This analysis of Hh signal response in the developing and mature GI tract provides a cellular basis for Hh function in this tissue and suggests new avenues for exploration. Novel findings include: a) shortly after gut tube formation, epithelial cells of the hindgut and tailgut express Ptch1, but not Gli1; b) serosal cells respond to Hh signals during fetal life; c) the developing MM contains Hh responsive cells at P10 and continues to receive Hh signals during adult life; d) Hh levels control the amount of smooth muscle in villus cores; e) enteric neurons are not responsive to Hh signals in fetal life; they express Ptch1, but not Gli1 in neonatal and adult life; f) Hh responding cells are concentrated at the pyloric border during formation of that sphincter and are prominent in the antrum, and much less so in the intestine after E16.5; g) epithelial cells of the antrum, small intestine and colon do not express Gli1 and therefore do not respond to Hh signals at any point during embryonic, fetal or adult life. This last finding is in conflict with previous reports that propose autocrine epithelial hedgehog signaling25
(discussed in Supplementary Information 6
). Here, we further enlarge upon the potential implications of these data in the context of gastrointestinal development and disease.
It is interesting that transient expression of Ptch1, but not Gli1 is detectable in E10.5 hindgut and tailgut epithelium as well as in postnatal enteric neurons. Ptch1 expression in the absence of Gli1 has also been seen in neural tissue33
. Although Ptch1 is normally a Hh target gene, it can also act as a dependence receptor, to promote apoptosis in the absence of a Hh signal. It has been proposed that such apoptotic activity may help to shape the neural tube during development34
. Perhaps transient expression of Ptch1 in these hindgut cells that clearly are not transducing Hh signals (since they are Gli1 negative) plays a role in preventing the apoptosis of these cells.
Hh signal transduction has not previously been demonstrated in the MM, but our data indicate that the cells of the forming MM are Hh responsive. Morphological studies indicate that the MM arises from the inner circular layer of ME39
. Indeed, we detected apparent connections between the MM and cells of the inner circular layer of ME () and the SM cells of the villus core. It will be important to lineage trace this developing SM network to confirm these apparent origins. Our transgenic studies further reveal that villus core SM is highly sensitive to the level of Hh ligand. Though MM did not appear amplified, this might be due to the fact that the strength of the villin promoter is greater in villus tips than in crypts31
. Alternatively, MM cells might be under separate control, even though these cells are also responsive to Hh signals as measured by their expression of Gli1.
Interestingly, from an early time point, the signaling properties of the inner circular and outer longitudinal muscles of the ME are different: only the former is Gli1 positive, indicative of active Hh signaling. Such different properties could potentially play a role in the differential response of these two muscles in disease. For example, familial type IV visceral myopathy presents with hypertophy of the inner circular and atrophy of the outer longitudinal muscle of the small bowel and is a rare cause of chronic intestinal pseudoobstruction40
. Given the ability of Hh signaling to modulate villus core smooth muscle as shown above and in our earlier studies17
, it will be important to examine whether increased Hedgehog signaling plays any role in the etiology of this rare but usually fatal pathological condition.
The finding that the serosal mesothelium responds to Hh signals throughout development suggests that a source of Hh ligand may lie in the peritoneal cavity and/or that these cells receive Hh signals as they migrate onto the surface of the gut tube at E11.542
. Recent studies indicate that mesothelial cells undergo epithelial to mesenchymal transition and migrate into the gut tube, differentiating into endothelial cells, vascular SM cells and pericytes42
. Whether the vasculogenic activity of gut serosal mesothelium requires Hh signaling has not been directly tested, but it is noteworthy that Smo null embryos, which lack Hh response, have major vascular defects43
and administration of Shh blocking antibodies or a chemical inhibitor of Hh signaling produces vascular malformations and impaired vascular remodeling44
. Confirmation of the connection between Hedgehog signaling and the serosal mesothelium of the gut that is suggested here could potentially have therapeutic implications in the context of a rare developmental anomaly called “apple peel bowel”, a syndrome of intestinal wasting that is associated with loss of the serosa and its associated blood vessels46
Our data also suggest a possible indirect role for Hh in the establishment of intestinal vs. stomach epithelial identity. Exactly at E16.5, the precise time when a clear-cut epithelial boundary is being generated between stomach and intestine32
, a dramatic difference in Hh signaling is generated in the mesenchyme: Hh signal transduction is robust in the antrum and much less prominent in small intestine. This difference is not visible at E14.5. This finding fits well with previous functional studies in Xenopus
, where Hh signaling is also downregulated during differentiation of the intestine48
. Indeed, constitutive activation of Hh signaling in the midgut results in arrested cytodifferentiation and poor growth of that tissue49
. Thus, there may be an evolutionarily conserved requirement for downregulation of Hh signaling to permit intestinal cell differentiation.
We show here that in contrast to earlier conclusions50
, Hh signal transduction is quite active in the antrum. Though its function in this adult tissue is not clear, a role for Hh in patterning the perinatal antral epithelium was previously suggested by the antral overgrowth phenotype seen in Gli3 and Shh null mice14
. Since signal transduction in the antrum is exclusively paracrine, this effect must represent an altered relay from epithelium to mesenchyme and back to epithelium. In this context, it is of interest to consider a common developmental abnormality of the pyloric stomach known as hypertrophic pyloric stenosis (HPS). In a study of over 100 infants with HPS, Hernanz-Schulman et al. found that robust overgrowth of the antral mucosa was responsible for obstruction of the pyloric opening51
. Interestingly, a survey of the literature shows that HPS is often associated with other anomalies51
, including: abdominal malrotation or volvulus53
, cardiac anomalies55
, imperforate anus, tracheal esophageal fistula56
and hydronephrosis of the kidney52
. Each of these malformations is seen in the VACTERL association, a constellation of abnormalities involving vertebral, anal, tracheal, esophageal, renal and limb development that are thought to be linked to defects in Hh signaling23
. Since both Shh null mice and humans with HPS exhibit antral epithelial overgrowth, HPS might represent yet another associated abnormality within the VACTERL spectrum.