Mechanics of stem cell lineage differentiation and survival : differential 3' cleavage of germline mRNAs and somatic septate junction function

To replace lost or damaged cells over the lifetime of an organism, many tissues harbor relatively undifferentiated adult stem cells, which retain the ability to proliferate and give rise to cells that differentiate into the functional cell types of that particular tissue. One well studied model adult stem cell lineage is the Drosophila male germline, where long-lived germline stem cells produce spermatogonia that first proliferate then switch to spermatocyte state in preparation for the two meiotic divisions and differentiation of the resulting spermatids into functional sperm. The mechanisms that control the ordered changes in gene expression during the sequential steps of differentiation are key for creation of the correct cell types and misregulation may lead to tissue degeneration or oncogenesis. In my PhD, I identified a mechanism based on developmentally regulated changes in the site of 3' cleavage of nascent mRNAs that regulates a switch in the proteins expressed when proliferating spermatogonia begin differentiate into spermatocytes. I also discovered that the function of Septate Junction components in somatic cyst cells is required for the survival of proliferating spermatogonia. Current research on the regulation of gene expression has focused predominantly on the role of transcriptional regulators to repress or activate expression of mRNAs at developmental transitions. However, it is clear that mRNAs are often translationally regulated through sequences in their 3' untranslated regions (3'UTRs) and these sequences can be altered by differential 3' cleavage of nascent mRNAs. I discovered developmentally regulated 3' cleavage of over 400 transcripts produced mRNA isoforms in proliferating spermatogonia with a long 3'UTR but mRNAs with a much shorter 3'UTR in differentiating spermatocytes. I found that this regulated 3' cleavage event occurred early during spermatocyte differentiation, correlated with changes in protein expression, either from ON in spermatogonia to OFF in spermatocytes or OFF in spermatogonia to ON in spermatocytes. I identified a motif signature present in differentially cleaved transcripts defined by strong polyadenylation signals (PASs) enriched at the distal cleavage site used in spermatogonia and weaker PASs were present at the proximal cleavage site used in spermatocytes. Strikingly, altering one base in a reporter containing the 3'UTR of dco, a gene that undergoes differential 3' cleavage, to create a strong PAS near the proximal cleavage site, altered 3' cleavage to produce an mRNA isoform with a shorter 3'UTR in spermatogonia and resulted in decreased protein expression. As differential 3' cleavage can act on nascent mRNAs already being transcribed, this may be a mechanism to rapidly switch protein expression to facilitate quick cell state transitions. My work has also implicated the surrounding somatic cyst cells as critical regulators of germ cell survival. I discovered that components of the occluding septate junction (SJ), including the gene Nrx-IV, are required in somatic cyst cells to promote the survival of TA cells beyond the 4-cell stage. Acute loss of SJ components resulted in spermatogonial death as early as 2 days following SJ knock down. Loss of SJ components in cyst cells did not appear to alter cyst cell differentiation and thus indicates the presence of a currently unknown signal from cyst cells that promotes germline survival.

Keywords: Developmental Biology.
Advisor: Fuller Margaret T.
Barna Maria (Professor of developmental biology) 
Simon Michael (Biology professor)
Villeneuve Anne