In this study we have used conditional gene targeting to dissect the functions of Dmrt1 in Sertoli cells and germ cells, comparing the phenotypes of animals lacking Dmrt1 function in each of these cell lineages to each other and to null mutant animals lacking Dmrt1 in all cells. This approach has uncovered cell type-autonomous requirements for Dmrt1 during early postnatal testis development in both Sertoli cells and germ cells, as well as a cell non-autonomous requirement for Dmrt1 in Sertoli cells, as summarized in and discussed below.
Deletion of Dmrt1 in Sertoli cells was particularly informative. We found that Dmrt1 mutant Sertoli cells in SCDmrt1KO animals failed to become closely associated with the tubule periphery. Instead they were distributed throughout the tubules, eventually filling the entire tubule. This closely resembles the Sertoli cell phenotype of Dmrt1 null mutants. Mutant Sertoli cells in SCDmrt1KO animals also failed to up-regulate the androgen receptor and polarize, indicating a developmental block at an immature stage, again very similar to the phenotype of Dmrt1 null mutants. We conclude that these defects are indicative of lineage-autonomous functions for Dmrt1 in Sertoli cells.
Although Sertoli cells in SCDmrt1KO
mice initially activated GATA1 expression by P21, suggesting that they had undergone partial maturation, this expression was lost by 14 weeks. We speculate that this might indicate “de-differentiation” of mutant Sertoli cells, which may help explain the disintegration of seminiferous tubules and changes in Sertoli cell morphology that occur in Dmrt1
null mutants between 6 weeks and about four months (Raymond et al., 2000
) (DZ, unpublished data). The basement membrane surrounding the semiferous tubules is produced jointly by peritubular myoid (PTM) cells and Sertoli cells (Skinner et al., 1985
; Tung and Fritz, 1987
). De-differentiation of mutant Sertoli cells in adults may result in failure to maintain the basement membrane and eventual loss of tubule integrity.
Sertoli cell-specific targeting also revealed that Dmrt1
is required in Sertoli cells for germ cell development. In SCDmrt1KO
mice, germ cells underwent radial migration and mitotic reactivation, and initiated meiosis, but they failed to complete meiosis and eventually died. This demonstrates that Dmrt1
activity in Sertoli cells is required for proper support of the germ line during meiosis, but apparently not for the events leading up to meiotic initiation. The eventual complete loss of germ cells, including spermatogonia, in SCDmrt1KO
mutants suggests that Dmrt1
activity in Sertoli cells is required to establish a functional niche capable of supporting spermatogenesis. Germ cell failure in SCDmrt1KO
mutants might result directly from the mutant Sertoli cells failing to produce a specific signal(s), such as a secreted signaling molecule, or from a more general failure of Sertoli cells to functionally associate with and nutritionally support germ cells, or both. Several candidate signaling pathways have been identified by mRNA expression profiling at P1 and P2, which suggested possible disruption in Dmrt1
null mutants of several signaling pathways, including GDNF, FSH, and retinoic acid (Fahrioglu et al., 2007
Deletion of the androgen receptor (AR) in Sertoli cells (in “SCARKO
” mutant mice), like deletion of Dmrt1
in Sertoli cells, has been shown to affect Sertoli cell maturation and germ cell development (Chang et al., 2004
; De Gendt et al., 2004
; Holdcraft and Braun, 2004
; Tan et al., 2005
). Thus the delayed activation of AR in Dmrt1
mutant Sertoli cells may partially account for the Sertoli cell and germ cell phenotypes of SCDmrt1KO
mutants. However, we observed additional defects in SCDmrt1KO
testes, including the failure of Sertoli cells to localize properly and to polarize, and a more severe disruption of germ cell development in SCDmrt1KO
mutants versus SCARKO
mutants. These phenotypic differences indicate that Dmrt1
cannot function exclusively via the AR pathway.
The germ cell defects in SCDmrt1KO mutants were less severe than in Dmrt1 null mutants, suggesting that Dmrt1 also is required autonomously in germ cells, and we confirmed this interpretation using GCDmrt1KO animals. Dmrt1 mutant germ cells in GCDmrt1KO mice were defective in radial migration and died between P7 and P14, just as in Dmrt1 null mutants. This contrasts with the wild type germ cells in SCDmrt1KO animals, which migrated and were still present at P14. The relatively low targeting efficiency of the TNAP-Cre transgene limited the analysis possible in GCDmrt1KO mutants. However, comparison of germ cells in Dmrt1 null mutants and GCDmrt1KO animals versus SCDmrt1KO animals clearly showed that Dmrt1 is autonomously required in germ cells for radial migration and survival. To our knowledge this is the first demonstration of a genetic requirement in germ cells for postnatal migration.
It is clear from SCDmrt1KO mutants that Dmrt1 activity is required in Sertoli cells for germ cell survival and differentiation, as discussed above. We did not observe any evidence of the converse: that Dmrt1 activity in germ cells is required for Sertoli cell differentiation. This conclusion is somewhat limited by the caveat that tubule sections in GCDmrt1KO mutants with all germ cells negative for Dmrt1 were uncommon, but in such tubules the wild type Sertoli cells were properly organized at the periphery and were polarized.
As mentioned earlier, Dmrt1
is unusual in being expressed in germ cells and Sertoli cells. These cell types are derived from unrelated progenitor populations in different parts of the early embryo and only come together later as a result of long-range migration by the germ cells. Thus it is perhaps unsurprising that most other testis regulators are expressed only in one cell type or the other. It is unknown in which cells the ancestral function of Dmrt1
likely arose, or even whether the expression pattern of Dmrt1
in the mouse is typical, because a variety of Dmrt1
expression patterns have been reported in vertebrates. In humans, fetal DMRT1
mRNA has been described only in Sertoli cells (Moniot et al., 2000
), while DMRT1 protein is expressed only in germ cells in the adult testis (Looijenga et al., 2006
). Similarly, in a lizard, Dmrt1
mRNA is expressed initially in pre-Sertoli cells and then in germ cells (Sreenivasulu et al., 2002
). In fish, Dmrt1
mRNA has been reported to be expressed only in Sertoli cells, or only in germ cells, or in both (Kobayashi et al., 2004
; Marchand et al., 2000
; Xia et al., 2007
). However, most of these studies examined limited developmental stages, so expression in both Sertoli cells and germ cells cannot be excluded in any group of vertebrates. Although it is unclear how common combined Sertoli and germ cell expression of Dmrt1
may be among vertebrates, our results demonstrate that Dmrt1
function is essential in both cell types for testicular differentiation and fertility in the mouse.
In conclusion, the work described here demonstrates that Dmrt1 function is required in Sertoli cells and germ cells for their respective postnatal development, and also has a function in Sertoli cells that is required for germ cell differentiation and survival. These data better define what this essential and highly conserved testicular regulator does in each cell type, which is important for eventually understanding how the remodeling and differentiation of the juvenile testis is controlled.