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LRIG1 Regulates Ontogeny of Smooth Muscle−Derived Subsets of Interstitial Cells of Cajal in Mice

      Background & Aims

      Interstitial cells of Cajal (ICC) control intestinal smooth muscle contraction to regulate gut motility. ICC within the plane of the myenteric plexus (ICC-MY) arise from KIT-positive progenitor cells during mouse embryogenesis. However, little is known about the ontogeny of ICC associated with the deep muscular plexus (ICC-DMP) in the small intestine and ICC associated with the submucosal plexus (ICC-SMP) in the colon. Leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) marks intestinal epithelial stem cells, but the role of LRIG1 in nonepithelial intestinal cells has not been identified. We sought to determine the ontogeny of ICC-DMP and ICC-SMP, and whether LRIG1 has a role in their development.

      Methods

      Lrig1-null mice (homozygous Lrig1-CreERT2) and wild-type mice were analyzed by immunofluorescence and transit assays. Transit was evaluated by passage of orally administered rhodamine B−conjugated dextran. Lrig1-CreERT2 mice or mice with CreERT2 under control of an inducible smooth muscle promoter (Myh11-CreERT2) were crossed with Rosa26-LSL-YFP mice for lineage tracing analysis.

      Results

      In immunofluorescence assays, ICC-DMP and ICC-SMP were found to express LRIG1. Based on lineage tracing, ICC-DMP and ICC-SMP each arose from LRIG1-positive smooth muscle progenitors. In Lrig1-null mice, there was loss of staining for KIT in DMP and SMP regions, as well as for 2 additional ICC markers (anoctamin-1 and neurokinin 1 receptor). Lrig1-null mice had significant delays in small intestinal transit compared with control mice.

      Conclusions

      LRIG1 regulates the postnatal development of ICC-DMP and ICC-SMP from smooth muscle progenitors in mice. Slowed small intestinal transit observed in Lrig1-null mice may be due, at least in part, to loss of the ICC-DMP population.

      Keywords

      Abbreviations used in this paper:

      ANO1 (anoctamin-1), DAPI (4',6-diamidino-2-phenylindole), DMP (deep muscular plexus), ICC (interstitial cells of Cajal), LRIG1 (leucine-rich repeats and immunoglobulin-like domains protein 1), MY (myenteric region), NK1R (neurokinin 1 receptor), PBS (phosphate-buffered saline), PDGFRA (platelet-derived growth factor receptor α), RFP (red fluorescent protein), SMA (smooth muscle actin), SMP (submucosal plexus), WT (wild-type), YFP (yellow fluorescent protein)
      See Covering the Cover synopsis on page 264; see editorial on page 283.
      Interstitial cells of Cajal (ICC) form networks within the musculature of the gastrointestinal tract and serve as pacemakers and transducers of neural inputs that control contraction of smooth muscle to regulate gut motility.
      • Torihashi S.
      • Ward S.M.
      • Nishikawa S.-I.
      • et al.
      c-kit-Dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract.
      • Thomson L.
      • Robinson T.L.
      • Lee J.C.F.
      • et al.
      Interstitial cells of Cajal generate a rhythmic pacemaker current.
      • Iino S.
      • Ward S.M.
      • Sanders K.M.
      Interstitial cells of Cajal are functionally innervated by excitatory motor neurones in the murine intestine.
      • Klein S.
      • Seidler B.
      • Kettenberger A.
      • et al.
      Interstitial cells of Cajal integrate excitatory and inhibitory neurotransmission with intestinal slow-wave activity.
      ICC dysfunction can contribute to the pathogenesis of a wide spectrum of intestinal disorders, including achalasia,
      • Gockel I.
      • Bohl J.R.
      • Eckardt V.F.
      • et al.
      Reduction of interstitial cells of Cajal (ICC) associated with neuronal nitric oxide synthase (n-NOS) in patients with achalasia.
      diabetic gastroparesis
      • Horváth V.J.
      • Vittal H.
      • Lörincz A.
      • et al.
      Reduced stem cell factor links smooth myopathy and loss of interstitial cells of Cajal in murine diabetic gastroparesis.
      and enteropathy,
      • Forrest A.
      • Huizinga J.D.
      • Wang X.-Y.
      • et al.
      Increase in stretch-induced rhythmic motor activity in the diabetic rat colon is associated with loss of ICC of the submuscular plexus.
      Crohn’s disease,
      • Porcher C.
      • Baldo M.
      • Henry M.
      • et al.
      Deficiency of interstitial cells of Cajal in the small intestine of patients with Crohn’s disease.
      and slow-transit constipation.
      • He C.-L.
      • Burgart L.
      • Wang L.
      • et al.
      Decreased interstitial cell of Cajal volume in patients with slow-transit constipation.
      • Kashyap P.
      • Gomez-Pinilla P.J.
      • Pozo M.J.
      • et al.
      Immunoreactivity for Ano1 detects depletion of Kit-positive interstitial cells of Cajal in patients with slow transit constipation.
      ICC are divided into several subpopulations according to their location. Myenteric ICC (ICC-MY) are found in the myenteric plexus of phasic muscle throughout the gut, and ICC associated with the deep muscular plexus (ICC-DMP) are located within the mucosal side of the circular muscle of the small intestine and ICC associated with the colonic submucosal plexus (ICC-SMP) are luminal to the circular muscle layer.
      • Iino S.
      • Horiguchi K.
      Interstitial cells of Cajal are involved in neurotransmission in the gastrointestinal tract.
      • Al-Sajee D.
      • Huizinga J.D.
      Interstitial cells of Cajal.
      Gastric, small intestinal, and colonic ICC-MY and colonic ICC-SMP are electrical pacemaker cells.
      • Torihashi S.
      • Ward S.M.
      • Nishikawa S.-I.
      • et al.
      c-kit-Dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract.
      • Ward S.M.
      • Burns A.J.
      • Torihashi S.
      • et al.
      Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.
      • Huizinga J.D.
      • Thuneberg L.
      • Klüppel M.
      • et al.
      W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.
      Small intestinal ICC-DMP are not thought to act as pacemakers,
      • Ward S.M.
      • Burns A.J.
      • Torihashi S.
      • et al.
      Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.
      but rather to mediate excitatory and inhibitory motor neural inputs.
      • Iino S.
      • Ward S.M.
      • Sanders K.M.
      Interstitial cells of Cajal are functionally innervated by excitatory motor neurones in the murine intestine.
      • Wang X.-Y.
      • Vannucchi M.-G.
      • Nieuwmeyer F.
      • et al.
      Changes in interstitial cells of Cajal at the deep muscular plexus are associated with loss of distention-induced burst-type muscle activity in mice infected by Trichinella spiralis.
      • Ward S.M.
      • McLaren G.J.
      • Sanders K.M.
      Interstitial cells of Cajal in the deep muscular plexus mediate enteric motor neurotransmission in the mouse small intestine.
      The murine colon also contains intramuscular and subserosal ICC populations (ICC-IM and ICC-SS, respectively).
      • Al-Sajee D.
      • Huizinga J.D.
      Interstitial cells of Cajal.
      KIT, a receptor tyrosine kinase that serves as the receptor for stem cell factor, is important for the proper development and function of ICCs.
      • Ward S.M.
      • Burns A.J.
      • Torihashi S.
      • et al.
      Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.
      • Huizinga J.D.
      • Thuneberg L.
      • Klüppel M.
      • et al.
      W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.
      • Torihashi S.
      • Ward S.M.
      • Sanders K.M.
      Development of c-Kit-positive cells and the onset of electrical rhythmicity in murine small intestine.
      Blockade of KIT activity with a neutralizing antibody in newborn mice results in loss of all ICC subsets, including ICC-DMP and ICC-SMP.
      • Torihashi S.
      • Ward S.M.
      • Nishikawa S.-I.
      • et al.
      c-kit-Dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract.
      • Torihashi S.
      • Nishi K.
      • Tokutomi Y.
      • et al.
      Blockade of kit signaling induces transdifferentiation of interstitial cells of Cajal to a smooth muscle phenotype.
      In contrast, ICC-DMP and ICC-SMP are present in Kit mutant mice with decreased KIT activity, such as W/Wv or Wsh/Wsh mice, and ICC-MY are grossly underdeveloped in the small intestine of these mice.
      • Ward S.M.
      • Burns A.J.
      • Torihashi S.
      • et al.
      Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.
      • Huizinga J.D.
      • Thuneberg L.
      • Klüppel M.
      • et al.
      W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.
      • Iino S.
      • Horiguchi K.
      • Nojyo Y.
      Wsh/Wsh c-Kit mutant mice possess interstitial cells of Cajal in the deep muscular plexus layer of the small intestine.
      These findings suggest ICC-DMP and ICC-MY in the small intestine may be differentially regulated and differentially dependent on KIT activity. Indeed, ICC-MY and ICC-IM development is regulated by the ETS family transcription factor, ETV1, but ICC-DMP and ICC-SMP development is not.
      • Chi P.
      • Chen Y.
      • Zhang L.
      • et al.
      ETV1 is a lineage-specific survival factor in GIST and cooperates with KIT in oncogenesis.
      However, factor(s) that selectively regulate the development and maintenance of ICC-DMP and ICC-SMP are unknown.
      In the mouse small intestine, both ICC-MY and intestinal smooth muscle cells emerge from common KIT-positive progenitors during mouse embryogenesis (E12.5 to E18).
      • Torihashi S.
      • Ward S.M.
      • Sanders K.M.
      Development of c-Kit-positive cells and the onset of electrical rhythmicity in murine small intestine.
      • Klüppel M.
      • Huizinga J.D.
      • Malysz J.
      • et al.
      Developmental origin and kit-dependent development of the interstitial cells of cajal in the mammalian small intestine.
      However, the origin of KIT-expressing ICC-DMP and ICC-SMP is uncertain; the former is present sparsely at birth in the mouse jejunum,
      • Ward S.M.
      • McLaren G.J.
      • Sanders K.M.
      Interstitial cells of Cajal in the deep muscular plexus mediate enteric motor neurotransmission in the mouse small intestine.
      • Torihashi S.
      • Ward S.M.
      • Sanders K.M.
      Development of c-Kit-positive cells and the onset of electrical rhythmicity in murine small intestine.
      and the latter does not appear until postnatal day 5 in the proximal colon.
      • Han J.
      • Shen W.-H.
      • Jiang Y.-Z.
      • et al.
      Distribution, development and proliferation of interstitial cells of Cajal in murine colon: an immunohistochemical study from neonatal to adult life.
      Both populations expand in number after birth to form functional cellular networks.
      • Ward S.M.
      • McLaren G.J.
      • Sanders K.M.
      Interstitial cells of Cajal in the deep muscular plexus mediate enteric motor neurotransmission in the mouse small intestine.
      • Han J.
      • Shen W.-H.
      • Jiang Y.-Z.
      • et al.
      Distribution, development and proliferation of interstitial cells of Cajal in murine colon: an immunohistochemical study from neonatal to adult life.
      Based on ultrastructual observations, it has been proposed that ICC-DMP emerges from undifferentiated cells, termed ICC blasts, that populate the DMP region;
      • Torihashi S.
      • Ward S.M.
      • Nishikawa S.-I.
      • et al.
      c-kit-Dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract.
      • Torihashi S.
      • Nishi K.
      • Tokutomi Y.
      • et al.
      Blockade of kit signaling induces transdifferentiation of interstitial cells of Cajal to a smooth muscle phenotype.
      • Pellegrini M.S.F.
      Morphogenesis of the special circular muscle layer and of the interstitial cells of Cajal related to the plexus muscularis profundus of mouse intestinal muscle coat.
      however, the origin of ICC blasts is unknown.
      Recently, we identified that leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1), a pan-ErbB−negative regulator,
      • Gur G.
      • Rubin C.
      • Katz M.
      • et al.
      LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation.
      • Laederich M.B.
      • Funes-Duran M.
      • Yen L.
      • et al.
      The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases.
      marks intestinal epithelial stem cells.
      • Powell A.E.
      • Wang Y.
      • Li Y.
      • et al.
      The Pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor.
      LRIG1 is also expressed in epidermal
      • Jensen K.B.
      • Collins C.A.
      • Nascimento E.
      • et al.
      Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis.
      and corneal stem cells.
      • Nakamura T.
      • Hamuro J.
      • Takaishi M.
      • et al.
      LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis.
      These tissues, which are maintained by LRIG1-expressing stem cells, turn over continuously. However, the distribution and role of LRIG1 in tissues where cells turnover or proliferate less frequently, such as muscle, are unclear.
      Here, we provide evidence that ICC-DMP and ICC-SMP arise postnatally from intestinal smooth muscle cells in murine small intestine and colon, respectively. We also show that ICC-DMP and ICC-SMP express LRIG1 and that LRIG1 regulates the differentiation of smooth muscle cells into ICC-DMP and ICC-SMP. In the absence of LRIG1, ICC-DMP and ICC-SMP are largely lost. In addition, Lrig1-null mice exhibit markedly delayed small intestinal motility. Taken together, these studies identify the ontogeny of ICC-DMP and ICC-SMP and assign a role for LRIG1 in their development.

      Materials and Methods

       Mice

      The generation of Lrig1tm1.(cre/ERT)Rjc (Lrig1-CreERT2/+) mice has been reported previously.
      • Powell A.E.
      • Wang Y.
      • Li Y.
      • et al.
      The Pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor.
      Lrig1-Apple/+ reporter mice, in which exon 1 of the Lrig1 gene was replaced by the apple fluorescent protein coding sequence, was generated in a strategy similar to Lrig1-CreERT2/+ mice.
      • Poulin E.J.
      • Powell A.E.
      • Wang Y.
      • et al.
      Using a new Lrig1 reporter mouse to assess differences between two Lrig1 antibodies in the intestine.
      Myh11-CreERT2 mice
      • Wirth A.
      • Benyó Z.
      • Lukasova M.
      • et al.
      G12-G13−LARG−mediated signaling in vascular smooth muscle is required for salt-induced hypertension.
      and Rosa26 (R26)-YFP mice
      • Srinivas S.
      • Watanabe T.
      • Lin C.-S.
      • et al.
      Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.
      were obtained from The Jackson Laboratory (Bar Harbor, ME). For developmental lineage tracing, Lrig1-CreERT2/+;R26-YFP/+ mice or Myh11-CreERT2/+;R26-YFP/+ mice were given a single intraperitoneal injection of tamoxifen (33 mg/kg; Sigma, St Louis, MO) at postnatal day 1 and analyzed at the time points indicated. Eight-week-old adult mice were used unless otherwise stated in figures and/or figure legends. All mouse experiments were approved by Institutional Animal Care and Use Committee at Vanderbilt University Medical Center.

       Human Samples

      Three freshly resected normal human duodenal specimens were obtained from the Cooperative Human Tissue Network (Vanderbilt University Medical Center). Deidentified tissues were collected with Institutional Review Board approval. The tissues supplied are not resected specifically for research, but are surgical waste tissues, which are left after the pathologist had taken tissues for diagnosis. Tissues were handled according to institutional ethical guidelines.

       Tissue Processing and Immunofluorescence

      For frozen sections, intestinal tissues were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde at 4°C, followed by consecutive 15% and 30% sucrose immersion before freezing in Optimal Cutting Temperature compound (Sakura Finetek, Torrance, CA). Cryosections were mounted onto glass slides and incubated at room temperature for 30 minutes in PBS containing 0.1% Triton X-100 and 2.5% normal donkey serum to reduce nonspecific immunostaining. Alternatively, for human samples, Protein Block Serum-Free (DAKO, Carpinteria, CA) was used to reduce nonspecific immunostaining. The sections were incubated with primary antibody at room temperature for 1 hour. Sections were then washed 3 times in PBS, and incubated with secondary antibodies for 30 minutes at room temperature. Sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI) for nuclear detection. Primary and secondary antibodies used are listed in Supplementary Table 1 (antibody list). Micrographic images were obtained using an Olympus IX-71 (Olympus, Center Valley, PA) for mouse tissue, and Axio Imager.M2 (Zeiss, Thornwood, NY) for human tissue. Images presented are representatives of 3 mouse or human samples.

       Whole-Mount Staining

      Whole intestinal tissues were fixed with ice-cold acetone and kept at −80°C less than 1 week until rehydration. Alternatively, for lineage-tracing experiments, tissues were fixed with 4% paraformaldehyde on ice for 15 minutes to preserve yellow fluorescent protein (YFP) immunoreactivity. After washing in PBS, the muscular layer was detached from the mucosa. After 1 hour of blocking in 2.5% donkey serum in PBS containing 0.1% Triton X-100, samples were incubated with primary antibodies at 4°C overnight. Samples were then washed with PBS and labeled with secondary antibodies at room temperature for 2 hours. Tissues were counterstained with DAPI and mounted on glass slides. Confocal images were obtained using an Olympus FluoView-1000. Images presented are representatives of 3 mouse samples.

       Quantification of Interstitial Cells of Cajal and Leucine-Rich Repeats and Immunoglobulin-Like Domains Protein 1−Expressing Cells

      The jejunum and colon of 8-week-old wild-type (WT) mice were used for quantification of adult mice. For quantification of lineage-traced cells, Lrig1-CreERT2/+;R26-YFP or Myh11-CreERT2;R26-YFP mice were given a single tamoxifen injection, as described here. Mice then were sacrificed 3 weeks after tamoxifen injection, and jejunum, ileum, and colon were used for quantification. The small intestine was divided into 3 equal-length segments rostral caudally, and the most proximal portion of the second segment was designated jejunum, and the most distal portion of the third segment was designated ileum. The colon was divided into 3 equal-length segments rostral-caudal to obtain proximal, middle, and distal segments. For image quantification, confocal z-stack images were obtained with no more than a 2-μm step between slices. Three mice were used for each genotype, and images were taken from 3 random fields per whole-mount preparation (40× magnification [0.1 mm2] for adult mice and 3D, and 60× magnification [0.045 mm2] for lineage-tracing expeiments. The number of positive cells with each label was counted with DAPI nuclear stain using ImageJ software (Cell Counter plug-in; National Institutes of Health, Bethesda, MD).

       Gastric Emptying and Intestinal Transit Assay

      Gastric emptying and small intestinal transit were measured by evaluating the intestinal location of rhodamine B−conjugated dextran (D1841; Molecular Probes Eugene, OR) as described elsewhere,
      • Miller M.S.
      • Galligan J.J.
      • Burks T.F.
      Accurate measurement of intestinal transit in the rat.
      with modification. Six-week-old mice were given 2.5 mg/mL rhodamine B−conjugated dextran in 5% methylcellulose (M7027; Sigma) via gavage. Ten minutes after administration for the gastric emptying assay and 60 minutes after administration for the small intestinal transit assay, the entire small bowel (unflushed) was divided into 10 equal segments and placed into tubes with 4 mL PBS. The stomach was prepared similarly. Tissues were homogenized and centrifuged at 2000 rpm for 10 minutes to separate out a pellet and supernatant. The fluorescence in each aliquot of the cleared supernatant was read by a Synergy4 plate reader (excitation 540 nm/emission 625 nm; BioTek, Winsooki, VT). Gastric emptying was calculated as the ratio of the total fluorescent intensity in small intestine divided by total fluorescent intensity in stomach and small intestine. For small intestinal transit, fluorescent intensity for each small intestinal segment (I1 to I10, numbered from oral side) was used to calculate the geometric center of delivered dextran by the formula.
      • Miller M.S.
      • Galligan J.J.
      • Burks T.F.
      Accurate measurement of intestinal transit in the rat.
      geometriccenter=n=110In×nI1+I2++I10


       Statistical Analysis

      For gastric emptying and intestinal transit assays, nonparametric Kruskal-Wallis analysis of variance was used to test for an overall difference among the 3 treatment groups at a 5% significance level. Then all possible pairwise tests were conducted using Wilcoxon rank-sum tests and reported only when the overall test was significant, and P < .05 was regarded as statistically significant. Data are displayed as mean ± SD.
      Additional experimental procedures are provided in Supplementary Methods.

      Results

       Leucine-Rich Repeats and Immunoglobulin-Like Domains Protein 1 Is Expressed by Deep Muscle Plexus and Submucosal Plexus Interstitial Cells of Cajal

      We previously reported that LRIG1, a pan-ErbB−negative regulator,
      • Laederich M.B.
      • Funes-Duran M.
      • Yen L.
      • et al.
      The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases.
      marks intestinal epithelial stem cells.
      • Powell A.E.
      • Wang Y.
      • Li Y.
      • et al.
      The Pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor.
      To characterize Lrig1 transcriptional activity in vivo, we recently generated Lrig1-Apple/+ mice into which a red fluorescent protein (RFP) reporter was inserted into the translational start site of the endogenous mouse Lrig1 locus.
      • Poulin E.J.
      • Powell A.E.
      • Wang Y.
      • et al.
      Using a new Lrig1 reporter mouse to assess differences between two Lrig1 antibodies in the intestine.
      As expected, RFP fluorescence was observed at the crypt base in the small intestine (Figure 1A) and colon (Supplementary Figure 1A). Unexpectedly, we observed fluorescence beneath the epithelium in both the small intestine (Figure 1A) and colon (Supplementary Figure 1A). To examine the specificity of LRIG1 expression in this region, we performed immunofluorescence using a commercially available LRIG1 antibody, and found a staining pattern identical to that of RFP, confirming that LRIG1 protein was also present in these subepithelial cells (Supplementary Figure 1B). LRIG1 immunoreactivity was not observed in Lrig1-CreERT2/CreERT2 (hereafter Lrig1-null) mice, confirming the specificity of the antibody (Supplementary Figure 1C). Because LRIG1 is also expressed in glial cells in mouse brain,
      • Suzuki Y.
      • Sato N.
      • Tohyama M.
      • et al.
      cDNA Cloning of a novel membrane glycoprotein that is expressed specifically in glial cells in the mouse brain LIG-1, a protein with leucine-rich repeats and immunoglobulin-like domains.
      we first examined whether these LRIG1-positive cells were components of the enteric nervous system. We co-stained with a glial cell marker, S100, and PGP9.5, a neuronal cell marker; the subepithelial LRIG1-positive cells did not express either marker (Supplementary Figure 1DG), suggesting that these LRIG1-positive cells are likely non-neuronal.
      Figure thumbnail gr1
      Figure 1ICC-DMP express LRIG1. (A) Immunofluorescent images of Lrig1-Apple/+ mouse small intestinal tissue sections stained for KIT (green). Endogenous apple fluorescence (RFP) is shown in red. ICC-DMP are enclosed with dotted boxes and enlarged in insets below. Double-headed arrow indicates circular muscular layer bound by the submucosa (upper side) and the myenteric region (lower side). Nuclei were stained with DAPI. Scale bar = 50 μm. (BD) Immunofluorescent images of WT small intestinal tissue sections stained for LRIG1 and KIT (B), or alternative ICC markers ANO1 (C) and NK1R (D). Double-headed arrow indicates circular muscular layer bound by the DMP region (upper side) and the myenteric region (lower side). Yellow arrowheads indicate cells that have positive staining for both LRIG1 and KIT. White arrowheads indicate LRIG1(+)/KIT(−) cell at the DMP region (B). White arrows indicate ICC-MY with weak LRIG1 staining (B, C). Nuclei were stained with DAPI. Scale bar = 50 μm.
      We next considered whether these cells might be ICCs. To investigate this possibility, we co-stained with the ICC marker, KIT. As expected, KIT immunoreactivity was detected in all ICC populations: ICC-MY and ICC-DMP in the small intestine, and ICC-MY, ICC-IM, ICC-SS, and ICC-SMP in the colon (Figure 1A and Supplementary Figure 1A). LRIG1 was co-expressed with KIT in ICC-DMP and ICC-SMP in the small intestine and colon, respectively (Figure 1A and B and Supplementary Figures 1A and H). In contrast, there was no LRIG1 staining in the colonic ICC-MY population (Supplementary Figure 1H) and only weak LRIG1 immunoreactivity in the small intestinal ICC-MY population (Figure 1B). We compared LRIG1 immunoreactivity with 2 other ICC markers: anoctamin-1 (ANO1), which is expressed in all ICC populations,
      • Gomez-Pinilla P.J.
      • Gibbons S.J.
      • Bardsley M.R.
      • et al.
      Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract.
      and the neurokinin 1 receptor (NK1R), which labels ICC-DMP,
      • Iino S.
      • Ward S.M.
      • Sanders K.M.
      Interstitial cells of Cajal are functionally innervated by excitatory motor neurones in the murine intestine.
      • Faussone-Pellegrini M.-S.
      Relationships between neurokinin receptor-expressing interstitial cells of Cajal and tachykininergic nerves in the gut.
      but not other ICC subsets. LRIG1 was co-expressed with these markers in ICC-DMP (Figure 1C and D) and ICC-SMP (Supplementary Figure 1I). As expected, ANO1 was expressed by ICC-MY, and LRIG1 staining was absent in this population. Thus, LRIG1 is expressed in ICC-DMP and ICC-SMP in both small intestine and colon, but not in ICC-MY in the colon and only weakly in ICC-MY in the small intestine. Interestingly, there was no ICC subpopulation in the stomach that exhibited LRIG1 immunoreactivity comparable with duodenal ICC-DMP (Supplementary Figure 1J and K). ICC-MY in the stomach had weak LRIG1 staining similar to what was seen in ICC-MY in the duodenum, and in contrast to absent LRIG1 immunoreactivity in colonic ICC-MY.
      In cross-sections of the small intestine, we noted that LRIG1 stained a number of cells in the DMP region of the small intestine that were KIT negative (Figure 1B). To evaluate whether this reflected a difference in intracellular distribution of LRIG1 and KIT, or the potential existence of 2 different populations, we performed whole-mount staining of the DMP region (Figure 2A). All KIT-positive ICC-DMP expressed LRIG1 (Supplementary Figure 2A and B), indicating LRIG1 is uniformly expressed in ICC-DMP. However, although 60% of LRIG1-positive cells also expressed KIT, 40% were KIT-negative (Figure 2B and Supplementary Figure 2B). We observed similar results when we quantified LRIG1- and ANO1-expressing populations, as well as LRIG1- and NK1R-expressing populations (Supplementary Figure 2C and D). In the colonic SMP region, such LRIG1(+)/KIT(−) cells were also observed, but less frequently (Supplementary Figure 2E). Similar to the small intestine, all ICC-SMP KIT-positive cells co-expressed LRIG1 (data not shown). In the DMP region of the small intestine, KIT-negative/platelet-derived growth factor receptor α (PDGFRA)-positive fibroblast-like cells with oval cell bodies and long processes run parallel with ICC-DMP and smooth muscle cells, while PDGFRA-positive fibroblast-like cells are absent in the colonic SMP region.
      • Iino S.
      • Horiguchi K.
      • Horiguchi S.
      • et al.
      c-Kit-negative fibroblast-like cells express platelet-derived growth factor receptor α in the murine gastrointestinal musculature.
      • Iino S.
      • Nojyo Y.
      Immunohistochemical demonstration of c-Kit-negative fibroblast-like cells in murine gastrointestinal musculature.
      We examined whether LRIG1(+)/KIT(−) cells were these KIT-negative fibroblast-like cells. We observed that LRIG1-positive cells and PDGFRA-expressing cells are adjacent, but nonoverlapping (Figure 2C and D). Overall, these results indicate that LRIG1 is expressed in ICC-DMP in the small intestine and ICC-SMP in the colon, and is also expressed in an unknown KIT-negative population, which is morphologically similar to its KIT-positive counterpart. Of note, human duodenum also exhibited both LRIG1-positive/KIT-positive and LRIG1-positive/KIT-negative cells in the DMP region of the circular muscular layer, while LRIG1 immunoreactivity was absent in ICC-MY (Figure 2E), suggesting the potential relevance of our murine findings to humans.
      Figure thumbnail gr2
      Figure 2LRIG1 is expressed in ICC-DMP, but not in PDGFRA-positive ICC-like fibroblasts. (A) Confocal images of the small intestinal DMP region. WT small intestinal whole mounts were stained for KIT (green) and LRIG1 (red). Nuclei were stained with DAPI. Scale bar = 50 μm. (B) KIT expression status in LRIG1-positive cells in the DMP region is shown as a percentage. Data are represented as mean ± SEM of 3 mice. (C) Immunofluorescent images of WT small intestine. Tissues were stained for PDGFRA (green) and LRIG1 (red). Double-headed arrow indicates circular muscular layer. Nuclei were stained with DAPI. Scale bar = 50 μm. (D) Confocal images of the DMP region of WT small intestine. WT small intestinal whole mounts were stained for PDGFRA (green) and LRIG1 (red). Nuclei were stained with DAPI. Scale bar = 20 μm. (E) Immunofluorescent images of human duodenum. Tissues were stained for KIT (green) and LRIG1 (red). White dotted line indicates circular muscular layer. Yellow arrowheads indicate ICC-MY. Confocal images of boxed DMP region is shown below enclosed with yellow arrows indicating cells stained for both LRIG1 and KIT, and white arrowheads with cells stained only for LRIG1. Nuclei were stained with DAPI. CM, circular muscular layer; LM, longitudinal muscular layer. Scale bar = 25 μm.

       Leucine-Rich Repeats and Immunoglobulin-Like Domains Protein 1 Expression Precedes Emergence of Deep Muscle Plexus and Submucosal Plexus Interstitial Cells of Cajal During Postnatal Development

      We next investigated the timing of LRIG1 expression beneath the epithelium during postnatal development. In the small intestine, ICC-DMP are rarely observed at postnatal day 0 (P0), but are detected shortly after birth and continue to develop until at least P10.
      • Ward S.M.
      • McLaren G.J.
      • Sanders K.M.
      Interstitial cells of Cajal in the deep muscular plexus mediate enteric motor neurotransmission in the mouse small intestine.
      In the colon, ICC-SMP are absent at birth; they emerge around P5 in the proximal colon, and shortly later in the distal colon.
      • Han J.
      • Shen W.-H.
      • Jiang Y.-Z.
      • et al.
      Distribution, development and proliferation of interstitial cells of Cajal in murine colon: an immunohistochemical study from neonatal to adult life.
      We examined a time course of the developing tunica muscularis in the ileum and proximal colon and observed that LRIG1 is expressed broadly in the smooth muscular layer from P0 to P5 (Figure 3Ai−iii and Supplementary Figure 3Ai−iii). LRIG1 exhibited a clear vertical gradient with strong expression at the mucosal side of the circular muscular layer and weaker expression at the outer border (Figure 3Ai−iii, Supplementary Figure 3Ai−iii). In the small intestine, some cells at the innermost surface of the circular muscular layer were negative for LRIG1 at P0 (Supplementary Figure 3D). At later time points, LRIG1-negative circular smooth muscle cells formed a thin inner layer (Supplementary Figure 3E and F); this layer was separated from the outer part of the circular muscular layer by ICC-DMP.
      • Pellegrini M.S.F.
      Morphogenesis of the special circular muscle layer and of the interstitial cells of Cajal related to the plexus muscularis profundus of mouse intestinal muscle coat.
      Strong LRIG1 expression in the circular muscular layer of the ileum gradually became dimmer, and was restricted to the mucosal side 3 weeks after birth; some LRIG1-positive cells also expressed the ICC-DMP marker KIT at this time (Figure 3A and B). We observed similar results in the proximal colon from P0 to 3 weeks (Supplementary Figure 3A and B). We also examined KIT immunoreactivity in the emerging ICC-DMP and ICC-SMP populations during this time course and observed a time-dependent increase in staining intensity (Figure 3A and C and Supplementary Figure 3A and C). Together, these data indicate that LRIG1 expression in the circular smooth muscle is widespread at P0 but, during the first 3 weeks of life, it becomes restricted to the region that contains KIT-expressing cells.
      Figure thumbnail gr3
      Figure 3Changes in LRIG1 and KIT expression during postnatal development of the muscular layer. (A) Immunofluorescent images of WT small intestinal tissue sections stained for LRIG1 (red) and KIT (green) during postnatal development from postnatal day 0 (i) to 3 weeks old (vi). Double-headed arrow indicates circular muscular layer. Nuclei were stained with DAPI. Scale bar = 50 μm. (B, C) Relative immunoreactivity of LRIG1 (B) and KIT (C) during postnatal development of the small intestinal muscular layer. Data are shown as relative intensity of LRIG1 in cells adjacent to the MY region compared with cells in the DMP region (B), or relative intensity of KIT in DMP region compared with MY region (C). Data are represented as mean ± SEM of 3 mice. (D, E) Confocal images of colon during postnatal development. ICC-SMP positive for both KIT (green) and the muscular marker SMA (D, red) at P5 and triple positive for KIT (green), calponin (E, red), and LRIG1 (E, white) at P7 were enclosed with dotted boxes and enlarged in insets below. Double-headed arrow indicates circular muscular layer. Nuclei were stained with DAPI. Scale bar = 10 μm.
      These observations imply a developmental link between smooth muscle cells and both ICC-DMP and ICC-SMP. To further investigate that possible link, we reasoned that smooth muscle markers should decrease within the inner region of the circular muscular layer, as KIT-positive ICC-DMP and ICC-SMP emerged. In fact, KIT expression in ICC-DMP and ICC-SMP gradually increased from P0 to P10 (Figure 3A and Supplementary Figure 3A), leading us to examine the transition from smooth muscle cells to ICCs during this time frame. At P5−7, some colonic ICC-SMP cells remained positive for smooth muscle actin (SMA) (P5) or calponin (P7), both of which are markers for smooth muscle cells (Figure 3D and E), suggesting that these cells might be in an intermediate state between smooth muscle cells and KIT-positive ICC-SMP cells. In addition, we observed coexpression of KIT, calponin, and LRIG1 at P7 (Figure 3E). Collectively, these results suggest that a subset of LRIG1-expressing smooth muscle progenitors give rise to KIT-expressing ICC-DMP and ICC-SMP.

       Leucine-Rich Repeats and Immunoglobulin-Like Domains Protein 1−Positive Smooth Muscle Cells Give Rise to Deep Muscle Plexus and Submucosal Plexus Interstitial Cells of Cajal

      To further substantiate the claim that LRIG1-expressing smooth muscle cells are the origin of ICC-DMP and ICC-SMP, we examined the dynamics of LRIG1 and SMA expression during early development. We observed that SMA expression was gradually lost from LRIG1-expressing cells at the surface of developing circular muscle of the colon from P0 to P9 (Figure 4AC). To ask directly if LRIG1-expressing cells were the cell of origin for ICC-SMP, we used Lrig1-CreERT2/+;R26-YFP mice
      • Powell A.E.
      • Wang Y.
      • Li Y.
      • et al.
      The Pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor.
      to perform lineage tracing. We administered a single injection of tamoxifen (33 mg/kg, intraperitoneal) at P1 before the emergence of ICC-SMP in the colon
      • Han J.
      • Shen W.-H.
      • Jiang Y.-Z.
      • et al.
      Distribution, development and proliferation of interstitial cells of Cajal in murine colon: an immunohistochemical study from neonatal to adult life.
      (Supplementary Figure 3Ai−ii) and when LRIG1 is expressed in smooth muscle cells beneath the epithelium (Figure 4A). When tissues were evaluated after development of ICC-SMP, YFP-positive cells were observed in the colonic KIT-expressing ICC-SMP population (Figure 4D−E), as in smooth muscle cells (Figure 4D). We quantified YFP positivity within ICC-SMP by confocal microscopic analysis and found that approximately 50% of ICC-SMP were lineage traced throughout the colon (Supplementary Figure 4C). In the small intestine, we also observed YFP positivity in ICC-DMP (Supplementary Figure 4AE). To directly confirm the smooth muscular origin of ICC-DMP and ICC-SMP, we also performed lineage-tracing experiments using inducible, smooth muscle-specific Cre-recombinase (smooth muscle myosin heavy chain 11, Myh11-CreERT2)
      • Wirth A.
      • Benyó Z.
      • Lukasova M.
      • et al.
      G12-G13−LARG−mediated signaling in vascular smooth muscle is required for salt-induced hypertension.
      crossed to R26-YFP mice.
      • Srinivas S.
      • Watanabe T.
      • Lin C.-S.
      • et al.
      Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.
      Tamoxifen was administered to Myh11-CreERT2/+;R26-YFP/+ mice at P1 and intestinal tissues were harvested and processed at later time points. We observed increasing ratios of lineage-traced YFP(+)/KIT(+) cells up to approximately 45% toward the distal part of the intestine (Figure 5AE), indicating ICC-SMP and ICC-DMP are the progeny of Myh11-expressing smooth muscle cells. This gradual increase in lineage-traced cells toward the distal intestine might correspond to a rostral-caudal gradient development of ICC-DMP
      • Ward S.M.
      • McLaren G.J.
      • Sanders K.M.
      Interstitial cells of Cajal in the deep muscular plexus mediate enteric motor neurotransmission in the mouse small intestine.
      and ICC-SMP,
      • Han J.
      • Shen W.-H.
      • Jiang Y.-Z.
      • et al.
      Distribution, development and proliferation of interstitial cells of Cajal in murine colon: an immunohistochemical study from neonatal to adult life.
      which might be related to inactivation of Myh11 transcription earlier in the more proximal parts of the intestine. These lineage-tracing results provide strong evidence that ICC-DMP and ICC-SMP arise from smooth muscle progenitors postnatally.
      Figure thumbnail gr4
      Figure 4ICC-SMP arise from LRIG1-positive cells during postnatal development. (A−C) Confocal images of the colonic tunica muscularis during postnatal development at indicated postnatal ages. LRIG1-positive cells (green) at the surface of the inner circular muscular layer (labeled by SMA, red) are enclosed in dotted boxes and enlarged in insets below. Nuclei were stained with DAPI. Scale bar = 15 μm. Immunofluorescent (D) and confocal (E) images of the colon of Lrig1-CreERT2/+;R26-LSL-YFP/+ mice. Tamoxifen was given at postnatal day 1 (P1) and tissues were collected at P9 (D) or P22 (E). Tissues were stained for YFP with an anti-EGFP antibody (green) and KIT (red). For confocal imaging, single optical slices of SMP region were acquired (E). Double-headed arrow indicates circular muscular layer. Nuclei were stained with DAPI. Arrowheads indicate ICC-SMP positive for YFP. Scale bar = 50 μm.
      Figure thumbnail gr5
      Figure 5ICC-DMP and ICC-SMP arise postnatally from smooth muscle progenitors. Immunofluorescent (A, C) and confocal (B, D) images of small intestine (A, B) and colon (CD) of Myh11-CreERT2/+;R26-LSL-YFP/+ mice. Tamoxifen was given at postnatal day 1 (P1) and tissues were collected at P10 (A, C) or P22 (B, D). Tissues were stained for YFP with an anti-EGFP antibody (green) and KIT (red). Double-headed arrow indicates circular muscular layer. Nuclei were stained with DAPI. Arrowheads indicate ICC-DMP (A, B) or ICC-SMP (C, D) cells that are positive for YFP. Scale bars = 50 μm. (E) Quantification of lineage-traced cells for confocal microscopic analysis. YFP expression status among KIT-positive cells in DMP and SMP region is shown as percentages. Data are represented as mean ± SEM of 3 mice.

       Leucine-Rich Repeats and Immunoglobulin-Like Domains Protein 1 Is Required for Development of Deep Muscle Plexus and Submucosal Plexus Interstitial Cells of Cajal

      We next examined whether the expression of LRIG1 influenced development of ICC. We examined WT and Lrig1-null mice and observed near-complete loss of KIT immunoreactivity in the DMP region in the small intestine and SMP region in the colon (Figure 6A, Supplementary Figure 5A). In Lrig1-null mice, additional markers for ICCs, ANO1 and NK1R, were also largely absent compared with WT mice (Figure 6B and C and Supplementary Figure 5B). Of note, SMA staining in the small intestine revealed that the DMP region does not develop in Lrig1-null mice (Supplementary Figure 6A and B), compared with WT mice of the same age. To quantify these findings, we counted total KIT-positive cells per visual field in the ICC-DMP region in WT, Lrig1-heterozygous (Lrig1-CreERT2/+, hereafter Lrig1-het) and Lrig1-null mice. Although Lrig1-het mice have only 1 WT Lrig1 allele, they have a similar number of LRIG1-positive and KIT-positive cells compared with WT mice (Figure 6D, left panel). We observed significantly fewer total KIT-positive cells in Lrig1-null mice, compared with the other 2 cohorts (Figure 6D, left panel). Together, our observations suggest that the ICC-DMP population is largely lost in Lrig1-null mice, and there appears to be no haploinsufficient effect.
      Figure thumbnail gr6
      Figure 6LRIG1 is required for development of ICC-DMP and ICC-SMP. (AC) Immunofluorescent images of WT (Ai, Bi, Ci) and Lrig1-null (Aii, Bii, Cii) small intestinal tissue sections stained for LRIG1 and ICC markers. (A) KIT, green; LRIG1, red. (B) LRIG1, green; ANO1, red. (C) KIT, green; NK1R, red. Dotted line indicates the submucosal side of the circular muscular layer where ICC-DMP should reside, but are lost in Lrig1-null mice. Double-headed arrow indicates circular muscular layer. Nuclei were stained with DAPI. Scale bar = 50 μm. (D) Quantification of cells in DMP (left panel) and MY (right panel) regions of the small intestine. Number of nuclei from KIT-positive cells per field (0.1 mm2) is shown. Data are represented as mean ± SEM of 3 mice. (E) Diagram of the postnatal development of ICC-DMP. (Left panel) At birth in the small intestine, circular smooth muscle cells express LRIG1 (red) and KIT expression (green) are observed in ICC-MY (upper left). During postnatal development, LRIG1 expression decreases in the outer side of the circular muscular layer, and cells at the most inner layer of LRIG1-positive circular smooth muscle cells show increased KIT expression (red-green stripe, upper middle). In the adult small intestine, sustained high LRIG1 expression is seen in ICC-DMP (red-green stripe) and LRIG1-positive ICC-like cells (red, upper right). When LRIG1 is lost, ICC-DMP are absent in the small intestine, while ICC-MY are preserved (lower). (Right panel) Localization of ICC-DMP and ICC-MY in the small intestine is shown. CM, circular muscular layer; Ep, epithelium; LM, longitudinal muscular layer; Sm, submucosa.
      In addition to LRIG1 expression in ICC-DMP and ICC-SMP, we also observed weak LRIG1 immunoreactivity in small intestinal ICC-MY (Figure 1B and Supplementary Figure 5C), but not in colonic ICC-MY in WT mice (Supplementary Figure 1H). However, ICC-MY were not altered in cell number or in gross morphology (Figure 6D, right panel, and Supplementary Figure 5D) in Lrig1-null mice. On the other hand, in the stomach that lacks a LRIG1-positive ICC population at the inner side of the circular muscle, no obvious changes in ICC populations were observed in Lrig1-null mice (data not shown). We also evaluated the postnatal development of ICC-DMP and ICC-SMP in Lrig1-null mice. Immunoflurorescent analyses using Lrig1-null tissue showed no emergence of ICC-DMP and ICC-SMP, confirming that lack of LRIG1 affects development of these cells, and that our results cannot be explained by regression of these cells after their emergence (Supplementary Figure 7 and data not shown). These results indicate that LRIG1 is not only a marker for ICC-DMP and ICC-SMP, but is also essential for development of these ICC subsets (Figure 6E).

       Leucine-Rich Repeats and Immunoglobulin-Like Domains Protein 1−Null Mice Exhibit Delayed Small Intestinal Transit

      To evaluate the functional consequence of ICC-DMP loss in Lrig1-null mice, we next assessed small intestinal transit, a process that is regulated by numerous factors,
      • Furness J.B.
      The enteric nervous system and neurogastroenterology.
      • Sanders K.M.
      • Koh S.D.
      • Ro S.
      • et al.
      Regulation of gastrointestinal motility—insights from smooth muscle biology.
      including ICC.
      • Huizinga J.D.
      • Lammers W.J.E.P.
      Gut peristalsis is governed by a multitude of cooperating mechanisms.
      • Yin J.
      • Chen J.D.Z.
      Roles of interstitial cells of Cajal in regulating gastrointestinal motility: in vitro versus in vivo studies.
      We administered rhodamine B−conjugated dextran via gavage and quantified segmental passage of the dextran within the small intestine. WT and Lrig1-het mice displayed a similar distribution of dextran throughout the small intestine, and Lrig1-null mice exhibited significantly slower transit (Figure 7A and B). To exclude the contribution of change in gastric emptying, an additional cohort of mice was given rhodamine B−conjugated dextran, and the amount of dye that left the stomach was evaluated. Of note, the ratio of the dextran retained in the stomach did not differ among genotypes (Figure 7C). In addition, the length of the small intestine was not significantly different in Lrig1-null mice compared with WT and Lrig1-het mice (Figure 7D), indicating that decreased segmental passage in Lrig1-null mice cannot be explained by longer segments than those in WT mice. Thus, Lrig1-null mice exhibit significantly delayed small intestinal transit, suggesting a possible functional consequence of the loss of this ICC population.
      Figure thumbnail gr7
      Figure 7Lrig1-null mice exhibit delayed small intestinal transit. (A) Geometric center of the distribution of delivered rhodamine B−dextran is plotted for each mouse examined. Black bar indicates mean value of each genotype group. (B) Representative data of the distribution of delivered rhodamine B-dextran for each mouse examined. Data from mice with a geometric center value closest to the mean value of each group is shown. The y-axis is presented as the percentage of total rhodamine B intensity. (C) Gastric emptying assay. Ratio of rhodamine B excluded from the stomach is plotted for each mouse examined. Black bar indicates mean value of each genotype group. (D) Length of small intestine of mice used for transit assay. Data are represented as mean ± SEM of 10 to 12 mice per group.

      Discussion

      In the current study, we show that ICC-DMP and ICC-SMP arise postnatally from circular smooth muscle cells, and that the pan-ErbB inhibitor, LRIG1, is a novel marker of these ICC subsets (Figure 6E). We also demonstrate that Lrig1-null mice exhibit delayed small intestinal transit. These findings identify the ontogeny of this subset of ICCs and implicate a role for LRIG1 in their development.
      There appears to be an intimate link between ICC-MY and smooth muscle cells. For example, ICC-MY and smooth muscle cells arise from a common KIT-positive progenitor in the mouse small intestine during embryogenesis.
      • Klüppel M.
      • Huizinga J.D.
      • Malysz J.
      • et al.
      Developmental origin and kit-dependent development of the interstitial cells of cajal in the mammalian small intestine.
      In addition, neutralizing antibody blockade of KIT activity in neonatal mice results in acquisition of ultrastructural features of smooth muscle cells by ICC-MY.
      • Ward S.M.
      • McLaren G.J.
      • Sanders K.M.
      Interstitial cells of Cajal in the deep muscular plexus mediate enteric motor neurotransmission in the mouse small intestine.
      However, a direct link between smooth muscle cells and ICC-DMP or ICC-SMP has not been shown previously. We now show by lineage tracing that both ICC-DMP in the small intestine and ICC-SMP in the colon arise postnatally from inner circular smooth muscle cells. LRIG1 expression in this region precedes KIT expression and the emergence of ICC-DMP and ICC-SMP. In early postnatal life, LRIG1 is expressed broadly in smooth muscle cells, but becomes more restricted to ICC-DMP and ICC-SMP over time. In addition, LRIG1 loss results in loss of ICC-DMP and ICC-SMP. Based on our observations, we propose a model in which LRIG1 promotes postnatal differentiation of smooth muscle cells to ICC-DMP and ICC-SMP. We suspect the non−lineage-traced cells in the ICC-DMP and ICC-SMP populations are due to inefficient recombination and/or a rostral-caudal developmental gradient. However, we cannot exclude that ICC-SMP and ICC-DMP are heterogeneous populations with different origins. We hypothesize that, in the absence of LRIG1, smooth muscle cells are unable to differentiate into ICC-DMP and ICC-SMP, similar to the observation that mice treated postnatally with a KIT-neutralizing antibody fail to exhibit differentiation of putative precursor cells.
      • Torihashi S.
      • Nishi K.
      • Tokutomi Y.
      • et al.
      Blockade of kit signaling induces transdifferentiation of interstitial cells of Cajal to a smooth muscle phenotype.
      Loss of ICC-DMP results in delayed small intestinal transit and it is tempting to speculate that loss of ICC-DMP is directly responsible for this delay. However, it is important to consider other possibilities that could affect gut motility in Lrig1-null mice, such as the potential contribution of LRIG1-positive/KIT-negative ICC-like cells. We found that approximately 40% of LRIG1-positive cells are KIT-negative in the small intestine, although they exhibit similar morphology to ICC-DMP. These LRIG1-positive/KIT-negative ICC-like cells, which have not been reported previously, form a network with ICC-DMP and are distinct from PDGFRA-positive fibroblast-like cells.
      • Iino S.
      • Horiguchi K.
      • Horiguchi S.
      • et al.
      c-Kit-negative fibroblast-like cells express platelet-derived growth factor receptor α in the murine gastrointestinal musculature.
      • Iino S.
      • Nojyo Y.
      Immunohistochemical demonstration of c-Kit-negative fibroblast-like cells in murine gastrointestinal musculature.
      In the colon, a rare population of LRIG1-positive ICC-like cells was also observed in the SMP region, where PDGFRA-positive fibroblast-like cells do not reside.
      • Iino S.
      • Horiguchi K.
      • Horiguchi S.
      • et al.
      c-Kit-negative fibroblast-like cells express platelet-derived growth factor receptor α in the murine gastrointestinal musculature.
      It will be important to determine whether these LRIG1-positive ICC-like cells have a functional role in smooth muscle, or if they represent an intermediate state preceding mature ICC-DMP or ICC-SMP. It is also possible that intestinal smooth muscle cells were affected due to loss of LRIG1, as LRIG1 is broadly expressed in smooth muscle of newborn mice. Ultimately, more detailed physiological studies will be required to fully understand which cell (or cells) are responsible for decreased gut motility in Lrig1-null mice.
      Our studies make use of LRIG1 deficiency in Lrig1-CreERT2/CreERT2 mice, which express Cre recombinase in place of the LRIG1 protein. In 2012, Wong et al
      • Wong V.W.Y.
      • Stange D.E.
      • Page M.E.
      • et al.
      Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling.
      reported the intestinal phenotype of Lrig1 germline knockout mice, which were generated using a different strategy and in a different genetic background. Of interest, this group observed a perinatal lethality in Lrig1-null mice related to marked abdominal distension. They proposed this phenotype was due to intestinal epithelial hyperproliferation, resulting from loss of this ErbB-negative regulator; however, the hyperproliferation was modest. We submit that an alternate explanation for the abdominal distension in these mice may be due to effects on ICC populations, in particular, ICC-DMP.
      We now report a genetically engineered mouse model in which there is loss of ICC-DMP and ICC-SMP with preservation of ICC-MY. This model will allow investigators to conduct experiments that will provide greater mechanistic and physiological understanding of the nature of ICC-DMP and ICC-SMP, and how these ICC subsets may influence gastrointestinal function in health and disease.

      Acknowledgments

      The authors thank Emily J. Poulin for editorial assistance, and acknowledge the generous support of Eileen and Mac Brown.

      Supplementary Methods

       Quantification of Immunoreactivity

      Supplementary Table 1Antibody list
      AntigenCloneSupplier (catalog number)DilutionHostConjugate
      LRIG1polyclonalR&D systems (AF3688)1:250Goat-
      KITACK2Millipore (CBL1360)1:250Rat-
      ANO1polyclonalAbcam (ab53212)1:100Rabbit-
      NK1RpolyclonalSigma (S8305)1:2000Rabbit-
      SMA1A4Sigma (C6198)1:2000MouseCy3
      calponinEP798YAbcam (ab46794)1:200Rabbit-
      PDGFRAAPA5e bioscience (16-1401)1:100Rat-
      PGP9.5polyclonalAbD Serotec (7863-0504)1:4000Rabbit-
      S100polyclonalDAKO (Z0311)1:400Rabbit-
      EGFPpolyclonallife technologies (A11122)1:500Rabbit-
      human LRIG1polyclonalR&D systems (AF7498)1:100Sheep-
      human KITK45Thermo Scientific (MA5-12944)1:100Mouse-
      Rat IgGpolyclonallife technologies (A21208)1:500DonkeyAlexa 488
      Rat IgGpolyclonallife technologies (A11077)1:500GoatAlexa 568
      Rat IgGpolyclonalJackson ImmunoResearch (712-165-153)1:400DonkeyCy3
      Rabbit IgGpolyclonallife technologies (A10042)1:500DonkeyAlexa 568
      Rabbit IgGpolyclonalJackson ImmunoResearch (711-165-152)1:400DonkeyCy3
      Goat-IgGpolyclonallife technologies (A11055)1:500DonkeyAlexa 488
      Goat-IgGpolyclonallife technologies (A11057)1:500DonkeyAlexa 568
      Goat-IgGpolyclonalJackson ImmunoResearch (705-165-147)1:400DonkeyCy3
      Mouse-IgGpolyclonallife technologies (A10037)1:500DonkeyAlexa 568
      Sheep-IgGpolyclonalJackson ImmunoResearch (713-175-147)1:400DonkeyCy5
      Figure thumbnail fx1
      Supplementary Figure 1LRIG1 is expressed in ICC-DMP and ICC-SMP. (A) Immunofluorescent images of Lrig1-Apple/+ colonic tissue sections stained for KIT (green). ICC-SMP is enclosed with yellow dotted boxes that are enlarged in insets. Endogenous Apple (RFP) fluorescence is shown in red. Double-headed arrow indicates the circular muscular layer bound by submucosa (upper side) and myenteric region (lower side). Nuclei were stained with DAPI. Scale bar = 50 μm. (B) Immunofluorescent images of WT small intestinal tissue sections stained for LRIG1 (green). Endogenous Apple (RFP) fluorescence is shown in red. Double-headed arrow indicates the circular muscular layer bound by submucosa (upper side) and myenteric region (lower side). Nuclei were stained with DAPI. Scale bar = 50 μm. (C) Immunofluorescent images of small intestinal tissue sections from WT and Lrig1-null mice stained for LRIG1 (green) and SMA (red). Nuclei were stained with DAPI. Scale bar = 50 μm. (D, E) Immunofluorescent images of Lrig1-Apple/+ small intestinal (SI) and colonic tissue sections stained for PGP9.5 (D, green) and S100 (E, green). Endogenous Apple (RFP) fluorescence is shown in red image. Nuclei were stained with DAPI. Double-headed arrow indicates circular muscular layer. Scale bar = 20 μm. (F, G) Immunofluorescent images of WT small intestinal and colonic tissue sections stained for LRIG1 (green) with PGP9.5 (F, red) and S100 (G, red). Nuclei were stained with DAPI. Double-headed arrow indicates circular muscular layer. Scale bar = 20 μm. (H, I) Immunofluorescent images of WT colonic tissue sections stained for KIT (A, green) and LRIG1 (H, red), or LRIG1 (I, green) and ANO1 (I, red). Double-headed arrow indicates the circular muscular layer. Yellow arrowheads indicate double-positive cells. Nuclei were stained with DAPI. Scale bar = 20 μm. (J, K) Immunofluorescent images of WT gastric and duodenal tissue sections stained for KIT (green) and LRIG1 (red). The stomach and duodenum were dissected, fixed, and mounted en bloc to ensure identical processing. Double-headed arrow indicates the circular muscular layer. Yellow arrowheads indicate LRIG1(+)/KIT(+) cells. Nuclei were stained with DAPI. Scale bar = 50 μm.
      Figure thumbnail fx2
      Supplementary Figure 2LRIG1 is expressed in ICC-DMP and LRIG1(+)/KIT(−)ICC-like cells. (A) LRIG1 expression status among KIT-positive cells in DMP region is shown as a percentage. Data are represented as mean ± SEM of 3 mice. (B) Expression status of LRIG1 and KIT among the cells in the DMP region of the small intestine. Data are represented as mean counts of nuclei ± SEM per field of confocal images (0.1 mm2). n = 3 mice. (C, D) Confocal images of the DMP region of WT small intestine. WT small intestinal whole mounts were double stained for ANO1 (C, green) or NK1R (D, green) with LRIG1 (red). Nuclei were stained with DAPI. Scale bar = 20 μm. (E) Confocal images of the SMP region of WT colon. WT colonic whole mounts were stained for KIT (green) and LRIG1 (red). Nuclei were stained with DAPI. White arrowheads indicate LRIG1(+)/KIT(−) cells. Scale bar = 20 μm.
      Figure thumbnail fx3
      Supplementary Figure 3ICC-SMP arise from LRIG1-positive cells during postnatal development. (A) Immunofluorescent images of WT colonic tissue sections stained for KIT (green) and LRIG1 (red) during postnatal development from postnatal day 0 (i) to 3 weeks old (vi). Nuclei were stained with DAPI. Double-headed arrows indicate the circular muscular layer. Scale bar = 50 μm. (B, C) Quantified relative immunoreactivity of LRIG1 (B) and KIT (C) during postnatal development of the colonic muscular layer. Data are shown as the relative intensity of LRIG1 in the cells adjacent to MY region compared with the cells in SMP region (B), or relative intensity of KIT in SMP region compared with MY region (C). Data are represented as mean ± SEM of 3 mice. (DF) Confocal images of the small intestinal tunica muscularis during postnatal development at indicated postnatal ages. Within the most inner layer of circular muscle, the cells positive for both LRIG1 (green) and SMA (red) are indicated by white arrows, while muscle cells without LRIG1 staining are indicated by yellow arrowheads. Double-headed arrows indicate circular muscular layer. Nuclei were stained with DAPI. Scale bar = 10 μm.
      Figure thumbnail fx4
      Supplementary Figure 4ICC-DMP and ICC-SMP arise from smooth muscle progenitors. Immunofluorescent (A) and confocal (B) images of the small intestine of Lrig1-CreERT2/+;R26-LSL-YFP/+ mice. Tamoxifen was given at postnatal day 1 (P1) and tissues were collected at P15 (A) or P22 (B). Tissues were stained for YFP with an anti−enhanced green fluorescent protein antibody (green) and KIT (red). For confocal imaging, single optical slices of DMP region were acquired. Double-headed arrow indicates circular muscular layer (A). Nuclei were stained with DAPI. Arrowheads indicate ICC-DMP positive for YFP. Scale bars = 25 μm. (C) Quantification of lineage-traced cells for confocal microscopic analysis. YFP expression status among KIT-positive cells in DMP or SMP region is shown as percentages. Data are represented as mean ± SEM of 3 mice. (D, E) Immunofluorescent images of WT small intestinal (D) and colonic (E) tissue sections stained with an anti-EGFP antibody at P15. Nuclei were stained with DAPI. Scale bar = 100 μm. These images indicate that no obvious autofluorescence or artificial immunoreactivity is observed with this anti-EGFP antibody under the same conditions as in A and C and A.
      Figure thumbnail fx5
      Supplementary Figure 5LRIG1 is required for the development of DMP and ICC-SMP, but not of ICC-MY. (A, B) Immunofluorescent images of WT colonic tissue sections (Ai, Bi) and Lrig1-null mice (Aii, Bii) stained for LRIG1 with KIT (A, green) and ANO1 (B, red). Double-headed arrow indicates circular muscular layer. Dotted line indicates submucosal side of circular muscular layer where ICC-SMP should reside, but are lost in Lrig1-null mice. Nuclei were stained with DAPI. Scale bar = 50 μm. (C) Confocal images of the MY region of small intestine. Whole-mount samples of WT mice were stained for KIT and LRIG1. Nuclei were stained with DAPI. Scale bar = 50 μm. (D) Confocal images of the MY region of small intestine. Whole-mount samples of WT mice (left) and Lrig1-null mouse (right) were stained for KIT. Scale bar = 20 μm.
      Figure thumbnail fx6
      Supplementary Figure 6Lrig1-null mice lack the regional space for ICC-DMP. (A, B) Immunofluorescent images of WT (A) and Lrig1-null (B) P9 mice stained for KIT (green) and SMA (red). Double-headed arrow indicates circular muscular layer. Yellow arrowheads indicate DMP region, which is distinguished by absent SMA staining, in WT mouse. Nuclei were stained with DAPI. Scale bar = 50 μm.
      Figure thumbnail fx7
      Supplementary Figure 7ICC-DMP fail to emerge during postnatal development in Lrig1-null mice. Immunofluorescent images of small intestinal tissue sections from Lrig1-null mice stained for KIT (green) and SMA (red) at indicated postnatal days. Double-headed arrow indicates the circular muscular layer. Dotted line indicates submucosal side of circular muscular layer where ICC-DMP should reside, but are lost. Nuclei were stained with DAPI. Scale bar = 50 μm.

      References

        • Torihashi S.
        • Ward S.M.
        • Nishikawa S.-I.
        • et al.
        c-kit-Dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract.
        Cell Tissue Res. 1995; 280: 97-111
        • Thomson L.
        • Robinson T.L.
        • Lee J.C.F.
        • et al.
        Interstitial cells of Cajal generate a rhythmic pacemaker current.
        Nat Med. 1998; 4: 848-851
        • Iino S.
        • Ward S.M.
        • Sanders K.M.
        Interstitial cells of Cajal are functionally innervated by excitatory motor neurones in the murine intestine.
        J Physiol. 2004; 556: 521-530
        • Klein S.
        • Seidler B.
        • Kettenberger A.
        • et al.
        Interstitial cells of Cajal integrate excitatory and inhibitory neurotransmission with intestinal slow-wave activity.
        Nat Commun. 2013; 4: 1630
        • Gockel I.
        • Bohl J.R.
        • Eckardt V.F.
        • et al.
        Reduction of interstitial cells of Cajal (ICC) associated with neuronal nitric oxide synthase (n-NOS) in patients with achalasia.
        Am J Gastroenterol. 2008; 103: 856-864
        • Horváth V.J.
        • Vittal H.
        • Lörincz A.
        • et al.
        Reduced stem cell factor links smooth myopathy and loss of interstitial cells of Cajal in murine diabetic gastroparesis.
        Gastroenterology. 2006; 130: 759-770
        • Forrest A.
        • Huizinga J.D.
        • Wang X.-Y.
        • et al.
        Increase in stretch-induced rhythmic motor activity in the diabetic rat colon is associated with loss of ICC of the submuscular plexus.
        Am J Physiol Gastrointest Liver Physiol. 2008; 294: G315-G326
        • Porcher C.
        • Baldo M.
        • Henry M.
        • et al.
        Deficiency of interstitial cells of Cajal in the small intestine of patients with Crohn’s disease.
        Am J Gastroenterol. 2002; 97: 118-125
        • He C.-L.
        • Burgart L.
        • Wang L.
        • et al.
        Decreased interstitial cell of Cajal volume in patients with slow-transit constipation.
        Gastroenterology. 2000; 118: 14-21
        • Kashyap P.
        • Gomez-Pinilla P.J.
        • Pozo M.J.
        • et al.
        Immunoreactivity for Ano1 detects depletion of Kit-positive interstitial cells of Cajal in patients with slow transit constipation.
        Neurogastroenterol Motil. 2011; 23: 760-765
        • Iino S.
        • Horiguchi K.
        Interstitial cells of Cajal are involved in neurotransmission in the gastrointestinal tract.
        Acta Histochem Cytochem. 2006; 39: 145-153
        • Al-Sajee D.
        • Huizinga J.D.
        Interstitial cells of Cajal.
        Sultan Qaboos Univ Med J. 2012; 12: 411-421
        • Ward S.M.
        • Burns A.J.
        • Torihashi S.
        • et al.
        Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.
        J Physiol. 1994; 480: 91-97
        • Huizinga J.D.
        • Thuneberg L.
        • Klüppel M.
        • et al.
        W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.
        Nature. 1995; 373: 347-349
        • Wang X.-Y.
        • Vannucchi M.-G.
        • Nieuwmeyer F.
        • et al.
        Changes in interstitial cells of Cajal at the deep muscular plexus are associated with loss of distention-induced burst-type muscle activity in mice infected by Trichinella spiralis.
        Am J Pathol. 2005; 167: 437-453
        • Ward S.M.
        • McLaren G.J.
        • Sanders K.M.
        Interstitial cells of Cajal in the deep muscular plexus mediate enteric motor neurotransmission in the mouse small intestine.
        J Physiol. 2006; 573: 147-159
        • Torihashi S.
        • Ward S.M.
        • Sanders K.M.
        Development of c-Kit-positive cells and the onset of electrical rhythmicity in murine small intestine.
        Gastroenterology. 1997; 112: 144-155
        • Torihashi S.
        • Nishi K.
        • Tokutomi Y.
        • et al.
        Blockade of kit signaling induces transdifferentiation of interstitial cells of Cajal to a smooth muscle phenotype.
        Gastroenterology. 1999; 117: 140-148
        • Iino S.
        • Horiguchi K.
        • Nojyo Y.
        Wsh/Wsh c-Kit mutant mice possess interstitial cells of Cajal in the deep muscular plexus layer of the small intestine.
        Neurosci Lett. 2009; 459: 123-126
        • Chi P.
        • Chen Y.
        • Zhang L.
        • et al.
        ETV1 is a lineage-specific survival factor in GIST and cooperates with KIT in oncogenesis.
        Nature. 2010; 467: 849-853
        • Klüppel M.
        • Huizinga J.D.
        • Malysz J.
        • et al.
        Developmental origin and kit-dependent development of the interstitial cells of cajal in the mammalian small intestine.
        Dev Dyn. 1998; 211: 60-71
        • Han J.
        • Shen W.-H.
        • Jiang Y.-Z.
        • et al.
        Distribution, development and proliferation of interstitial cells of Cajal in murine colon: an immunohistochemical study from neonatal to adult life.
        Histochem Cell Biol. 2010; 133: 163-175
        • Pellegrini M.S.F.
        Morphogenesis of the special circular muscle layer and of the interstitial cells of Cajal related to the plexus muscularis profundus of mouse intestinal muscle coat.
        Anat Embryol (Berl). 1984; 169: 151-158
        • Gur G.
        • Rubin C.
        • Katz M.
        • et al.
        LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation.
        EMBO J. 2004; 23: 3270-3281
        • Laederich M.B.
        • Funes-Duran M.
        • Yen L.
        • et al.
        The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases.
        J Biol Chem. 2004; 279: 47050-47056
        • Powell A.E.
        • Wang Y.
        • Li Y.
        • et al.
        The Pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor.
        Cell. 2012; 149: 146-158
        • Jensen K.B.
        • Collins C.A.
        • Nascimento E.
        • et al.
        Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis.
        Cell Stem Cell. 2009; 4: 427-439
        • Nakamura T.
        • Hamuro J.
        • Takaishi M.
        • et al.
        LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis.
        J Clin Invest. 2014; 124: 385-397
        • Poulin E.J.
        • Powell A.E.
        • Wang Y.
        • et al.
        Using a new Lrig1 reporter mouse to assess differences between two Lrig1 antibodies in the intestine.
        Stem Cell Res. 2014; 13: 422-430
        • Wirth A.
        • Benyó Z.
        • Lukasova M.
        • et al.
        G12-G13−LARG−mediated signaling in vascular smooth muscle is required for salt-induced hypertension.
        Nat Med. 2008; 14: 64-68
        • Srinivas S.
        • Watanabe T.
        • Lin C.-S.
        • et al.
        Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.
        BMC Dev Biol. 2001; 1: 4
        • Miller M.S.
        • Galligan J.J.
        • Burks T.F.
        Accurate measurement of intestinal transit in the rat.
        J Pharmacol Methods. 1981; 6: 211-217
        • Suzuki Y.
        • Sato N.
        • Tohyama M.
        • et al.
        cDNA Cloning of a novel membrane glycoprotein that is expressed specifically in glial cells in the mouse brain LIG-1, a protein with leucine-rich repeats and immunoglobulin-like domains.
        J Biol Chem. 1996; 271: 22522-22527
        • Gomez-Pinilla P.J.
        • Gibbons S.J.
        • Bardsley M.R.
        • et al.
        Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract.
        Am J Physiol Gastrointest Liver Physiol. 2009; 296: G1370-G1381
        • Faussone-Pellegrini M.-S.
        Relationships between neurokinin receptor-expressing interstitial cells of Cajal and tachykininergic nerves in the gut.
        J Cell Mol Med. 2006; 10: 20-32
        • Iino S.
        • Horiguchi K.
        • Horiguchi S.
        • et al.
        c-Kit-negative fibroblast-like cells express platelet-derived growth factor receptor α in the murine gastrointestinal musculature.
        Histochem Cell Biol. 2009; 131: 691-702
        • Iino S.
        • Nojyo Y.
        Immunohistochemical demonstration of c-Kit-negative fibroblast-like cells in murine gastrointestinal musculature.
        Arch Histol Cytol. 2009; 72: 107-115
        • Furness J.B.
        The enteric nervous system and neurogastroenterology.
        Nat Rev Gastroenterol Hepatol. 2012; 9: 286-294
        • Sanders K.M.
        • Koh S.D.
        • Ro S.
        • et al.
        Regulation of gastrointestinal motility—insights from smooth muscle biology.
        Nat Rev Gastroenterol Hepatol. 2012; 9: 633-645
        • Huizinga J.D.
        • Lammers W.J.E.P.
        Gut peristalsis is governed by a multitude of cooperating mechanisms.
        Am J Physiol Gastrointest Liver Physiol. 2009; 296: G1-G8
        • Yin J.
        • Chen J.D.Z.
        Roles of interstitial cells of Cajal in regulating gastrointestinal motility: in vitro versus in vivo studies.
        J Cell Mol Med. 2008; 12: 1118-1129
        • Wong V.W.Y.
        • Stange D.E.
        • Page M.E.
        • et al.
        Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling.
        Nat Cell Biol. 2012; 14: 401-408