The African clawed frog, Xenopus laevis, has a ZZ/ZW-type sex-determination system. We previously reported that a W-linked gene, Dm-W, can determine development as a female. However, the mechanisms of early sex differentiation remain unclear. We used microarrays to screen for genes with sexually dimorphic expression in ZZ and ZW gonads during early sex differentiation in X laevis and found several steroidogenic genes. Importantly, the steroid 17α-hydroxylase gene Cyp17a1 and the aromatase gene Cyp19a1 were highly expressed in ZZ and ZW gonads, respectively, just after sex determination. At this stage, we found that Cyp17a1, Cyp19a1, or both were expressed in the ZZ and ZW gonads in a unique mass-in-line structure, in which several masses of cells, each surrounded by a basement membrane, were aligned along the anteroposterior axis. In fact, during sex differentiation, ovarian cavities formed inside each mass of Cyp17a1- and Cyp19a1-positive cells in the ZW gonads. However, the mass-in-line structure disappeared during testicular development in the ZZ testes. These results suggested that the mass-in-line structure found in both ZZ and ZW gonads just after sex determination might be formed in advance to produce ovarian cavities and then oocytes. Consequently, we propose a view that the default sex may be female in the morphological aspect of gonads in X laevis.

Elucidation of the genes regulating the critical (sensitive) period of imprinting behavior may shed light on the mechanism underlying neural plasticity in early childhood learning. We focused on the family of natriuretic peptides (NPs) as candidates involved in the regulation of the critical period. In avians, several structurally related molecules comprised the NP family, including renal NP (RNP), B-type NP (BNP) and C-type NP (CNP1, CNP3 and CNPP). To understand the functional roles of NPs in neural plastic changes, we aimed to characterize NPs and their receptors in chick brain. We found that CNP3 mRNA was expressed in several regions in the telencephalon, including the visual Wulst (VW, considered as mammalian visual cortex) and amygdala. CNP1 mRNA was expressed throughout the telencephalon. Using real-time PCR, the gene expression levels of NPs and their receptors (NPR1 and NPR2) were studied during and after the critical period of imprinting (post-hatching day [P]1 and P7). CNP3 mRNA was found to show higher expression in the VW of P1 chicks than in VW of P7 chicks. Moreover, the ability of these peptides to stimulate chicken NPR1 or NPR2 was tested in HEK293 cells expressing either of the receptors. The activation of NPR1 was stronger with CNP3 than with other subtypes of CNP. In the VW, CNP3-expressing cells were negative for NPR1, but they resided in the vicinity of NPR1-expressing cells. These results suggest that CNP3 and its receptor NPR1 in the VW may have functional roles in the early learning.

The transcription factor DMRT1 (doublesex and mab-3 related transcription factor) has two distinct functions, somatic-cell masculinization and germ-cell development in some vertebrate species, including mouse and the African clawed frog Xenopus laevis. However, its transcriptional regulation remains unclear. We tried to identify DMRT1-interacting proteins from X. laevis testes by immunoprecipitation with an anti-DMRT1 antibody and MS/MS analysis, and selected three proteins, including PACT/PRKRA (Interferon-inducible double-stranded RNA dependent protein kinase activator A) derived from testes. Next, we examined the effects of PACT/PRKRA and/or p53 on the transcriptional activity of DMRT1. In transfected 293T cells, PACT/PRKRA and p53 significantly enhanced and repressed DMRT1-driven luciferase activity, respectively. We also observed that the enhanced activity by PACT/PRKRA was strongly attenuated by p53. Moreover, in situ hybridization analysis of Pact/Prkra mRNA in tadpole gonads indicated high expression in female and male germline stem cells. Taken together, these findings suggest that PACT/PRKRA and p53 might positively and negatively regulate the activity of DMRT1, respectively, for germline stem cell fate.

Tracheal stenosis is a refractory and recurrent disease induced by excessive cell proliferation within the restricted tracheal space. We investigated the role of extracellular signal-regulated kinase (ERK), which mediates a broad range of intracellular signal transduction processes in tracheal stenosis and the therapeutic effect of the MEK inhibitor which is the upstream kinase of ERK. We histologically analyzed cauterized tracheas to evaluate stenosis using a tracheal stenosis mouse model. Using Western blot, we analyzed the phosphorylation rate of ERK1/2 after cauterization with or without MEK inhibitor. MEK inhibitor was intraperitoneally injected 30 min prior to cauterization (single treatment) or 30 min prior to and 24, 48, 72, and 96 hours after cauterization (daily treatment). We compared the stenosis of non-inhibitor treatment, single treatment, and daily treatment group. We successfully established a novel mouse model of tracheal stenosis. The cauterized trachea increased the rate of stenosis compared with the normal control trachea. The phosphorylation rate of ERK1 and ERK2 was significantly increased at 5 min after the cauterization compared with the normal controls. After 5 min, the rates decreased over time. The daily treatment group had suppressed stenosis compared with the non-inhibitor treatment group. p-ERK1/2 activation after cauterization could play an important role in the tracheal wound healing process. Consecutive inhibition of ERK phosphorylation is a potentially useful therapeutic strategy for tracheal stenosis.