1232 dislocations, the position of the dentate line does not necessarily allow conclusions concerning developmental processes in the anorectum. Nevertheless, the border between ectodermal- and entodermal-derived anorectal epithelia is still assumed to lie in the area of the dentate line [7–9]. Recently we have revealed the fact that differentiation of the anorectal layers starts after disappear- ance of the cloacal membrane at the beginning of the seventh week post conceptionem (p.c.). Indications for an interaction between epithelium and mesenchymal structures were shown, whereas an ingrowth of surface ectoderm was not observed. The epithelial differentiation was described as a local process, probably induced by a cross-talk between epithelium and mesenchyme [10]. Up to now, specific markers for the very early embryonic ectodermal and entodermal epithelia are missing. The solution of this question may be a subject of cell biological and experimental studies. From the clinical point of view, however, it is of much more relevance to know which kind of tumour may derive from the different heights of the anorectum. Thus, to get new insights into the developmen- tal differentiation of the anorectal epithelia and probable similarities between the embryonic situation and tumour development in the adult, we used different cytokeratins (CKs) and CDX2 as markers for immunohistochemistry. The expression of cytokeratins is cell-type specific. During malignant transformation their specificity remains con- served in large parts [11–13]. Furthermore, the expression of cytokeratins is specific for different types of epithelial differentiation [14–16]. CK 7 and CK 18/20 are commonly described as markers for simple and columnar epithelium and thus believed to belong to entodermal-derived tissues, whereas CK 14 is a marker for squamous epithelium and thus considered to belong to ectodermal-derived tissue [17]. They have already been applied in several investigations concerning intestinal epithelia of the adult. In our study these cytokeratins were first examined in regard to their ability to demarcate different epithelial sections within the developing anorectum. The results were compared with the normal situation in the adult and with cases of both low rectal and anal carcinoma. CDX2 belongs to the family of parahox genes and encodes an intestine-specific transcription factor which is important for the regulation of proliferation and differenti- ation of intestinal epithelial cells [18]. It is expressed in the nuclei of entodermal-derived epithelial cells from duode- num to rectum. Previous studies showed CDX2 expression in normal colonic mucosa and in most colorectal adeno- carcinomas and metastases, but it is rarely seen in tumours of other sites [19–21]. In the present study, we examined CDX2 expression in the human anorectal epithelia during development, in the adult and in carcinomas of both the low rectal and anal region. Int J Colorectal Dis (2010) 25:1231–1242 It was the purpose of this study to create an actual basis for cellular origin and differentiation of the anorectal region including carcinomas. The hypothesis of the underlying study is that the prenatal situation indicates the origin of the different types of carcinomas in the adult anorectum. Materials and methods Differentiation and spatiotemporal distribution of the different epithelial sections of the anorectum were examined by means of conventional histology and immunohistochemistry using monoclonal antibodies directed against cytokeratins (CK) 18, 20, 7 and 14 as well as CDX2. Thirty-three human embryos and fetuses (8th–33rd week p.c.), sections of a 2-year-old child and of four adults (36 years old male, 65 years old male, 76 years old male and 74 years old female) were studied microscopically. The embryonic and fetal specimens were provided by the collection of the Department of Anatomy, Histology and Embryology, Innsbruck, or obtained from legal abortions collected from different gynecologists having received parental consent. The adult specimens were also taken from the Department's collection and had no intestinal disorders or malformations. Specimens were fixed in 7% formaldehyde in PBS (0.2 M). After paraffin embedding in a routine histological infiltration processor (Miles Scientific Inc., Naperville, IL, USA), specimens were cut in 4-μm sections in sagittal or axial planes by means of a Microm ERGO Star Rotations- microtome (Microm, Walldorf, Germany). The sections were dried overnight, dewaxed with xylene, rehydrated in graded alcohol series, and every 10th to 20th section of a series was stained with haematoxylin and eosin (HE). For immunohistochemical study the Discovery XT automated staining system by Ventana Medical Systems Inc. (Ventana, Strasbourg, France) was used. After dewax- ing and rehydrating, the antigen retrieval was started by heat (for CK 18, 20 and 14 staining) or protease (for CK 7 staining) induced epitope unmasking while the sections were immersed in citrate buffer. The slides were incubated with prediluted monoclonal antibodies recognizing CK 7, 14, 18, 20 (Linaris, Germany) for 60 minutes and biotinylated secondary antibody (universal, IGG antimouse, antirabbit) (Ventana, Strasbourg, France) for 30 minutes. Subsequently, the antigen–antibody complex was fixed with 0.05 % glutaraldehyde in 0.9% sodium chloride. Antibody binding sites were visualised by means of a 3.3-diaminobenzidine (DAB) detection kit (according to the manufacturer's protocols). For CDX2 staining, we used Histoprime® Cdx2, a monoclonal mouse antibody, ready-to-use clone Cdx2-88 (Linaris, Wertheim-Bettingen, Germany). Subsequent to