Int J Colorectal Dis (2010) 25:1231–1242 R Fig. 5 a–m Series of sagittal sections through the anorectum of a 25-week- old human fetus. a-d RZ, e–g ARZ, h–j ATZ and DL, k–m SZ and PS with the fate of the cloaca and the cloacal membrane— mainly contributed to the debate of the entodermal or ectodermal origin of the different epithelia [22–24]. All these publications have the same weakness: they are only based on normal HE histology. Thus, the results are purely descriptive, mostly speculative and therefore, in regard to their contents, without any connection to new molecular biological findings in laboratory animals or to clinical pathological aspects. To be able to gain new aspects as to the heterogeneity of the different anorectal epithelia, we are the first to include immunohistochemistry of different cytokeratins and of CDX2 expression in all human prenatal stages. Based on Fenger's [4] clearly arranged subdivision of the anorectum into five epithelial zones, the results of our study can be summarized as follows: As everywhere in the large intestine, the rectal mucosa has transitional folds in early fetal life [25]. Starting in the eighth week, the epithelium of the RZ is positive for CK 18 and CDX2 during all following stages of human development, and for CK 20 from the tenth week onwards. Rectal CK 7 positivity is obvious only in the ARZ where CK 20 and CDX2 staining vanishes. It is the most important finding that the ARZ already exists in prenatal life. This is why the ARZ must be considered as an area of epithelial changes and not as a metaplastic one. It differs from the RZ as to CK 7 expression. The epithelium of the ATZ mostly consists of stratified cuboidal epithelium in prenatal stages [24] and is characterized by positive labelling for CK 7. Anal valves are covered with squamous epithelium and thus show CK 14-positive reaction. According to our results and to former studies [5, 26], the rectal columns differentiate in the late embryonic and early fetal period and arise from the entoderm [10, 22]. The height of the columns is covered with non-keratinizing stratified squamous epithelium only in the postnatal stages as was already shown by Hollinshead [27]. Both anal stratified epithelia are positive for CK18 and CK7 in early stages before gaining CK14 positivity from the ninth week onwards. Our results clearly show that cell differentiation in the anorectal region does occur craniocaudally, regardless of where the cells take their origin from, and this is also true for the differentiation of the anal squamous epithelium. This backs up the results of our last study where we supposed that “an ingrowth of surface ectoderm is not evident … and that the epithelial differentiation is a locally organized process and induced by the interaction with the mesenchyme …” [10]. Contrary to another study [28] whose authors only elaborate on the introversive transfer of the epithelium and on that basis speculate that the epithelium is growing inwards from the outside, we are 1239 able to prove beyond doubt that the differentiation starts inside and proceeds externally. When comparing the results of the human fetuses and adults with the cases of low rectal and anal tumours, we expected to find a cytokeratin profile of the carcinoma cells that is similar to its native epithelium, i.e. we expected CK 18/CK 20- and CDX2-positive labelling in the low rectal carcinomas and CK 14 positivity in tumours deriving from the squamous epithelium of the anal canal below the DL. The rectal tumour probes we included in the present study showed the expected labelling for CK 18/20 and CDX2. In three cases we found an additional positivity for CK 7 which—in contrast to former studies postulating that the presence of CK 7 can be used to exclude a rectal origin of tumours and metastases—is not astonishing as the tumours are characterized as ultralow rectal ones that originate in the ARZ where positive labelling for CK 7 normally occurs. These important findings are in accordance with a recent study [29] which revealed that CK 7 can be expressed in rectal adenocarcinomas, and that the combination of CK 20/7 with CDX2 is an excellent one securely to identify metastatic rectal carcinomas. This might be helpful in discriminating rare anal malignant conditions like adeno- carcinoma of the anal canal from the more common squamous cell carcinoma on the one hand and ultralow adenocarcinoma of the rectum on the other hand. Adeno- carcinoma of the anal canal is rare and there is often diagnostic doubt between true anal canal cancer, most likely arising from the anal glands that develop from the rectal epithelium [10] and direct invasion from very low rectal cancers [30]. Changes in the expression of certain CKs provide an indication of the differentiation stage (dedifferentiation) of a tumour which is comparable to prenatal stages of differentiation and the respective expres- sion pattern [31]. Our study successfully aimed to show that the demarca- tion line between the five epithelial zones is more distinct in some places than in others: whereas it is rather transient between the RZ and the ARZ, on the DL it is clearly delimited in all stages of development. Below the DL the demarcation between the keratinizing stratified squamous epithelium of the anal canal and the non-keratinizing stratified squamous epithelium of the rectum is clearly visible from the 16th week of development. According to our qualitative results, the extent of the different anorectal sections, especially of the ATZ, is variable and differs interindividually [4]. Therefore, in the case of a tumour, it is most important to document the exact location macroscopi- cally as well as microscopically. CDX2 discontinues to be expressed at the borderline between the RZ and the ATZ, i.e. in the ARZ. Cranial to the ARZ, transitional folds—typical for the intestinal identity— appear in early fetal life. Thus, up to this line, the intestine