Stem cells harbor the potential to produce healthy cells to replace lost, damaged or diseased cells because they have the ability to both self-renew and give rise to daughter cells that differentiate. It is often necessary to establish a large number of stem cells for therapeutic purposes, but identifying, isolating, and maintaining these cells have proven difficult. Furthermore, stem cells that have been successfully isolated and maintained in culture may fail to integrate properly when transferred into damaged tissues. Understanding how stem cells function in their cellular microenvironment, or niche, is critical to generating methods for manipulating them. One of the underlying aspects of stem cell biology that remains largely unexplored is how and when adult stem cells are established during normal development.
Stem cell establishment is defined as the process by which a non-self-renewing precursor cell becomes an asymmetrically dividing cell that both self-renews and also produces differentiating daughters. This process has previously been studied in a limited number of systems including hematopoiesis, neural, and epithelial development (reviewed in Chia et al., 2008
; Fuchs, 2008
; Slack, 2008
). In these systems, the formation of stem cells involves dramatic changes in cell signaling, extensive cellular migration, and is closely coordinated with organogenesis as niche formation occurs with stem cell establishment. Factors required for stem cell establishment are often, but not always, essential in maintaining self-renewing adult stem cells depending on the stem cell type. For example, while SCL/tal-
, and Runx1
are all required for mouse blood lineage specification, inactivation of these genes in adult HSCs does not abolish maintenance or self-renewal (Orkin and Zon, 2008
). In contrast, bulge stem cells in the hair follicle require Sox9 for their specification as well as maintenance (Nowak et al., 2008
; Slack, 2008
Spermatogenesis is one of the most accessible systems used to study stem cell formation as germline stem cells (GSCs) can often be assayed functionally. Primodial germ cells (PGCs) are the precursors of GSCs, and their development is remarkably similar between vertebrates and invertebrates (Seydoux and Braun, 2006
). In many organisms, PGCs are specified early in embryogenesis, divide and migrate extensively before assimilating with somatic cells in the gonad (Santos and Lehmann, 2004
). In Drosophila,
PGCs form at the posterior of the syncytial embryo, migrate through the epithelium after gastrulation, split into two groups and finally coalesce with the somatic gonad in parasegment 10 (reviewed in Dansereau and Lasko, 2008
). Female GSCs are formed at the larval to pupal transition (Gilboa et al., 2003
; Zhu and Xie, 2003
), preferentially from PGCs at the anterior of the gonad (Asaoka and Lin, 2004
). Dpp signaling maintains GSCs in the adult ovary, and is similarly required in the larval gonad during the PGC to GSC transition (Gilboa et al., 2003
; Zhu and Xie, 2003
). In contrast, male GSCs are thought to be specified much earlier in development.
While previous studies have indicated that male GSCs may be formed in Drosophila
at the end of embryogenesis (Aboim, 1945
; Kerkis, 1931
), the exact timing and cellular behavior of PGCs transitioning to GSCs have not been examined. In the adult Drosophila
testis, GSCs are maintained by Jak-STAT signaling initiated from a group of somatic cells at the testis apex called the hub (Kiger et al., 2001
; Tulina and Matunis, 2001
). 5–9 GSCs are anchored to the hub by cell adhesion molecules at the hub-GSC interface, and the orientation of their division is regulated by cortically localized Adenomatous Polyposis Coli tumor suppressor (APC) proteins (Yamashita et al., 2003
). It is believed that physical displacement of the stem cell daughter from the hub causes it to initiate differentiation.
As the gonialblast moves away from the hub, it is enveloped by two cyst cells produced by cyst progenitor cells (CPCs, also referred to as somatic stem cells) also docked at the hub, and undergoes four rounds of cell division to produce a 16-cell spermatogonial cyst. Spermatogonial divisions are marked by incomplete cytokinesis, and result in the step-wise development of 2-, 4-, 8- and 16- cell syncytia with stabilized ring canals that serve as intracellular bridges. Specialized organelles known as fusomes extend through the cytoplasm of interconnected spermatogonia (Fuller, 1993
; Hardy et al., 1979
). Furthermore, late 2-cell to early 16-cell spermatogonia express the differentiation factor Bag-of-marbles (Bam), which is required for spermatogonia to mature (Chen and McKearin, 2003
; Song et al., 2004
). However, whether any of these mechanisms of adult GSC maintenance and regulation are observed in nascent GSCs remains to be determined.
Recently, hub formation in male embryonic gonads has been characterized during embryogenesis (Le Bras and Van Doren, 2006
). Initially, the gonad is formed from the coalescence of PGCs and somatic gonadal precursors (SGPs) at stage 14 of embryogenesis. While a group of Abdominal-B
-specified male specific SGPs (msSGPs) is maintained in male gonads at the posterior (DeFalco et al., 2003
), a subset of escargot
expressing SGPs coalesces at the anterior of the gonad to form the embryonic hub, which expresses adult hub markers and is associated closely with a rosette of germ cells at the anterior of the gonad by the end of embryogenesis. These anterior germ cells may be specified to self-renew by the hub at this time, although no direct method has been used to determine this.
Here, we directly examine the timing and regulation of GSC establishment in developing Drosophila testes. We show that a subset of PGCs associated with the hub begin to function as GSCs by the end of embryogenesis. Furthermore, we find that the Jak-STAT pathway is required to prevent differentiation of these hub-associated PGCs, and is likely required for the initial establishment of the GSC population. Taken together, these data show that niche formation is closely associated with stem cell establishment, and that mechanisms regulating stem cell maintenance in the adult are likely to control the process of stem cell establishment.