SOX10 is abnormally expressed in aganglionic bowel of Hirschsprung's disease infants

Abstract

Background: The primary pathology of Hirschsprung's disease (HD) is a congenital absence of ganglion cells in the caudal most gut. The spastic aganglionic bowel is often innervated by a network of hypertrophied nerve fibres. Recently, mutations of SOX10 have been identified in patients with HD but only in those with Waardenburg-Shah syndrome.

Aims: To understand the molecular basis for the pathogenesis of HD we intended to determine the specific cell lineages in the enteric nervous system which normally express SOX10 but are affected in disease conditions.

Methods: We studied colon biopsies from 10 non-syndromic HD patients, aged three months to four years, and 10 age matched patients without HD as normal controls. The absence of mutation in the SOX10 gene of HD patients was confirmed by DNA sequencing. Expression and cellular distribution of SOX10 in bowel segments of normal and HD infants were examined by reverse transcription-polymerase chain reaction and in situ hybridisation.

Results: We found that in normal infants and normoganglionic bowel segments of HD patients, SOX10 was expressed in both neurones and glia of the enteric plexuses and in the nerves among the musculature in normal colon. In the aganglionic bowel segments of patients, SOX10 expression was consistently lower and was found to be associated with the hypertrophic nerve trunks in the muscle and extrinsic nerves in the serosa.

Conclusion: We conclude that SOX10 is normally required postnatally in the functional maintenance of the entire enteric nervous system, including neurones and glia. In non-syndromic HD patients who do not have the SOX10 mutation, the SOX10 gene expressed in the sacral region may be involved in the pathogenesis of the abnormal nerve trunks through interaction with other factors.

Figure 1

Expression of SOX10 in both neurones and glia of enteric plexuses in the colon of normal infants. (A) Dark field illumination of a cross section of normal colon showing strong in situ hybridisation signals in myenteric plexuses and in the nerves in the longitudinal and circular muscular layers. (B) Bright field illumination of an adjacent section showing S100 immunoreactivity in the SOX10 positive cells. (C) Dark field illumination and (F, I) bright field illumination of a myenteric plexus hybridised in situ with a SOX10 probe. (D, G, J) Immunohistochemistry of an adjacent section with S100 antibody showing that the glial cells present in the plexus are stained. (E, H, K) Immunohistochemistry of another adjacent section with RET antibody showing that the neurones and nerves in the plexus are stained. For comparison, in I, J, and K, the same glial cells are marked with arrows and the neurones are circled by broken lines. Both glial cells immunoreactive for S100 and neurones immunoreactive for RET had positive SOX10 hybridisation signals. Original magnification: (A, B) ×100; (C, D, E) ×400; (F, G, H) ×500; (I, J, K) ×1000. Sm, submucosa; cm, circular muscle; lm, longitudinal muscle.

Figure 2

Expression of SOX10 in the aganglionic (A), hypoganglionic (H), and normoganglionic (N) colon segments of a Hirschsprung's disease (HD) patient examined by reverse transcription-polymerase chain reaction (RT-PCR) in the presence (+) and absence (−) of reverse transcriptase. Expression of SOX10 in normal control colon tissue (C) is also shown. The expected SOX10 fragment of about 250 bp was amplified from normal control and HD patient samples. As a control the β-actin band of about 300 bp was also amplified from all samples. No DNA fragment could be amplified from cDNA samples that lacked reverse transcriptase (−) indicating that the amplified bands were derived from mRNA.

Figure 3

Expression of SOX10 in the normoganglionic (A, B) and aganglionic (C-L) colon segments of a Hirschsprung's disease patient examined by in situ hybridisation. SOX10 is expressed in myenteric plexuses (arrowhead) and nerves in the circular and longitudinal muscles in the normoganglionic colon (A, bright field; B, dark field). In the aganglionic colon, SOX10 is expressed in the hypertrophic nerve trunks and in nerves in the musculature (C, K, bright field; D, L, dark field). Immunohistochemistry on adjacent sections using S100 (H, I) and RET (J) antibodies confirms that the hypertrophic nerves contain nerve fibres and glial cells but no neurones. The SOX10 positive signals are found in the glia and nerve bundles. In addition, SOX10 is also weakly expressed in extrinsic nerves (F, bright field; G, dark field; arrowhead, blood vessel) in the serosa. Immunohistochemistry on an adjacent section shows that the extrinsic nerve is immunoreactive for S100 antibody (E). Original magnification: (A-D) ×100; (E) ×200; (F, G, I-L) ×500; (H) ×100. Sm, submucosa; cm, circular muscle; lm, longitudinal muscle; s, serosa.