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Our investigation so far has established that the newly identified Notch positive cells are indeed multi-potent. Previous studies (Jung et al., 2005) have demonstrated that dome expressing progenitor cells are also multi-potent. Therefore, the next important question was to determine the hierarchical relationship between Notch positive cells and dome positive cells.

To address this issue we employed expression studies, detailed characterization of Notch lineage-traced cells at different developmental time points as well as genetic ablation of the Notch cells. Our investigation revealed that by late 16 stage of embryogenesis, expression of Notch becomes restricted to 4–5 cells in the embryonic lymph gland visualized by Odd skipped (Odd) expression (arrows, N-Gal4, 2XEGFP; Figure 4A–A': lateral view and Figure 4B–B': dorsal view). Odd is a transcription factor and is known to be the earliest marker of lymph gland (Ward and Coulter, 2000; Mandal et al., 2004). We were then intrigued to know the expression of dome in embryonic lymph gland. Interestingly, while mesodermal dome expression can be seen in the gut (arrow; dome-MESO-LacZ;Figure 4C: dorsal view and Figure 4E: dorso-lateral view), we could not detect any expression of dome in the embryonic lymph gland (Figure 4D–D': dorsal view and Figure 4F–F': dorso-lateral view). Likewise, (Mandal et al., 2004) expression of tepIV (tepIV-Gal4, 2xEGFP), a JAK/STAT responsive thioester containing protein 4-coding gene (Irving et al., 2005), was also absent from the lymph gland in a similarly staged embryo (Figure 4G–H': dorsal view and Figure 4I–J': lateral view). It is thus evident from these results that although Notch signaling is active in the late embryonic lymph gland, JAK/STAT pathway has not yet been elicited.

To investigate whether these Notch cells, encountered in embryos, eventually turn on dome, we adopted the following strategy: a fly line harboring dome-MESO-EBF2 (Evans et al., 2014) was brought in the background of N-Gal4 and then lineage traced following the scheme shown in Figure 5A. In order to trace the relationship between these two cell types, we assayed the larval lymph gland of the above genotype, not only in late third instar but also earlier at different developmental time windows. The stages were selected on basis of the proliferation profile discussed in Figure 2 . In the above genotype, in addition to red (Notch real time expression) and green (Notch-lineage traced), magenta cells are seen that represent dome-MESO expression (Figure 5B).

At 8 hr AEH, we detected two kinds of cells: the first group consisted of 4–5 Notch expressing (stars, Red: Figure 5C) lineage traced (stars, Green: Figure 5C') cells that were negative for domeless (magenta) (Figure 5C''–C''') and the other group expressed only domeless (magenta: Figure 5C''). Interestingly, the dome cells were not lineage traced for Notch (Figure 5C"–C'"), validating that indeed at this time point, none of the Notch expressing cells have undergone cell division. However, on analyzing 15 hr AEH three different cell types were observed. As before, the first type of cells expressed Notch (red and green thus yellow, stars) but lacked domeless (magenta) expression. The number of these cells was now reduced to 3 (stars, Figure 5D–D'"'). We have previously showed that cell division in Notch expressing cells starts from 13 hr onwards (Figure 2I–I", N and Figure 2—figure supplement 1G). Interestingly, the second cell type observed was Notch lineage traced and dome positive (magenta) which also validated that indeed cell division has happened during this time. This group thus was the clonal expansion of Notch expressing cells that have now turned on domeless expression. In addition, we also detected few cells that were not Notch lineage traced but expressed dome (magenta: arrowheads). This group we believe, arose from the dome expressing cells that co-existed with Notch cells during the time of lineage tracing.

The numbers of Notch expressing cells were further reduced by 18 hr AEH (stars) with a concomitant increase of Notch lineage traced dome expressing cells (Figure 5E–E'"'), clearly supporting our previous conclusion (arrows indicate cells that have low levels of Notch but have now up regulated dome expression). Remarkably, at 30 hr AEH the lymph gland was substantially populated by dome (magenta) expressing cells that were lineage traced for Notch (Figure 5F–F'"). Quite interestingly, none of the Notch (red: Figure 5F) expressing cells were seen from this time onwards, undoubtedly strengthening the fact that Notch positive cells eventually become dome positive cells. When this genotype was analyzed at late third instar (following the same regime of lineage tracing as in Figure 5A), this transient labeling of the Notch positive cells only during first instar resulted in labeling of almost all cells of the lymph gland including dome expressing progenitors (Figure 5G–G'", also see Figure 3C–C").

Put together, the above results clearly establish the hierarchical relationship of dome positive cells with respect to Notch positive cells.

We further endorsed the above finding by transient-lineage tracing of dome and tepIV expressing progenitors, following the same scheme that was employed for Notch (Figure 5—figure supplement 1A and Figure 5A). In case of N-Gal4, the transient activation of the lineage-tracing construct led to substantial labeling of the lymph gland (Figure 5—figure supplement 1B–B"and Figure 3C–C') including the dome positive and Cubitus interuptus+ (Ci+) hemocyte progenitors. Compared to this, activation of the lineage-tracing cassette with dome-Gal4 within the same short window resulted in generation of extremely restricted clonal expansion of dome expressing cells (Figure 5—figure supplement 1D–D"). This clearly showed that not enough multi-potent progenitors were born at that time to render labeling of the entire gland and that the potency of Notch expressing cells are higher than the dome positive ones. This was further validated by Ci expression (a specific marker for progenitors) that marked a large population of progenitors that were not lineage traced (Ci: red; Figure 5—figure supplement 1D–D"). Using tepIV-Gal4, another independent driver for hemocyte progenitors (Figure 5—figure supplement 1E–E'), a result akin to transient domeless activation was obtained (Figure 5—figure supplement 1F–H).

In contrast, the few multi-potent Notch expressing cells present at time had the potential to substantially label the almost entire lymph gland including domeless and Ci expressing progenitors.

In order to complement the lineage tracing experiment and provide a functional correlation, we induced genetic ablation of Notch expressing cells. This was achieved by the activation of reaper (rpr, pro-apoptotic gene) specifically in the HSCs by Notch-Gal4. As Notch is required in several development processes, we encountered early lethality on activation of rpr. A narrow window of four hours activation of reaper post emergence of larvae yielded few individuals that were not lethal at late third instar stage. To assay, if Notch expressing cells were successfully ablated, dome-MESO-LacZ was brought in the background of N-Gal4. On restricted activation of UAS-rpr, few individuals of every batch were analyzed at first instar stages to ensure that the lymph gland contain only dome-MESO-LacZ and no Notch expressing cells, confirming a successful ablation (Compare Figure 6A with Figure 6C). The siblings of the batch that had only dome expressing progenitors in the first instar lymph gland and made it to third instar stage, were analyzed. We found that genetic ablation of Notch expressing cells caused a massive reduction in the lymph gland size at third instar stage (Compare Figure 6B with Figure 6D). Quantitative analyses revealed a 5-fold reduction in the total size of the lymph gland (Figure 6E). Moreover, the ratio of area of progenitor cell population versus the total lymph gland area had dropped by more than 2 fold (Figure 6F) indicating that by killing the Notch expressing founder cells, the reserve population of progenitors arising from them were also eliminated. This in turn resulted in a massive reduction in the total lymph gland size that now housed the clonal expansion of those progenitors that coexisted with the HSCs.

Put together, we can conclude that the Notch positive cells are higher in the order to Dome expressing progenitors and that the transient Notch expressing cells of first instar lymph gland are the multi-potent founder cells or the HSCs (Figure 6—figure supplement 1).


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