Making Germ Cells from Human Embryonic Stem Cells - Male Germline Stem Cells Developmental and Regenerative Potential - page 56

signaling of a group of cells located in the proximal epiblast at the base of the
allantois (Santos and Lehmann 2004 ; Hayashi et al. 2007 ; McLaren 2003 ). These
PGCs express specific mRNA and protein markers including the cell membrane
protein tissue nonspecific alkaline phosphatase (TNAP), Oct4 (or Pou5f1, POU-
domain class-5 transcription factor 1), and Stella (Dppa3), markers also expressed
in human ES cells (Clark et al. 2004b ; Niwa et al. 2000 ; Saitou et al. 2002 ; Scholer
et al. 1990 ). Recent work has found that specific bone morphogenetic proteins
(BMPs) are released by the extraembryonic ectoderm and visceral endoderm to
induce the neighboring cells of the proximal epiblast to adopt this primordial germ
cell fate (Santos and Lehmann 2004 ; Hayashi et al. 2007 ). Specifically, null alleles
of Bmp4 , Bmp7, Bmp8b , Bmp2 , the TGF-b type I receptor Alk2 , and the BMP sig-
naling transduction molecules Smad1 , Smad4 , and Smad5 all result in greatly
reduced or absent primordial germ cells (Chang and Matzuk 2001 ; Chu et al. 2004 ;
de Sousa Lopes et al. 2004 ; Tremblay et al. 2001 ; Winnier et al. 1995 ; Ying and
Zhao 2001 ; Ying et al. 2000 ; Zhao et al. 1996, 2001 ). Most notably, these findings
emphasize the important role of BMP signaling in early germ cell development and
maintenance.
Signaling by the transcriptional repressor, Blimp1/Prdm1 is also critical in the
specification and maintenance of the early primordial germ cell precursors. Animals
lacking normal Blimp1/Prdm1 genes have reduced numbers of PGCs and misex-
pression of several somatic genes in their germ cells (Hayashi et al. 2007 ; Chuva
de Sousa Lopes and Roelen 2008 ; Ohinata et al . 2005 ). Blimp1 expression is first
detected at E5.5 in a few proximal-posterior epiblast cells, which are the lineage-
restricted PGC precursor cells. Blimp1 represses the somatic program in these cells
and promotes their progression toward the germ cell fate. Other studies, using
neutralizing antibodies during specification to block E-cadherin and hence cell-to-
cell-contact, found a reduction in the number of founder PGCs, emphasizing the
importance of intercellular interactions among these initial PGCs (Okamura et al.
2003 ). Formation of the initial cohort of approximately 40 identifiable founder
PGCs, expressing the germ cell marker Stella , occurs at E7.25 (Hayashi et al. 2007 ;
Saitou et al. 2002 ; Ohinata et al. 2005 ).
During the time that the PGCs migrate into the embryo and along the hindgut to
the genital ridges near the mesonephros, an entirely different developmental pro-
gram occurs and other distinct molecular events are critical. It is during this migra-
tion, from ~E7.25 to E10.5, that massive proliferation and reprogramming of the
PGCs occurs, including erasure of genomic methylation at both imprinted (sex-
specific) and non-imprinted loci as well as changes in histone modifications and
chromatin structure. Subsequent colonization and proliferation of the PGCs, now
termed gonocytes, in the gonad occurs around E10.5. The gonia then become devel-
opmentally arrested at E15.5 (McLaren 2000, 2003 ; Hayashi et al. 2007 ). Several
molecules, especially those that are involved in the transduction of extracellular
signals and interaction with the surrounding somatic environment are critical dur-
ing the process of migration and colonization. Mouse knockout models lacking the
genes for the Kit (kit oncogene) ligand, ckit receptor, Sdf1 ligand, the chemokine
receptor Cxcr4 , and the b1 integrin receptor all have great reductions in the number
of migrating PGCs before reaching the gonads at E9.5 (Anderson et al. 1999 ;