A possible role of Reproductive homeobox 6 in primordial germ cell differentiation

Abstract

Rhox6 is one of the Reproductive Homeobox genes on the X chromosome (Rhox) that is expressed in the placenta and the post-migratory primordial germ cells (PGCs) in the nascent gonad. Despite its novel expression pattern, the significance of Rhox6 expression in the differentiation of these cell types remains unknown. To investigate the role that Rhox6 plays in PGCs, cDNA encoding Rhox6 and short-hairpin (sh) RNA directed against Rhox6 transcripts were introduced by unique expression vectors into a genetically engineered mouse embryonic stem cell (ESC) line. This ESC line expresses enhanced green fluorescent protein (EGFP) under the Oct3/4 promoter, thereby allowing us to monitor the presence of undifferentiated ESCs and PGCs in culture in real time. This ESC line was used to isolate clones that stably expressed Rhox6 cDNA, shRNA against Rhox6 transcripts, or controls. Quantitative RT-PCR results validated that overexpression had been achieved, as well as knockdown of Rhox6 transcripts in these ESC clones. However, these clones exhibited a normal appearance of undifferentiated ESCs and expressed EGFP. Next, these ESC clones were induced to differentiate into PGCs by generating embryoid bodies (EBs) in culture medium without leukemia inhibitory factor. Detection of EGFP expression by fluorescence microscopy and germ cell markers by RT-PCR validated the differentiation of PGCs in EBs. The Rhox6 transgene had little, if any, effect on EGFP expression in EBs, whereas Rhox6 knockdown significantly decreased EGFP expression in EBs. Thus, it is suggested with these results that Rhox6 is necessary for determination of the germ cell lineage.

Generation of organized germ layers from a single mouse embryonic stem cell

“…This modification of Venus was carried out by PCR with Phusion DNA polymerase and the following primers: forward 5′-ATGATATCGCCACCATGGTGAGCA-3′; reverse 5′-TCGAATCTGTACAGCTCGTCC-3′ (ref. 59). The resulting pBS_IVpA with modified Venus was digested with EcoRI and XbaI to clone the puromycin-resistant gene ( Puro r ) with the self-cleaving peptide T2A60 at the 5′ end of Puro r , which was obtained from pCAG_T2APuro59.…”

“…59). The resulting pBS_IVpA with modified Venus was digested with EcoRI and XbaI to clone the puromycin-resistant gene ( Puro r ) with the self-cleaving peptide T2A60 at the 5′ end of Puro r , which was obtained from pCAG_T2APuro59. The resulting vector, namely pBS_IV2AP, was digested with NotI and ClaI and subsequently ligated with self-annealed oligonucleotides that encode restriction enzyme sites of NotI, EcoRV, PmeI, AflII, PacI, NheI and HpaI (sense: 5′-GGCCGCGATATCGTTTAAACTTAAGTTAATTAAGCTAGCGTTAAC-3′; antisense 5′-CGGTTAACGCTAGCTTAATTAACTTAAGTTTAAACGATATCGC-3′; Integrated DNA Technologies (IDT), Coralville, IA, USA).…”

“…Then, Venus in pBS_MCSIV2AP was removed by digestion with NcoI and EcoRI and replaced with DsRedT3 (ref. 61) obtained from pCAG_DsRedT2AP (refs 59, 62). The resulting vector was named pBS_MCSIR2AP.…”

“…The purified resulting vector, namely pTIRPPB, was transfected to mouse ES cells that express EGFP under the Oct3/4 promoter (OGR1)1259626465 by FuGene HD (Roche Applied Science, Indianapolis, IN, USA) essentially as previously described59. Blasticidin-resistant clones, namely OGTR1, were isolated and expanded for analysis.…”

Regulated Expression of Rhox8 in the Mouse Ovary: Evidence for the Role of Progesterone and RHOX5 in Granulosa Cells1

“…Our expression analysis indicates that Rhox6 and Rhox9 may have independent functions in the ovary as their peak expression falls in different windows. To this end, recent in vitro studies using embryonic stem cell and primordial germ cell models indicate that RHOX6 and RHOX9 may not be redundant as they possess distinct abilities to promote germ cell differentiation [56]. Thus, if the primary site of action of RHOX6 and RHOX9 is in oocytes, then the differential expression patterns we observe in the total ovary may be superfluous and compensation by RHOX6 in the oocytes may still explain the lack of ovarian phenotype in Rhox9-null mice.…”