Stem cell populations are maintained in a number of ways, but most importantly by 1) physical attachment to somatic niche cells 2) recognition of short-range proliferative signals, and 3) prevention of accumulation of differentiation-related factors. It is becoming increasingly clear that negative regulators of gene expression play an important role in maintaining many different stem cell populations by repressing the activity of differentiation factors 
. In many cases multiple regulatory mechanisms may repress a single gene and its mRNA and protein products, in order to maintain tight, developmental control over the stem cell pool while still allowing the capacity to respond to physiological cues. Here we have shown that in the female germline, as in the male germline, the RNA-binding protein HOW is required for maintenance of GSCs and exhibits genetic repression of bam
While the phenotypes that we observed when HOW levels were upregulated or downregulated in the female germline were not the same as in the male germline, these differences appear to be explained by the differential role of Bam in the two sexes (). We previously showed that HOW binds very strongly to bam
mRNA from in vivo
lysates, and as bam
contains a five nucleotide HOW recognition element 
in its 3′-UTR, HOW was a good candidate to be a repressor of bam
expression. One isoform of HOW, HOW(L), has been previously demonstrated as a negative regulator of target mRNAs, by binding to the 3′-UTR of its target and preventing export from the nucleus 
. As in the male germline, expression of HOW in the female germline appeared to be nuclear, indicating the prevalence of the HOW(L) isoform. Despite the expression domain of HOW being slightly more restricted in the female germline (being downregulated by the 2-cell stage compared to the 4-cell stage in males), the complementary staining pattern exhibited by Bam was also conserved in the female germline, as Bam is first detectable by immunostaining at the 2-cell stage in females. This is further indicative of HOW playing a role as a negative regulator of bam
expression in the germline of both sexes.
Model for HOW action in the male and female germline.
Genetic evidence also suggests that HOW represses bam
expression. Despite being termed a germ cell “differentiation factor”, Bam plays different roles in males and females. In males, Bam is required for terminal differentiation of spermatogonial cells into spermatocytes 
. Differentiation is dependent on levels of Bam reaching a certain threshold 
. In females, Bam is required in cystoblasts to ensure transition from the stem cell state to mitotically active cystocytes after asymmetric GSC division 
In both sexes, germline overexpression of HOW(L) resulted in a delay in Bam accumulation. In the male germline, some 8-cell cysts did not show detectable levels of Bam 
, while in the female germline, we observed excess early germ cells prior to the domain of Bam expression. This could be explained by the delay in bam
expression due to increased HOW(L) expression resulting in a failure to specify cystoblast-fate during asymmetric stem cell division. As we also observed higher numbers of GSC-like cells when HOW(L) was expressed, it appears as though both possibilities may be occurring. The observation that overexpression of HOW(L) in both sexes resulted in a very similar phenotype to what has been observed in bam
heterozygotes supports the theory that HOW represses bam
expression. In females, bam/+
germaria have previously been shown to contain an increased number of GSC-like cells 
, which we have also observed in this study.
The effect of losing HOW function in GSCs is also indicative of a role for HOW in regulating bam
levels. In females, ectopic Bam expression in GSCs results in premature GSC differentiation without self renewal, whereas in males, this results in germ cell death 
. In females, how
GSCs are lost, however, unlike in males, this may be due to premature differentiation of GSCs into cystoblasts. In females, we did not observe Bam protein in how
mutant GSCs, however Bam is required at different levels in males and females. In the male germline, Bam gradually accumulates to an observable threshold value in order to carry out its main role, initiation of terminal differentiation of spermatogonia into spermatocytes 
. In females, Bam is required in the cystoblast for differentiation; however Bam protein levels are undetectable at the cystoblast stage, indicating that in females, Bam can exert its effect on cystoblast differentiation at very low levels, beyond those which are observable by immunohistochemistry. As female germ cells lacking HOW were able to complete the mitotic amplification program, unlike in the male, and no how
GSCs were observed to undergo apoptosis, it is unlikely that these cells are being lost due to cell death, suggesting that how
GSCs are lost from the niche due to premature differentiation. Similarly, how
GSCs were not lost as quickly from the female germline as in the male germline, which may be consistent with them surviving and differentiating as opposed to the apoptotic loss that was observed in males. While Dpp has been shown to be required for transcriptional repression of bam
in GSCs 
, this repression may not be absolute. Chen and McKearin (2005) suggest that there are extremely low levels of Bam present in GSC spectrosomes; however they could not detect bam
transcripts in GSCs. Therefore it is possible that a main role of HOW in female GSCs is to post-transcriptionally repress bam
mRNA by preventing its export from the nucleus and initiating its degradation, in turn maintaining Bam protein at very low levels. Loss of HOW, therefore, would increase bam
mRNA translation, and hence Bam protein, resulting in premature differentiation of GSCs into cystoblasts.
In females, Dpp is required in GSCs for bam
, while in males Dpp and Gbb act cooperatively in GSCs to repress bam 
. In the female germline, the short-range Dpp signal is believed to act on GSCs, but not in any cells further than one cell diameter from the niche. This could further explain why loss of HOW in GSCs does not show detectable upregulation of Bam, as bam
is transcriptionally repressed to a large extent by Dpp and Gbb in these cells.
One feature of HOW function which has not been conserved in the female germline, is that HOW appears to have no interaction with cycB
. In the male germline, loss of HOW led to a failure to accumulate the G2 cyclin, CycB, which is the only G2 cyclin required for mitosis in the germline 
. The cell cycle stalled in the G2 phase of the cell cycle, cells grew abnormally large, and were removed from the germline via apoptosis. This was the prime reason for GSC loss in male how
. In females, germ cells lacking HOW displayed no growth abnormalities, were able to progress through the mitotic amplification period through to the egg chamber stage, and importantly, showed no difference in CycB accumulation. As female how
germ cells displayed no mitotic defects, this again highlights the likely possibility that GSCs lacking how
function are lost due to premature differentiation instead of cell death, which is what was observed in male GSCs.
Therefore in the female germline, in contrast to the male, HOW does not play any role in regulating TA divisions. This suggests that the main function of HOW in the female germline is repression of bam mRNA as Bam is only required for cystoblast-fate specification (). This explains why altering levels of HOW in females had no affect on TA mitoses as it does in the male.
Sex-specific germ cell cycle control has previously been demonstrated in the Drosophila
germline. Two families which have been shown to activate the APC/C complex (targets cell cycle related proteins for degradation by the proteasome in mitosis and meiosis) are Fizzy (Fzy) and Fizzy-related (Fzr) 
. Recently, a member of the Fzr family, Fizzy-related 2 (Fzr2) has been discovered which is detected specifically in the male germline. fzr2
can substitute for fzr
function when ectopically expressed in other tissue types. In fzr
mutant embryos, there is a failure to degrade the mitotic cyclins A, B, and B3 
, however forced Fzr2 expression can rescue this defect 
. Fzr2 is primarily detected in pre-meiotic spermatocytes, suggesting that Fzr2 plays a meiotic role, specifically in the male germline 
. Therefore, male-specific cell regulation of the germ cell cycle by the RNA-binding protein HOW is a possible scenario.
The RNA-binding proteins Nanos (Nos) and Pumilio (Pum) act as part of a protein complex to repress translation of cycB
mRNA in pole cells as they migrate to the presumptive gonad during embryonic development 
. In the female germline, Nos and Pum are expressed in GSCs and act together to prevent cystoblast differentiation 
. The mode of action and downstream targets of this complex remain unclear but it has recently been shown that in the cystoblast, Nos is post-transcriptionally repressed by Bam 
, allowing expression of differentiation genes. Pum, despite its role in the maintaining proliferative GSCs 
, plays a different role in the cystoblast. Pum functions together with a potent differentiation gene, brain tumor
, to repress self-renewal targets such as Mad
and dMyc 
. While Nos expression is present in the male germline, Pum is expressed at very low levels (or essentially not at all) in the male. While a number of Pum mutant alleles exhibit female sterility 
no functional role for Pum has been demonstrated in the male germline. This is another example of differential use of RNA-binding proteins in the male and female germline.
The exact mechanism by which HOW regulates cycB in the male germline is yet to be elucidated, but since loss of HOW function results in loss of cycB expression it could be predicted that HOW is repressing a negative regulator of cycB expression.
The role of HOW in regulation of Bam appears to have been conserved between the sexes. It is now apparent that bam
expression is regulated at various levels (see below). As reproduction is of critical importance for the survival of the species, it is not surprising that key regulators of this process have evolved tight controls on their expression and function. Proper expression of Bam is vital for maintaining tissue homeostasis, and misexpression has serious outcomes. bam
has previously been shown to be transcriptionally repressed by the Dpp pathway in both sexes 
, however recent studies have also shown that a number of other factors, such as ISW1 
, Otefin 
, EIF4A 
, and Piwi 
also play various roles in maintaining bam
repression in GSCs. Here we show that HOW is responsible for bam
mRNA regulation at a post-transcriptional level in both sexes. This complex and redundant regulation of stem cell proliferation highlights how important tight control of stem cell behavior is for the organism.