No abstract

Most gastrointestinal tract and associated gland epithelia originate from the endoderm germ layer discovered by Pander in 1817. The recent surge in stem cell concepts revived interest in the findings of 30 years ago that the endoderm layer itself originates from the epiblast (which since Pander’s time had been held to be the forerunner of the ectoderm and mesoderm germ layers only). However, the question as to which parts of the mammalian gastrulation-stage embryonic disc generate endoderm cells is still unresolved. Therefore, the expression of the gene coding for the transcription factor Sox17, a key transcription factor involved in endoderm formation in mouse, chick, frog, and zebrafish, was analyzed in the rabbit, a model organism for mammalian gastrulation morphology, using whole-mount in situ hybridization and high-resolution histological analysis of embryos at gastrulation and early neurulation stages. Sox17 mRNA in the mesoderm and lower layer (hypoblast) compartments within and adjacent to Hensen’s node and the anterior segment of the primitive streak confirmed the validity of this approach, as this region had previously been shown to form endoderm in mouse and chick. However, Sox17 expression in central and posterior epiblast at pregastrulation stages together with a transient expression at the posterior extremity of the primitive streak suggest that endoderm (possibly hindgut) may be formed close to the emerging cloacal membrane, as well.

Relative to recent advances in understanding molecular requirements for endoderm differentiation, the dynamics of germ layer morphology and the topographical distribution of molecular factors involved in endoderm formation at the caudal pole of the embryonic disc are still poorly defined. To discover common principles of mammalian germ layer development, pig and rabbit embryos at late gastrulation and early neurulation stages were analysed as species with a human-like embryonic disc morphology, using correlative light and electron microscopy. Close intercellular contact but no direct structural evidence of endoderm formation such as mesenchymal-epithelial transition between posterior primitive streak mesoderm and the emerging posterior endoderm were found. However, a two-step process closely related to posterior germ layer differentiation emerged for the formation of the cloacal membrane: (i) a continuous mesoderm layer and numerous patches of electron-dense flocculent extracellular matrix mark the prospective region of cloacal membrane formation; and (ii) mesoderm cells and all extracellular matrix including the basement membrane are lost locally and close intercellular contact between the endoderm and ectoderm is established. The latter process involves single cells at first and then gradually spreads to form a longitudinally oriented seam-like cloacal membrane. These gradual changes were found from gastrulation to early somite stages in the pig, whereas they were found from early somite to mid-somite stages in the rabbit; in both species cloacal membrane formation is complete prior to secondary neurulation. The results highlight the structural requirements for endoderm formation during development of the hindgut and suggest new mechanisms for the pathogenesis of common urogenital and anorectal malformations.

transient expression at the posterior extremity of the primitive streak suggest that endoderm (possibly hindgut) may be formed close to the emerging cloacal membrane, as well.