Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

The mature pancreas is composed of exocrine (acinar and duct cells) and endocrine (α-, β-, δ-, ε-, and PP-cells) compartments. The differentiation of these distinct cell types is regulated by the coordinated expression of numerous transcription factors (1,–3). Among these transcription factors, neurogenin 3 (Neurog3), a member of the basic helix-loop-helix transcription factor family, plays essential roles in initiating endocrine differentiation during embryonic development, regeneration, and transdifferentiation into functional insulin-producing cells (4,,,,–9). In addition, the transient nature of Neurog3 expression makes it a useful marker for uniquely identifying endocrine progenitor cells—cells that have committed to the endocrine lineage but have not yet differentiated into hormone-producing endocrine cells (10,11).

Mouse models expressing fluorescent reporter proteins have been used to sort specific cell populations. For example, cells sorted from Ngn3-eGFP mouse lines generated by different groups have been used to examine gene expression profiles during pancreatic endocrine differentiation (12,13). However, because of the long half-life (14), fluorescent reporter proteins persist after the Neurog3 gene itself has shut off; thus, the fluorescent cell population includes cells at different stages of differentiation. Destabilized fluorescent proteins have shorter half-lives but lower fluorescence (15). In addition, sorting cells at earlier time points may decrease the overlap with more differentiated cells as described previously (13); however, this approach cannot be used at later time points or for distinguishing more mature cells.

To solve this problem, we developed a novel transgenic mouse model (Ngn3-Timer) in which human NEUROG3 upstream and downstream sequences were used within a bacterial artificial chromosome (BAC) to drive expression of DsRed-E5, a variant of the Discosoma sp. red fluorescent protein that shifts its fluorescence emission peak from green to red in a time-dependent manner (16). Using fluorescence microscopy, green fluorescence could be detected in developing pancreata of Ngn3-Timer embryos as early as embryonic day 9.5 (E9.5) (data not shown). Both green and red fluorescent signals were readily detected in developing pancreata of Ngn3-Timer embryos from E12.5 to E18.5, whereas predominantly red fluorescence was observed at postnatal day 7 (P7) (supplemental Fig. 1, available in the online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0390/DC1), consistent with previous reports that few Neurog3-expressing cells persist after birth (5). At E17.5, histological analyses detected green-dominant and green/red double-positive fluorescent cells in close apposition with the ductal lumen, whereas red-dominant cells appeared in islet-like clusters (Fig. 1 A), supporting a model whereby cells in the endocrine lineage emerge from ductal regions as Neurog3-positive cells and migrate away from the ductal region as differentiation progresses. Consistent with this model, staining for Neurog3 protein overlapped with predominantly green-dominant cells and some green/red double-positive cells (supplemental Fig. 2), whereas insulin staining overlapped with green/red double-positive cells and red-dominant cells (supplemental Fig. 3).

To quantify the shift in fluorescence during endocrine cell differentiation, flow cytometric analyses were performed with dissociated cells isolated from Ngn3-Timer mice. Labeled cells isolated from E13.5 pancreata emitted fluorescence predominantly in the green channel; however, a progressive increase in red fluorescence was observed in older embryos until P0, when the number of green-dominant cells dramatically declined compared with the earlier time points and most labeled cells fluoresced in the green/red double-positive area (Fig. 1 B). These data support the hypothesis that the DsRed-E5 protein initially expressed in the Neurog3 lineage emits in the green spectrum and that as the cells turn off Neurog3 expression, age, and differentiate, it shifts to the red emission spectrum.

To verify this hypothesis and estimate the temporal resolution of this model, fluorescent cells were sorted by a fluorescence-activated cell sorter (FACS) into four different populations, placed in culture, and then reanalyzed by flow cytometry at various time points following culture. Green-dominant cells, sorted from gate A in Fig. 2, converted to green/red double-positive within 6 h, whereas green/red double-positive cells in gate B converted to the lower green/red ratio (red dominant) of gate C cells within 12 h. Therefore, the green-dominant cells were within a 6-h time window after initial DsRed-E5 expression and green/red double-positive cells within a 12-h time window. On the other hand, the fluorescent cells sorted using gate C changed little over 12 h, presumably because of the long half-life of DsRed-E5.

Real-time PCR analyses performed on RNA from cells sorted by FACS into the four populations defined in Fig. 2 confirmed the sequential maturation of these cells. The expression level of Neurog3 was highest in green-dominant cells and dramatically decreased in green/red double-positive cells (Fig. 3,A), reflecting the rapid downregulation of Neurog3 once it has initiated endocrine differentiation. Expression of the islet hormones peaked later (Fig. 3 B–D), with the mRNA encoding insulin and glucagon highest in cells from gate C, the most mature population.

To gauge the expression of known pancreatic transcriptional regulators (13,17) in these differentiating cell populations, TaqMan RT-PCR assays were performed with RNA from cells isolated by FACS from E17.5 Ngn3-Timer pancreata. The expression levels of 145 genes were normalized to β-glucuronidase, and the relative level of mRNA from each gate was determined with respect to the value in nonfluorescent cells (gate N). For 21 genes, expression in the earliest progenitor cells (gate A) exceeded expression in the nonfluorescent cells by more than 10-fold (gate [A]/gate [N] >10; Fig. 3 E–J and supplemental Table).

Among these genes, three (Neurog3, Pax4, and Mycl1) demonstrated a sharp expression peak in gate A, with a subsequent decline of more than 10-fold from gate A to gate C (Fig. 3 J)—which is consistent with the conclusion that these genes have expression restricted to the islet progenitor cells. These data confirm previous evidence that Neurog3 directly activates transient Pax4 gene expression followed by repression by Pax4 (18). The profile of Mycl1 paralleled those of Neurog3 and Pax4; therefore, it may also play a specific role in the transient endocrine progenitor cells.

Several of the other genes, including NeuroD, Nkx2. 2, Myt1, and Insm1, are also known targets of Neurog3 but persist in mature islet cells (1,–3). In addition, this approach identified several factors with known expression in the pancreas (13,17) but without known roles in the Neurog3 pathway, including Mycl1, Mnt, Nfatc2, Pou2f2, Sim1, and Fev. Fev, which encodes an ETS transcription factor and showed the highest relative induction in the gate A cells (Fig. 3 E), and we have recently confirmed its expression and function in the islet lineage (Y. Ohta and M.S.G., unpublished data). Finally, Rfx6, a member of the Rfx family of winged-helix transcription factors, has not been studied in the pancreas previously, but these data place it in the Neurog3 pathway and islet lineage.

Of the 145 genes only three, Mafa, Hopx, and Myt1l, showed a robust increase from gate A to gate C (Fig. 3,K) that paralleled the expression profiles of Ins1 and Ins2 (Fig. 3 B and C). MafA appears late in pancreatic development specifically in mature β-cells (19) and directly transactivates the insulin gene in conjunction with NeuroD1 and Pdx1, which are expressed earlier. Therefore, MafA may play a critical role in the final maturation of β-cells. It follows that Hopx and Myt1l potentially also contribute to the maturation of the endocrine cells.

As expected, Ptf1a and Rbpjl, known exocrine genes, were confined to gate N (Fig. 3,L). On the other hand, mRNA encoding Hes1, Notch2, and Onecut1 (Hnf6), which regulate the expression of Neurog3 (4,20,21), persisted in gate A but rapidly declined as cells matured (Fig. 3 M).

In summary, the Ngn3-Timer mouse provides a useful in vivo tool for temporal dissection of the differentiation processes. The model allowed us to accurately isolate cells from distinct narrow time points along the differentiation pathway and also to study the characteristics of those cells. We have used this tool to identify genes that likely play unique roles at these different steps of differentiation. The availability of this tool also permits relatively facile methods for studying signals that impact the initiation or completion of differentiation, the rate of differentiation, or the proliferation or death of differentiating cells. Likewise, similar transgenic models could be used to study the temporal features of differentiation in other cell populations.