growth, developing enteric neurons treated with RA have much shorter neurites than those not exposed to RA during mid-gestation. We think this occurs because RA reduces levels of the E3 ubiquitin ligase SMURF1 in ENS precursors at this stage of development. Our cultured ENS precursors were on the journal cover for Developmental Biology.
2. RA signaling is also very important for ENS precursor migration (Fu RA migration paper). Studies in vivo employed Rbp4 mutant mice generously provided by Dr. William Blaner to show that fetal vitamin A deficiency causes Hirschsprung disease-like distal bowel aganglionosis (See Figure below). Combining Ret heterozygosity and vitamin A deficiency causes a more severe defect in bowel colonization by ENS precursors.

3. ENS precursor migration is complex since a "wave" of cells migrate in chains down the fetal bowel. These cells interact with each other and with their environment. Cells at the "wavefront" appear to migrate more vigorously than cells behind the wavefront. Our work on RA signaling suggest that this occurs because RA reduces protein levels for PTEN in the ENS precursor cells at the leading edge of the wavefront (Fu RA migration paper). Remarkably, ENS precursors just behind the wavefront have much higher levels of PTEN, a change that may reduce migration, trigger cell cycle exit and encourage differentiation.
4. RALDH1, RALDH2 and RALDH3 are all expressed in developing bowel and each enzyme influences ENS development (Wright-Jin et. al. RALDH paper).  The effect of RALDH1 or RALDH3 mutations was more subtle than the effect of RALDH2 mutation previously demonstrated by Niederreither et. al.. Even when bowel was colonized by ENS precursors, RALDH mutations led to a reduced neuron to glia ratio in the ENS and an increased ratio of nerve fibers to nerve cell bodies in the colon consistent with our results using cultured cells.  Unexpectedly, RALDH1KORALDH2HetRALDH3KO mice did not have delayed colonization of the distal bowel, in contrast to the effect of vitamin A deficiency or a complete RALDH2 mutation.  
Ongoing work: These findings raise many new questions that we are investigating.
1. We need to know more about the mechanisms through which RA alters ENS development.  Although we discovered effects of RA deprivation on PTEN and SMURF1 protein abundance, we do not know if these effects are direct.  We also know there is much more to discover.
2. To understand how RA affects ENS development in vivo, we need to determine what role RA plays within ENS precursors. This is important since our prior studies in vivo do not distinguish between RA effects on ENS precursors or surrounding cells. To understand disease mechanisms we are determining the effect of blocking RA signaling within ENS precursors using RAR dominant negative mice (Rosselot RARDN paper) generously provided by Dr. Cathy Mendelsohn.
4. We need to understand why cells at the leading edge of the "wavefront" of migrating ENS precursors respond differently to RA than cells behind the wavefront. If we could figure this out, we might be able to develop new strategies to enhance ENS precursor migration.