Paired-related homeodomain proteins Prx1 and Prx2 are expressed in embryonic pituitary stem/progenitor cells and may be involved in the early stage of pituitary differentiation.

We recently cloned a paired-related homeodomain protein Prx2 as a novel factor in the pituitary. In the present study, we investigated the ontogenic profiles of Prx2 and another cognate Prx1 in the rat embryonic pituitary. Quantitative real-time polymerase chain reaction showed low expression of Prx2 and a marked increase of Prx1 on rat embryonic day (E)20.5. Immunohistochemical analyses using an antibody that recognises both proteins, with the aim of investigating their roles in pituitary organogenesis, demonstrated that PRXs first appear in the Rathke's pouch on E13.5 in the pituitary stem/progenitor cells expressing Prop1 and Sox2. After E16.5, the number of Prx-expressing cells was increased in both anterior and intermediate lobes. SOX2(+) stem/progenitor cells in the intermediate lobe started to produce PRXs, and PRX(+) /SOX2(+) /PROP1(+) -cells were present on the anterior side of the marginal cell layer and were scattered in the parenchyma of the anterior lobe. On the other hand, PRX(+) -cells negative for PROP1 and SOX2 were located in the anterior lobe. Analysis of the relationship with pituitary endocrine cells revealed that a part of PRX(+) /PROP1(-) /SOX2(-) -cells in the anterior lobe co-expressed all types of hormones. The proportion of co-localisation of PRXs and hormones was highest on the day each hormone first appeared. These data indicate that PRXs are produced in the pituitary progenitor cells and may play roles in the process of terminal differentiation during early pituitary organogenesis. An in vitro small interfering RNA-knockdown experiment in the pituitary-derived cell line, TtT/GF, revealed that PRX1 and PRX2 play roles in proliferation by different mechanisms because knockdown of Prx2, but not Prx1, induced the p21 expression. Furthermore, immunohistochemical analysis demonstrated that 76% of PRXs(+) cells were positive for a cell proliferation marker Ki67 in the E18.5 pituitary. This is the first report of the involvement of PRX1 and PRX2 in organogenesis of tissue originating from the ectoderm other than the mesoderm.