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Hirschsprung Disease: Exploring Its Causes, Consequences, and Novel Treatments

Massachusetts General
Allan Goldstein, MD

The enteric nervous system (ENS) is an intricate network of neurons and glial cells present in the wall of the intestine and responsible for regulating critical functions of the gastrointestinal (GI) tract, including peristalsis, absorption, and secretion. This intestinal “brain” contains as many neuronal cells and subtypes as the central nervous system (CNS), and is capable of autonomous function without CNS input. Given its complexity and importance, it is not surprising that abnormalities of ENS structure and function lead to serious neurointestinal diseases, including Hirschsprung disease, irritable bowel syndrome, chronic intestinal pseudo-obstruction, slow transit constipation, and esophageal achalasia, which together affect a large number of individuals. My primary research focus is on understanding the causes and consequences of congenital enteric neuropathies and on developing innovative approaches to their treatment.

The cells that comprise the ENS arise from the embryonic neural crest at the level of the hindbrain. These cells migrate to the foregut at week 4 of gestation, continue migrating proximo-distally along the entire length of the GI tract, and reach the distal end of the colon by week 7, subsequently maturing into two ganglionated plexus containing the neuronal and glial cell bodies that supply the gut (Fig. 1). Failure of these neural crest-derived cells to the reach the end of the intestine results in Hirschsprung disease, which is characterized by a variable length of distal aganglionosis. The disease typically presents in newborns as a bowel obstruction, leading to the failure to pass meconium. The diagnosis is made by identifying the absence of ganglion cells in a suction rectal biopsy, and treatment consists of resecting the aganglionic segment. Despite removing the aganglionic segment, however, complications of the disease are common, with at least 50% of patients suffering from severe constipation, diarrhea, fecal incontinence, or Hirschsprung-associated eneterocolitis (HAEC), which is the major cause of morbidity and mortality in this disease. Our research aims to understand the molecular mechanisms that cause Hirschsprung disease, to identify the consequences of aganglionosis and how they contribute to HAEC, and to apply these observations to designing novel treatments for Hirschsprung disease and its complications.

Development of the ENS: understanding the causes of distal aganglionosis

In order for neural crest-derived cells to populate successfully the entire GI tract with neurons and glial cells, they must proliferate extensively, migrate appropriately, and differentiate at the proper times into neuronal and glial subtypes. Using avian embryos, we have characterized the role of several signaling pathways during ENS development. Chick and quail embryos are excellent model systems for this work as they offer the unique opportunity to combine classic embryologic techniques with modern molecular methods, allowing us to manipulate the developing ENS at the cellular and genetic levels at any stage of development (Fig. 2). Despite the importance of normal ENS development to gut formation and function, the mechanisms regulating this process are poorly understood. We believe that characterizing the molecular and cellular process involved is essential to designing targeted treatments for human ENS disorders.

Endothelin-3(Et3) is a polypeptide present in the developing embryonic gut. It binds to its receptor, endothelin receptor B (Ednrb), which is expressed by migrating enteric neural crest-derived cells in the intestine. We found that inhibition of Et3 activity in chick embryos leads to colonic aganglionosis, as is observed in mice and humans with mutations in Et3 pathway. Using a variety of techniques in the avian embryo, we found that Et3 signaling promotes neural crest cell proliferation in the gut and keeps the cells in an undifferentiated state. As a result, inhibition of this pathway in the embryonic gut leads to insufficient cell proliferation and their premature differentiation into neurons, leaving the cells unable to complete their journey to the bottom of the gut. In addition to mechanistic data, this study also provided a useful model of human aganglionosis that we are exploiting to test potential treatment interventions aimed at enhancing the extent of ENS colonization

Perhaps the most important gene in Hirschsprung disease is Ret, a receptor tyrosine kinase that is mutated in the majority of patients with the disease. Ret is expressed by the neural crest cells in the gut, and its ligand (Gdnf, glial-derived neurotrophic factor) is expressed by the gut wall. We studied this pathway’s mechanism of action by injecting avian embryos with retrovirus vectors to overexpress the Gdnf gene or an siRNA designed to silence its expression. We found that Gdnf-Ret signaling is essentially for promoting the migration, proliferation, and differentiation of neural crest cells as they populate the developing gut. Moreover, Gdnf is also chemoattractive to neural crest cells, promoting their directional migration along the GI tract. This multifactorial role of the Gdnf-Ret pathway explains its essential function during ENS formation and the high prevalence of Ret mutations identified in human Hirschsprung disease.

Recently we identified a novel role for an extracellular matrix (ECM) protein in ENS development. Using a variety of techniques, including rat-chick coelomic explants, gut organ cultures, and neural crest migration assays, we found that tenascin-C, an ECM glycoprotein, is produced by enteric neural crest-derived cells in the gut and, furthermore, that it promotes the migration of these cells. This observation supports a novel mechanisms whereby neural crest cells are able to modify their own microenvironment through ECM expression and thereby help to regulate their own migration. Current studies are aimed at defining the roles of various other ECM components to establish the optimal microenvironmental milieu for these cells.

Hirschsprung-associated enterocolitis (HAEC): causes and possible treatments

HAEC is associated with significant mortality and morbidity, yet its pathogenesis remains unknown. Changes in the colonic epithelium related to goblet cells and the luminal mucus layer have been postulated to play a key role. We recently found that the colonic epithelium of both aganglionic and ganglionic segments are altered both in patients with Hirschsprung disease and in Endothelin receptor B knockout (Ednrb-/-) mice, a model of human Hirschsprung disease (Fig. 3). Structurally, goblet cells are altered with increased goblet cell number and reduced intracellular mucins in the distal colon of biopsies from Hirschsprung’s patients. Ednrb-/- mice have increased goblet cell number and size as compared to wild-type mice in the aganglionic segments, and reduced goblet cell size and number in the ganglionic segments. Functionally Ednrb-/- mice showed increased transepithelial resistance and reduced stool water content. Interestingly, goblet cell differentiation factors SPDEF and Math1 are increased in the distal colon of Ednrb-/- mice, suggesting that aganglionosis perturbs normal goblet cell maturation in the colonic epithelium. Both Ednrb-/- mice distal colon and biopsies from HD patients show reduced MUC4 expression when compared to normal controls. Further supporting these results, multiple-particle tracking studies show that mucus from Ednrb-/- mice exhibits significantly hindered diffusion of 200nm nanoparticles as compared to their wild-type littermates. These results suggest that aganglionosis is associated with increased goblet cell proliferation and differentiation and subsequent altered surface mucus properties, prior to any significant inflammation in the distal colonic epithelium in Hirschsprung disease. We believe that restoring normal goblet cell function and mucus layer properties in the colonic epithelium of children with Hirschsprung disease may be a viable therapeutic strategy for preventing HAEC.

Neuronal cell transplantation: a novel treatment approach for Hirschsprung disease

We are working on strategies to establish cell replacement therapy as a novel treatment approach for Hirschsprung disease and other conditions in which enteric neurons are either absent or abnormal. We have successfully isolated and expanded neuronal stem cells from the intestine of pre- and post-natal mice in vitro (Fig. 4). We have also transplanted these cells into the colon of wild-type mice and shown that they are able to survive, migrate, and differentiate into a functional neuronal network. This preliminary work, combined with that from other labs, supports the notion that intestinal biopsies from the ganglionated proximal intestine of patients with Hirschsprung disease can serve as a source of enteric neurons for autologous cell replacement. However, repopulating an entire aganglionic segment of human colon requires a large number of cells capable of migrating long distances. To achieve this, we are currently exploring approaches to modifying the neuronal stem cells in vitro by introducing viral vectors expressing Gdnf to promote their proliferation and migration, or co-transplanting them with serotonin, which both in vivo and in vitro has a significant neurogenic effect on these cells. Once we can generate a large population of transplanted cells that produce a dense neuroglial network within the gut wall, the next hurdle is to demonstrate improved colorectal function in the the transplanted mice using assays including fecal pellet counts, rectal bead expulsion time, and anorectal manometry. These studies promise to change the paradigm of how we currently treat infants with Hirschsprung disease and how we manage post-operative colorectal dysfunction.

Summary

The experiments described above will continue to further our understanding of ENS development, give us new insights into the etiology and pathophysiology of Hirschsprung disease and its complications, and lead to an improved understanding of the embryologic causes and clinical manifestations of other enteric neuropathies. We hope that through our work at MGHfC, and together with our collaborators within the institution and around the world, we will be able to offer new hope for children with these very challenging conditions.

References

 1. Doyle AM, Roberts DJ, Goldstein AM. Enteric nervous system patterning in the avian hindgut. Developmental Dynamics 2004; 229:708-712.

 2. Goldstein AM, Brewer KC, Doyle AM, Nagy N, Roberts DJ. BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system. Mechanisms of Development 2005; 122:821-833.

 3. de Santa Barbara P, Williams J, Goldstein AM, Doyle AM, Nielsen C, Winfield S, Faure S, Roberts DJ. BMP signaling pathway plays multiple roles during gastrointestinal tract development. Developmental Dynamics 2005; 234:312-322.

 4. Brewer KC, Mwizerva O, Goldstein AM. BMPRIA is a promising marker for evaluating ganglion cells in the enteric nervous system – a pilot study. Human Pathology 2005; 36:1120-1126.

 5. Nagy N, Goldstein AM. Endothelin-3 regulates neural crest cell proliferation and differentiation in the hindgut enteric nervous system. Developmental Biology 2006; 293:203-217.

 6. Nagy N, Goldstein AM. Intestinal coelomic transplants: a novel method for studying enteric nervous system development. Cell and Tissue Research 2006; 326:43-55.

 7. Nagy N, Brewer KC, Mwizerwa O, Goldstein AM. The pelvic plexus contributes ganglion cells to the hindgut enteric nervous system. Developmental Dynamics 2007; 236:73-83.

 8. Irani K, Rogriduez L, Doody DP, Goldstein AM. Botulinum toxin for the treatment of chronic constipation in children with internal anal sphincter dysfunction. Pediatric Surgery International 2008;24:779-783.

 9. Goldstein AM, Nagy N. A bird’s eye view of enteric nervous system development: lessons from the avian embryo. Pediatric Research 2008;64:326-333.

 10. Field HA, Kelley KA, Martell L, Goldstein AM, Serluca FC. Analysis of gastrointestinal physiology using a novel intestinal transit assay in zebrafish. Neurogastroenterology & Motility 2009;21:304-312.

 11. Nagy N, Mwizerwa O, Yaniv K, Carmel L, Pieretti-Vanmarcke R, Weinstein BM, Goldstein AM. Endothelial cells promote migration and proliferation of enteric neural crest cells via b1 integrin signaling. Developmental Biology 2009;330:263-272. (provided cover image for this issue)

 12. Christison-Lagay ER, Rodriguez L, Kurtz M, St. Pierre K, Doody DP, Goldstein AM. Antegrade colonic enemas and intestinal diversion are highly effective in the management of children with intractable constipation. Journal of Pediatric Surgery 2010;45:213-219.

 13. Rodriguez L, Flores A, Gilchrist BF, Goldstein AM. Laparoscopic-assisted percutaneous endoscopic cecostomy (LAPEC). Gastrointestinal Endoscopy 2011;73:98-102.

 14. Mwizerwa O, Das P, Nagy N, Akbareian SE, Mably JD, Goldstein AM. Gdnf is mitogenic, neurotrophic, and chemoattractive to enteric neural crest cells in the embryonic colon. Developmental Dynamics 2011;240:1402-1411.

 15. Rodriguez L, Irani K, Jiang H, Goldstein AM. Clinical presentation, response to therapy, and outcome of gastroparesis in children. Journal of Pediatric Gastroenterology & Nutrition 2012;55:185-190.

 16. Auksorius E, Bromberg Y, Motiejunaite R, Pieretti A, Liu L, Coron E, Aranda J, Goldstein AM, Bouma BE, Kazlauskas A, Tearney GJ. Dual-modality fluorescence and full-field optical coherence microscopy for biomedical imaging applications. Biomedical Optics Express 2012;3:661-666.

 17. Nagy N, Burns AJ, Goldstein AM. Immunophenotypic characterisation of enteric neural crest cells in the developing avian colorectum. Developmental Dynamics 2012;241:842-851

 18. Ward NL, Pieretti A, Dowd SE, Cox SB, Goldstein AM. Intestinal aganglionosis is associated with early and sustained disruption of the colonic microbiome. Neurogastroenterology & Motility 2012;24:874-e400.

 19. Coron E, Pieretti A, Auksorius E, Mahe MM, Liu L, Steiger C, Bromberg Y, Bouma B, Tearney G, Neunlist M, Goldstein AM. Full-field optical coherence microscopy (FFOCM) is a novel technique for imaging enteric ganglia in the gastrointestinal tract. Neurogastroenterology & Motility 2012;24:e611-621.

 20. Belkind-Gerson J, Goldstein AM, Kuo B. Balloon expulsion test as a screen for outlet obstruction in children with chronic constipation. Journal of Pediatric Gastroenterology & Nutrition 2013;56:23-26.

 21. Belkind-Gerson J, Carreon A, Benedict LA, Steiger C, Pieretti A, Nagy N, Dietrich J, Goldstein AM. Nestin-expressing cells in the gut give rise to enteric neurons and glial cells. Neurogastroenterology & Motility 2013;25:61-69.

 22. Goldstein AM, Hofstra RMW, Burns AJ. Building a brain in the gut: development of the enteric nervous system. Clinical Genetics 2013;83:307-316.

 23. Rodriguez L, Roberts LD, LaRosa J, Heinz N, Gerszten R, Nurko S, Goldstein AM. Relationship between postprandial metabolomics and colon motility in children with constipation. Neurogastroenterology & Motility 2013;25:420-426.

 24. Belkind-Gerson J,Surjanhata B, Kuo B, Goldstein AM. The bear-down maneuver is a useful adjunct in the evaluation of children with chronic constipation. Journal of Pediatric Gastroenterology & Nutrition 2013 (in press).

 25. Akbareian SE, Nagy N, Steiger CE, Mably JD, Miller SA, Hotta R, Molnar D, Goldstein, AM. Enteric neural crest-derived cells promote their migration by modifying their microenvironment through tenascin-C production. Developmental Biology 2013 (in press).

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