Volume 141, Issue 2, Pages 576-587.e6 (August 2011) Expression Level of Hand2 Affects Specification of Enteric Neurons and Gastrointestinal Function in Mice  Fabien D'Autréaux, Kara G. Margolis, Jane Roberts, Korey Stevanovic, Gary Mawe, Zhishan Li, Nima Karamooz, Ankur Ahuja, Yuka Morikawa, Peter Cserjesi, Wanda Setlick, Michael D. Gershon  Gastroenterology  Volume 141, Issue 2, Pages 576-587.e6 (August 2011) DOI: 10.1053/j.gastro.2011.04.059 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Hand2+/− and Hand2LoxP/− mice are hypomorphic. (A−D) Real-time PCR was used to quantify transcripts encoding Hand2, HuB, HuC, and HuD in the gut of Hand2+/+, Hand2LoxP/+, Hand2+/−, and Hand2LoxP/− mice at E13. (A) The abundance of transcripts encoding Hand2 in the gut of Hand2+/+ mice > Hand2LoxP/+ mice > Hand2+/− mice > Hand2LoxP/− mice. (B) No significant differences are found in abundance of enteric transcripts encoding HuB in Hand2+/+, Hand2LoxP/+, Hand2+/−, and Hand2LoxP/− mice. (C) The abundance of enteric transcripts encoding HuC is significantly decreased in Hand2LoxP/− mice. (D) The abundance of enteric transcripts encoding HuD in Hand2+/+ > Hand2LoxP/+ > Hand2+/− > Hand2LoxP/−. Each parameter (A−D) was analyzed in 3 mice of each genotype. Gastroenterology 2011 141, 576-587.e6DOI: (10.1053/j.gastro.2011.04.059) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Neuronal numbers are reduced in the ENS of Hand2+/− mice. HuD immunoreactivity in the myenteric plexus of the colon of (A) a Hand2+/+ mouse and (B) a Hand2+/− littermate. (C–F) Numbers of HuD-immunoreactive neurons were quantified and compared in Hand2+/+ and Hand2+/− bowel as a function of ganglia and area (C). Significantly more neurons are found in the myenteric plexus of the Hand2+/+ than in the Hand2+/− gut, both counted as neurons per ganglia and neurons/mm2 (D). Neurons are significantly smaller in Hand2+/− than in the Hand2+/+ colon. (E, F) The relative abundance of neuritis and connectives, evaluated by β3 tubulin immunoreactivity, is lower in the myenteric plexsus of the Hand2+/− than in the Hand2+/+ mice. Gastroenterology 2011 141, 576-587.e6DOI: (10.1053/j.gastro.2011.04.059) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 nNOS+ neurons are Hand2-dependent. (A−F) The density (pixels/mm gut length, normalized to the density of Hu+ neurons) of nNOS+ neurons was analyzed in the intestine of Hand2+/+, Hand2LoxP/+, Hand2+/−, and Hand2LoxP/− mice at E17. (A) Although the density of nNOS+ neurons in Hand2LoxP/+ was similar to that of Hand2+/+ controls, nNOS+ neuronal density was significantly reduced in Hand2+/−, and Hand2LoxP/− fetuses. (Bi−Biv) The myenteric plexus, doubly immunostained to demonstrate nNOS (red) and Hu immunoreactivities (green). (Bi) Hand2+/+. (Bii) Hand2LoxP/+. (Biii) Hand2+/−. (Biv) Hand2LoxP/−. The fluorescence of nitrergic neurons is yellow because antibodies to Hu and nNOS doubly immunostain these cells. The proportion of nNOS+ neurons to Hu+ neurons is about the same in Hand2+/+ and Hand2LoxP/+ gut but is reduced in Hand2+/− and especially in Hand2LoxP/− bowel. (C, D) The density of nNOS immunoreactivity in Hand2LoxP/+ and Hand2LoxP/− mice, normalized to that of Hu, was compared in the stomach, proximal small intestine (SI prox), distal small intestine (SI dist), cecum, and colon. (C) The myenteric plexus, doubly immunostained to demonstrate nNOS (red) and Hu (green) is illustrated for each of the regions analyzed in (D). The regional specificity of effect of genotype on nNOS+ neuronal density is particularly evident in the panel illustrating the distal small intestine and cecum of the Hand2LoxP/− mouse where a loop from each of these regions from the same animal appear in a single section; nNOS+ neurons are virtually absent in the distal small intestine but abundant in the cecum. (D) Quantitation of the ratios of the regional density of nNOS to Hu immunoreactivities in the bowel of Hand2LoxP/+ and Hand2LoxP/− mice. The nNOS+ neuronal density in the stomach and cecum was similar in Hand2LoxP/+ and Hand2LoxP/− fetuses; however, in SI prox, SI dist, and colon the nNOS+ neuronal density in Hand2LoxP/+ was significantly greater than that in Hand2LoxP/−. (E) The density of Substance P (SP) immunoreactivity/mm length of bowel in Hand2LoxP/+ proximal small intestine does not differ significantly from that in the gut of E17 Hand2LoxP/− mice. (F) The ratios of the densities of SP to nNOS immunoreactivity were measured in proximal small intestine. Because of the decrease in nNOS without a corresponding decrease in SP, the SP/nNOS ratio is significantly greater in Hand2LoxP− than Hand2LoxP+ animals. Each parameter (A−D) was analyzed in 3 mice of each genotype. Five additional Hand2LoxP+ and 4 Hand2LoxP/− animals were analyzed in (E, F). Scale bars = 50 mm. Gastroenterology 2011 141, 576-587.e6DOI: (10.1053/j.gastro.2011.04.059) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Regional effects of Hand2 haploinsufficiency on enteric calretinin+ neurons and nNOS+ are similar. Representative sections are shown at low magnification to facilitate comparisons between nNOS+ and calretinin+ neurons at various levels of the E18 bowel of Hand2LoxP/+ (Ai, Aiii, Bi, Biii, Ci, Ciii, Di, Diii) and Hand2LoxP/− (Aii, Aiv, Bii, Biv, Cii, Civ, Dii, Div) mice. Two mice of each genotype are illustrated. (A) Stomach. Numbers of nNOS+ and calretinin+ neurons are similar in Hand2LoxP/+ (Ai, Aiii) and Hand2LoxP/− (Aii, Aiv) mice. (B) Cecum. Numbers of nNOS+ and calretinin+ neurons are similar in Hand2LoxP/+ (Bi, Biii) and Hand2LoxP/− (Bii, Biv) mice. The section in (Civ) contains a segment of colon, which can be compared to the segment of cecum in the same field of view. There is little calretinin immunoreactivity in the colon, but calretinin immunoreactivity is prominent in the cecum of the same Hand2LoxP/− animal. (C) Small intestine. The immunoreactivities of nNOS and calretinin are prominent in the developing myenteric plexus in Hand2LoxP/+(Ci, Ciii) mice, but deficient in that of Hand2LoxP/− (Cii, Civ) littermates. (D) Colon. nNOS and calretinin immunoreactivities are prominent in the developing myenteric plexus of Hand2LoxP/+(Di, Diii) mice, but lacking in that of Hand2LoxP/− (Dii, Div) mice. (Div) Contains cecum, which can be compared to colon in the same field. Calretinin immunoreactivity is evident in the cecum, but lacking in the colon of the same Hand2LoxP/− animal. Bars = 50 μm. Gastroenterology 2011 141, 576-587.e6DOI: (10.1053/j.gastro.2011.04.059) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Little or no apoptosis of myenteric neurons is seen in either Hand2+/+ or Hand2+/− mice. Laminar whole mounts of longitudinal muscle with adherent myenteric plexus were prepared from the bowel of 3-week-old Hand2+/+ and Hand2+/− mice. The TUNEL procedure (red) was carried out to detect apoptosis in neurons identified as Hu+ cells (green). DNA was stained with bisbenzimide (blue). (Ai−Aiv) A field from a Hand2+/+ mouse, imaged to show the TUNEL reaction (Ai), DNA (Aii), Hu immunoreactivity (Aiii), and merged images (Aiv). No structures show TUNEL staining (Ai); therefore, no red fluorescence appears in the merged image (Aiv). The blue fluorescence of smooth muscle nuclei (Aii) forms a bed to which the myenteric ganglia adhere (Aiv). (Bi, Bii) A field from a Hand2+/− mouse, imaged to show the TUNEL reaction (Bi), DNA (Bii), Hu immunoreactivity (Biii), and the merged images (Biv). Again, no structures show TUNEL staining (Bi) and no red fluorescence appears in the merged image (Biv). More neurons are evident in the ganglia of Hand2+/+ mice (Aiii, Aiv) than in those of Hand2+/− animals (Biii, Biv). (C) The preparation was treated with DNAase to provide a positive control for the TUNEL reaction. A high-power merged image is shown in which the DNA fragmentation is made evident by the superimposed merged red fluorescence of the TUNEL reaction and the blue fluorescence of DNA. Hu+ neurons are marked by their green fluorescence. (D) Terminal transferase was omitted to provide a negative control for the TUNEL reaction. A merged image is shown at the same magnification as that of the positive control (C). There is no red fluorescence, therefore, nuclei display unmodified blue fluorescence in the merged image. Again, Hu+ neurons are marked by their green fluorescence. (E) A high-power merged image of a myenteric ganglion from a Hand2+/+ mouse subjected to the TUNEL procedure. No red fluorescence indicative of DNA fragmentation has been produced by the TUNEL assay. (F) A high-power merged image of myenteric ganglion from a Hand2+/− mouse subjected to the TUNEL procedure. Again, no red fluorescence indicative of apoptosis has been produced by the TUNEL assay. The markers (A, B) = 100 μm; (C−F) = 20 μm. Gastroenterology 2011 141, 576-587.e6DOI: (10.1053/j.gastro.2011.04.059) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Hand2 haploinsufficiency slows total gastrointestinal transit and colonic motility. (A) Total gastrointestinal transit time in Hand2+/− mice is significantly greater than that of their Hand2+/+ littermates. (B) Colonic motility in Hand2+/− mice is significantly slower than in their Hand2+/+ littermates. n = 4 (Hand2+/+), n = 4 (Hand2+/−). (C, D) The neurochemistry of inhibitory junction potentials from circular muscle in Hand2+/+ mice is different from that in Hand2+/− animals. (C) Intracellular recordings (above) and graph (below) demonstrating that in Hand2+/+ mice, the NOS inhibitor Nω-nitro-l-arginine (LNNA) did not affect the IJP; nevertheless, the IJP was almost completely eliminated by apamin, which inhibits purinergic neuromuscular signals. (B) Intracellular recordings (above) and graph (below) demonstrating that LNNA significantly reduced the amplitude of the IJP in Hand2+/− mice and the IJP was eliminated in the presence of LNNA plus apamin. Under basal conditions, the amplitude of the IJP was approximately the same in Hand2+/+ and +/− mice. *P < .01 vs basal; **P < .0001 vs basal. Gastroenterology 2011 141, 576-587.e6DOI: (10.1053/j.gastro.2011.04.059) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Histological signs of inflammation are greater in Hand2+/+ than Hand2+/− mice. TNBS was used to induce colitis in Hand2+/+ and Hand2+/− mice. The colons were fixed 7 days later for histological evaluation. (A) Hand2+/+ colon. Note the presence of mucosal ulceration and infiltration of the underlying tissue with leukocytes. (B) Hand2+/− colon. Ulceration is absent and only moderate edema and leukocyte infiltration are evident. (C) The severity of inflammation-induced tissue damage was quantified by assignment of histological scores on a scale of 0 to 3. The total score and component scores for ulceration, crypt damage, and leukocyte infiltration are illustrated. Total scores and each of the component scores were significantly higher in Hand2+/+ (green) than in Hand2+/− mice (red). *P < .01; **P < .05. Gastroenterology 2011 141, 576-587.e6DOI: (10.1053/j.gastro.2011.04.059) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Neurites and connectives are denser in the myenteric plexus of Hand2+/+ than in Hand2+/− mice. Acetylcholinesterase (AChe) was demonstrated histochemically in 6-week-old Hand2+/+ (A) and Hand2+/− mice (B). Note the primary and secondary elements of the myenteric plexus are thinner in Hand2+/− than in Hand2+/+ mice. Note also that the space between ganglia and connective is larger in the Hand2+/− animals. Marker = 100 μm. Gastroenterology 2011 141, 576-587.e6DOI: (10.1053/j.gastro.2011.04.059) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 More neurons can be visualized with antibodies to β3-tubulin in myenteric ganglia of Hand2+/+ than in that of Hand2+/− mice; however, no decrease in numbers of glia can be detected in the Hand2+/− animals. (A) Neurons visualized in a myenteric ganglion of a Hand2+/+ mouse. Note that the cell bodies are well-filled with β3-tubulin immunoreactivity and many are large in diameter. (B) Neurons visualized in a myenteric ganglion of a Hand2+/− mouse. Note that the cell bodies are not well-filled with β3-tubulin immunoreactivity and are smaller in diameter than their counterparts in a Hand2+/+ mouse [compare with (A)]. (C) Glia visualized in a myenteric ganglion of a Hand2+/+ mouse. Note that the cell bodies are well-outlined with glial fibrillary acidic protein (GFAP) immunoreactivity. (D) Glia visualized with GFAP immunoreactivity in a myenteric ganglion of a Hand2+/− mouse. Glia in Hand2+/+ and Hand2+/− mice are similar in size and shape. The markers (A−D) = 25 μm. (E) Numbers of β3-tubulin-immunoreactive myenteric neurons are significantly greater in Hand2+/+ than in Hand2+/− mice. (F) Numbers of GFAP-immunoreactive myenteric glia are not significantly different in Hand2+/+ from those in Hand2+/− mice. (G) The glia to neuron ratio is significantly greater in Hand2+/− than in Hand2+/+ mice. Gastroenterology 2011 141, 576-587.e6DOI: (10.1053/j.gastro.2011.04.059) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 The numbers of neurons expressing nNOS or calretinin immunoreactivities in 6- to 8-week-old mice are significantly greater in Hand2+/+ than in Hand2+/− mice. The severity of the difference is about the same in the submucosal and myenteric plexuses. Neurons were visualized immunocytochemically and counted in whole mounts of laminar preparations of submucosa (above) and longitudinal muscle with attached myenteric plexus (below). Gastroenterology 2011 141, 576-587.e6DOI: (10.1053/j.gastro.2011.04.059) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 VIP is expressed in neurons of the Hand2LoxP/− bowel despite its deficiency in nNOS. (A, B) In situ hybridization was used to locate transcripts encoding VIP in E18 Hand2LoxP/+ (A) and Hand2LoxP/− gut (B). VIP transcripts were identified in ganglia of the primordial myenteric plexus in both types of mouse. The number of VIP-expressing neurons in Hand2LoxP/− mice is not less than that in Hand2LoxP/+ animals. The immunoreactivities of VIP (red fluorescence) and nNOS (blue fluorescence) and coincident in Hand2LoxP/+ mice (A, inset) but VIP occurs in neurons that lack nNOS in Hand2LoxP/− animals (B, inset). Cells containing VIP and/or nNOS were identified as neurons by their coincident expression of the immunoreactivity of HuD (green fluorescence). (C, D) In situ hybridization was used to identify VIP-expressing neurons, which were quantified along with nNOS in the proximal small intestines of the same animals. Because the numbers of VIP-expressing neurons were not significantly different in Hand2LoxP/+ and Hand2LoxP/− mice (C), the significant decrease in nNOS-immunoreactive neurons in Hand2LoxP/− mice (C) caused the VIP/nNOS ratio to be significantly greater in Hand2LoxP/− than in Hand2LoxP/+ animals (D). Markers A and B = 250 μm; insets = 20 μm. Gastroenterology 2011 141, 576-587.e6DOI: (10.1053/j.gastro.2011.04.059) Copyright © 2011 AGA Institute Terms and Conditions