Expression pattern of the homeobox gene Hox-3.5 during mouse development, as revealed by a simplified in situ hybridization method

Improved procedures for a simplified in situ hybridization using paraffinembedded tissues and riboprobes were described. We found that when tissues were fixed with 4% paraformaldehyde and embedded in paraffin, harsh pretreatments so far employed, such as proteolytic, heat, or HCl treatment each damaging cell morphology to some degree, are not essential to expose mRNAs on the surface of the sections subjected. Typical results are shown for the detection of NGF and EGF mRNAs in mouse submandibular glands. Furthermore, we demonstrated that our method is also applicable to tissues decalcified with 5% EDTA, as shown in the detection of elastin mRNAs in the calcified tissues in the mouse oral region. We applied our method to determine expression patterns of a new mouse homeobox-containing gene, designated Hox-3.5. Hox-2.1 and Hox-1.3 in embryos. The Hox-3.5 gene is expressed intensively in the rhombencephalon and spinal cord of 12 day embryos, and in the three (at least) cervical ganglion near the occipital bone of 13 day embryos. In the 14 day old embryos, expression of the Hox-3.5 gene is spatially limited at least to the epithelium of both lung and intestine, derived from endoderm. These results suggest that spatial expression of the Hox-3.5 gene may play an important role in formation of ectodermal tissues in 12 day embryos and then in generation of the digestive and respiratory tubes from endoderm in 14 day embryos.