TGFbeta signaling in male germ cells regulates gonocyte quiescence and fertility in mice.

During testis development, proliferation and death of gonocytes are highly regulated to establish a standard population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with time. Using an organ culture system mimicking in vivo development, we show higher proportions of proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence. These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis.

Control of beta-cell differentiation by the pancreatic mesenchyme.

The importance of mesenchymal-epithelial interactions for normal development of the pancreas was recognized in the early 1960s, and mesenchymal signals have been shown to control the proliferation of early pancreatic progenitor cells. The mechanisms by which the mesenchyme coordinates cell proliferation and differentiation to produce the normal number of differentiated pancreatic cells are not fully understood. Here, we demonstrate that the mesenchyme positively controls the final number of beta-cells that develop from early pancreatic progenitor cells. In vitro, the number of beta-cells that developed from rat embryonic pancreatic epithelia was larger in cultures with mesenchyme than without mesenchyme. The effect of mesenchyme was not due to an increase in beta-cell proliferation but was due to increased proliferation of early pancreatic duodenal homeobox-1 (PDX1)-positive progenitor cells, as confirmed by bromodeoxyuridine incorporation. Consequently, the window during which early PDX1(+) pancreatic progenitor cells differentiated into endocrine progenitor cells expressing Ngn3 was extended. Fibroblast growth factor 10 mimicked mesenchyme effects on proliferation of early PDX1(+) progenitor cells and induction of Ngn3 expression. Taken together, our results indicate that expansion of early PDX1(+) pancreatic progenitor cells represents a way to increase the final number of beta-cells developing from early embryonic pancreas.

The mesenchyme controls the timing of pancreatic beta-cell differentiation.

The importance of mesenchymal-epithelial interactions in the proliferation of pancreatic progenitor cells is well established. Here, we provide evidence that the mesenchyme also controls the timing of beta-cell differentiation. When rat embryonic pancreatic epithelium was cultured without mesenchyme, we found first rapid induction in epithelial progenitor cells of the transcription factor neurogenin3 (Ngn3), a master gene controlling endocrine cell-fate decisions in progenitor cells; then beta-cell differentiation occurred. In the presence of mesenchyme, Ngn3 induction was delayed, and few beta-cells developed. This effect of the mesenchyme on Ngn3 induction was mediated by cell-cell contacts and required a functional Notch pathway. We then showed that associating Ngn3-expressing epithelial cells with mesenchyme resulted in poor beta-cell development via a mechanism mediated by soluble factors. Thus, in addition to its effect upstream of Ngn3, the mesenchyme regulated beta-cell differentiation downstream of Ngn3. In conclusion, these data indicate that the mesenchyme controls the timing of beta-cell differentiation both upstream and downstream of Ngn3.

Label-retaining cells in the rat pancreas: location and differentiation potential in vitro.

Islets of Langerhans are micro-organs scattered throughout the pancreas that contain insulin-producing cells, called beta-cells. Although new light has been recently shed on beta-cell development, information on the phenotype and location of beta-stem cells remains scarce. Here, we provide evidence that beta-stem cells are slow-cycling cells located within and around the islets of Langerhans. First, using a bromodeoxyuridine (BrdU) pulse/chase approach, we detected BrdU-retaining cells in vivo in the islet area of rat pancreata. These cells were negative for endocrine markers but expressed Pdx1, a marker for pancreatic stem cells. Next, using an in vitro model that mimicked endocrine cell development, we found that BrdU-retaining cells were capable of differentiating into beta-cells. Taken together, these observations demonstrate that BrdU retention is a property of beta-stem cells.