Dual role of Bnl/Fgf signaling in proliferation and endoreplication of Drosophila tracheal adult progenitor cells

Abstract Adult progenitor cells activation is a key event in the formation of adult organs during development. The initiation of proliferation of these progenitor cells requires specific temporal signals, mostly of them still unknown. In Drosophila, formation of adult tracheal system depends on the activation of tracheal adult progenitors (tracheoblasts) of Tr4 and Tr5 tracheal metamers specific spiracular branches (SB) during the last larval stage. The mitotic activity of these tracheoblasts generate a pool of tracheal differentiated cells that migrate during pupal development along the larval trachea by the activation of the Branchless (Bnl)/Fibroblast growth factor (FGF) signaling to form the abdominal adult tracheal system. In here, we found that, in addition to migration, Bnl/FGF signaling, mediated by the transcription factor Pointed, is also required for adult progenitor cell proliferation in the SBs. Moreover, we found that tracheoblast proliferation in Tr4 and Tr5 SBs relies on the specific expression of the FGF ligand Bnl in their nearby transverse connective branches. Finally, we also show that, in absence of the transcription factor Cut (Ct), Bnl/FGF signaling induces endoreplication of differentiated tracheoblast daughter cells by in part promoting Fizzy-related (Fzr) expression. Altogether, our results suggest a dual role of Bnl/FGF signaling in tracheal adult progenitors, inducing both proliferation and endoreplication of tracheoblasts in late larval development, depending on the presence or absence of the transcription factor ct, respectively. Author summary The generation of adult organs and tissue renewal are complex processes that depend on the proliferation and posterior differentiation of undifferentiated progenitor cells in a temporal coordinated manner. Although many signals that regulate the activity of progenitor cells have been identified, the characterization of the mechanisms underlying the temporal and spatial control of such events remain unknown. The tracheal system of Drosophila, the respiratory organ, forms during embryogenesis and it is remodeled during metamorphosis from quiescent adult progenitor cells that proliferate. We have discovered that this proliferation depends on the activation of the FGF signaling as mutations that either inactivate or over-activate the pathway blocks cell division or induced over-proliferation of progenitor cells, respectively. Interestingly, we have found that the same signaling pathway also controls tracheal progenitor cells differentiation by promoting endoreplication. We found that this dual role of FGF signaling in adult progenitor cells, depends on the presence or absence of the transcription factor Cut. Altogether, our results, reveal the mechanism that control the division and differentiation of progenitor cells and open the possibility that analogous signaling pathway may play a similar role in vertebrate stem cell regulation and tumor growth.


Introduction
The formation of adult organs depends on the activation of progenitor undifferentiated cells during development. Temporal regulation and level of activity of adult progenitor cells are critical to coordinate their proliferation and differentiation in order to form an adult functional tissue. Although great progress has been achieved in the identification of signals that regulate the activity of progenitor cells, the characterization of the mechanisms underlying the temporal and spatial control of such events remain far from understood. Here, we use the formation of the adult tracheal system of Drosophila, the tubular organ responsible for oxygen transport [1,2], to address this issue.
The embryonic trachea of Drosophila develops from 10 bilaterally symmetric clusters (Tr1-Tr10) of ~80 cells that invaginate to form epithelial sacs that remain connected to the epidermis through the spiracular branches (SBs) [3]. These cells migrate and differentiate under the control of the Fibroblast growth factor (FGF)/branchless (Bnl) signaling pathway during embryogenesis to generate a network of interconnected tubes that will function as the larval tracheal system. This larval tracheal network is then heavily remodeled during pupal metamorphosis from a reduced number of different adult precursors cells, called tracheoblasts [2,[4][5][6][7][8][9][10][11][12][13][14]. One type of these cells are the abdominal SB tracheoblasts, which are multipotent undifferentiated cells that are specified in the embryo and remain quiescent until the third larval instar (L3), when they proliferate and differentiate to form the definitive adult abdominal tracheal system [2,5,6,[13][14][15]. Remarkably, although SB tracheoblasts are present in all abdominal metameres of the larvae (Tr4-Tr9), only those from the Tr4 and Tr5 metameres proliferate and differentiate during metamorphosis to generate the definitive adult abdominal airways [13,15]. However, the molecular mechanisms underlying such spatially-restricted SB tracheoblast proliferation remains elusive.
Upon activation, tracheoblasts in the Tr4 and Tr5 SBs start to proliferate.
However, tracheoblast mitotic activity does not occur uniformly. Instead, four cell populations with different proliferation rates can be distinguish in the SB [13,15].
The tracheoblasts located in the intermediated SB zone, called zone 2, presents the higher rate of proliferation. After division, these cells move towards zone 1, the most dorsal part of the SB closest to the DT, and instead of mitosis, initiate one round of DNA replication by activation of the anaphase promoting complex/cyclosome (APC/C) activator Fizzy related (Fzr) to become 4C at the wandering stage [13]. Finally, tracheoblasts at zone 3 presents a very low mitotic rate, while those located at zone 4, at the most ventral tip of the SB, do not proliferate [13]. Previous work has shown that the difference in the proliferation rate of the SB tracheoblasts depends on the relative abundance of the homeobox transcription factor Cut (Ct). Thus, whereas the highly proliferative zone 2 requires intermediate Ct amounts, the non-proliferative zone 1 demands the complete absence of Ct [15]. This work also shows that the different levels of Ct result from the positive and negative regulatory activity of Wingless and Notch signaling pathways, respectively [15]. This particular expression pattern of Ct, however, is not only detected in the Tr4 and Tr5 SBs, but in all abdominal SBs, from Tr4 to Tr9, thus suggesting that other factors must act for the spatial control of the proliferation and differentiation of abdominal SB tracheoblasts.
To address this question, we focus our attention to the Fgf/Bnl signaling pathway, as it has been shown that the FGF receptor Breathless (Btl) is expressed in the endoreplicative cells of zone 1 and in the proliferative growth zone 2 of all SBs [13]. This expression of Btl has been linked to the migration of the Tr4 and Tr5 tracheal progenitors into the posterior part of the abdomen later on pupal development [16]. Here, we found that Fgf/Bnl signaling also exerts a dual regulatory role in the control of tracheoblast development. First, we showed that activation of Bnl/Fgf signaling in the Tr4 and Tr5 SBs is required to initiate and promote tracheoblast proliferation at zone 2. Remarkably, we show that the spatially restricted tracheoblast proliferation to Tr4 and Tr5 SBs is due to specific expression of the Fgf ligand Bnl in those metameres. In addition, we showed that Fgf/Bnl signaling also promotes endoreplication in differentiated SB progenitor cells that express Fzr at zone 1. Finally, we demonstrate that the dual regulatory effect induced by Bnl/Fgf is transduced via the transcription factor pointed (Pnt).
Altogether, our results demonstrate that the Fgf/Bnl pathway is critical in SB tracheoblasts development, playing a dual role on promoting mitotic cell division as well as cell growth through endoreplication.

Bnl/Fgf signaling promotes cell proliferation in SB cells.
In order to analyze the role of Bnl/Fgf signaling in SB development we either overactivate or inactivate the pathway in adult progenitor cells at L3. Depletion of the FGF ligand Bnl in all tracheal cells using the btlGAL4 driver completely abolished proliferation of SB progenitor cells (Fig. 1A-B). Consistently, overexpression of Bnl using the same driver promoted over-proliferation of SB progenitor cells and the overgrowth of the SB ( Fig. 1 A and D). Interestingly, we found that Then, we investigated whether Bnl/Fgf signaling in tracheoblast proliferation requires transcriptional regulation. To address that, we analyzed the expression of pnt, the Ets domain transcription factor that mediates Fgf/Bnl signaling transcription activity in embryonic and larval tracheal cells [11]. Using a specific enhancer trap pnt-lacz, we found that pnt was specifically expressed in the cells of zone 2 and 3 of the SB, where Bnl/Fgf signaling presumably was active ( Fig.   3 A-A'). To confirm that the expression of Pnt is related to Fgf/Bnl signaling activation, we overexpressed pnt RNAi in SB progenitor cells under the control of CiGal4, a specific driver of SB cells. Interestingly, we found that depletion of pnt impaired SB growth by reducing cell proliferation (Figure 3 B-C'). Similar results were obtained when overexpressing pnt RNAi flip-out clones were generated in the SB, as a low number of small clones were detected, suggesting that Pnt is required to mediate FGF signaling in the SB (data not shown). It is important to note, however, that Pnt also transduces the activation of the Epidermal Growth Factor (EGF) pathway, inducing mitotic division of the Air Sac Primourdium (ASP) tracheal cells through the phosphorylated isoform of Pnt, PntP2 [7,11]. To see whether this mechanism also operates in the SBs, UAS-EGFR RNAi was overexpressed under the control of the SB specific driver CiGal4. Interestingly, depletion of EGFR in the SB did not impair proliferation (

Bnl/Fgf signaling acts independently of the transcription factor ct
Our results above provide compelling evidence for the role of Bnl/Fgf signaling in promoting tracheoblast proliferation in the SBs. Previous studies, however, have proposed the transcription factor ct as the main factor that coordinates cell proliferation in the SB [13,15]. Therefore, one possibility is that Bnl/Fgf signaling may control proliferation by regulating ct expression. To solve this question, we over-activate or inactivate the Bnl/Fgf signaling pathway in adult progenitor cells of the SB and analysed the expression pattern of ct in those cells. Interestingly, under any of these conditions, ct expression was unaffected ( Supplementary Fig   2), suggesting that Bnl/Fgf signaling promotes proliferation in Tr4 and Tr5 SBs without disturbing ct expression.
Then, we investigate whether Ct expression is necessary for Bnl/Fgf signaling activity to maintain proliferation of the SB cells. In fact, it has been shown that Ct restricts the expression of the FGF receptor Btl to cells in zone 1 and 2 of the SB [13,15]. According to this regulation, a reduction of Ct expression would induce an expansion of Btl expression into zone 3, promoting ectopic cell proliferation.
Conversely, overexpression of Ct would repress Btl expression in zone 1 and 2, diminishing cell division. To verify this hypothesis, we either overexpressed or depleted Ct in the SB under the control of btlGal4; tubGAL80 ts driver, rearing the animals at 25°C to avoid cell death as Ct acts as a cell survival factor in the SB [15]. As predicted, overexpression of Ct in SB cells reduced the total number of SB cells, probably due to reduction of Btl protein (Fig. 4 B Bnl/Fgf signaling in Drosophila has been described as the main pathway that guide and differentiate tracheal cells during embryogenesis to form the larval trachea and also during metamorphosis to remodel and form the definitive adult trachea [2,3,5,[7][8][9][10][11][12][13]. In addition to these well established roles, our results describe for the first time a role of Bnl/Fgf signaling in SB development by promoting cell proliferation and endoreplication. To date, the transcription factor Ct had been considered the factor that promotes either cell proliferation or differentiation in the SB depending on its abundance [15]. Our work, however, provides several lines of evidence demonstrating that it is not Ct but rather Bnl/Fgf signaling activity that is responsible for promoting SB cell proliferation and endoreplication: (1) SB cells proliferate even when Ct is depleted in these cells ( Fig. 3 D); (2) absence of Bnl/Fgf signaling activity abolished SB development even in the presence of Ct expression; (3) ectopic activation of the Bnl/Fgf pathway induces SB growth by the dramatic increase of tracheoblast proliferation; (4) whereas Ct is similarly expressed in the SB of every tracheal metamere, Bnl is specifically expressed only in the Tr4 and Tr5 metameres, the only two metameres that will grow and develop (Fig. 1 F-G'); and (5) overexpression of bnl in all tracheal cells initiates the SB development of every SB (Fig 1 F). The factor that activates and restricts bnl expression in Tr4 and Tr5 is still unknown. The regulation of bnl expression is very complex as its expression in the ectoderm and tracheal cells is very dynamic during development [12,16,20]. Nevertheless, it is likely that Hox genes control the expression of bnl in the larva tracheal system. In this sense, Tr4 and Tr5 are specified by the expression of low levels of Abdominal A and Ultrabithorax [8,10,14], a hox code that may allow the expression of Bnl. Further experiments are needed to check this hypothesis.
Although Btl had been originally related to the proliferation of the ASP tracheoblasts [12], recent works have shown that Bnl/Fgf signaling promotes tracheoblast mitosis indirectly through the activation of the EGF ligand vein [7,11].
In contrast, our data shows that in the SB, Bnl/Fgf signaling promotes proliferation directly via the transcription factor pnt, and independently of the EGF pathway ( Fig. 3). Therefore, our data show a direct role of Bnl/Fgf signalling in proliferation in Drosophila, in a similar way that occurs during the mammary gland development, where FGF signalling stimulates cell proliferation to generate cells both at the branching epithelium tips and cells in the subtending duct [21,22].
Contrary to previous reports, we also show that the main role of Ct in the SB is to determine the cell mode of tracheoblast by regulating the expression of Fzr. In this sense, our data indicate that Bnl/Fgf signaling induces cell proliferation or endoreplication depending on the presence or absence of Ct, respectively. Thus, Ct acts in the SB like in the ovary follicular epithelium where it also regulates the switch from mitotic cycles to endoreplication (Sun, 2005). The requirement for Ct in maintaining the mitotic cell cycle in Drosophila tracheoblast echoes its role in mammalian systems. The data in mammals suggest that CDP/Cut expression or activity might be restricted to proliferating cells [23]. Interestingly, the expression of the mouse CDP/Cut protein, Cux-1, in the kidney was found to be inversely related to the degree of cellular differentiation [24]. In addition, it has been shown that depletion of Cux-1 resulted in a significant increase in binucleate hepatocytes [25].
Our data indicates that Bnl/Fgf pathway not only initiates SB development and promotes cell proliferation of tracheoblast but also promotes cell endoreplication.
In zone 1 of the SB, the activation of Notch signaling represses Ct expression thus allowing the initiation of endoreplication through the upregulation of the APC activator Fzr/Cdh1 [26]. Once activated, the successive endocycles are regulated by an intrinsic oscillator that consist of alternate APC activator and the levels of Cyc E [27]. Depending on the number of the times that the oscillator is on give rise to cells with 4C, 8C, 16C, 32C, etc. The activity of the Bnl/Fgf pathway seems to regulate the oscillator in SB differentiated cells, as inactivation of the pathway reduces the DNA content, whereas its overactivation increases the number of endocycles. Interestingly, this different effect of the signaling pathway promoting cell differentiation or endoreplication depending on the cell context is reminiscent of the EGFR signaling in the adult gut. After gut epithelial damage, EGFR signaling drives proliferation of intestinal stem cells (ISC), as well as endocycling in differentiated enterocits [28]. As EGFR and Bnl/Fgf pathway share many downstream components, including the transcription factor pnt, it is conceivable that the mechanism to promote endoreplication might be very similar. Another example is found in the oncogene Dmyc, which stimulates cell proliferation of ISCs in the Drosophila adult midgut [29] as well as endoreplication of fat body cells [30]. The hippo pathway has also been involved in promoting cell proliferation and endoreplication in larval tracheal cells depending on the expression of fzr [14]. However, the role of Bnl/Fgf pathway in SB development contrasts with the effect of the FGF4 in mammals. In this case FGF is only required to maintain trophoblast stem cells and therefore cell proliferation, as its inactivation drive the formation of Trophoblast giant cells that growth by endoreplication [31].
Our observations of the Drosophila tracheal system reveal that one signaling pathway can be used in a specific developmental process to induce both cell proliferation and endocycling, and that this capacity may be more common than has been generally appreciated not only in development but also in cancer. In certain contexts, cancerous cells use endoreplication as a path to drug resistance [32,33]. Interestingly, different evidences point to upregulation of the FGF/FGFR signaling as a mechanism of chemoresistance and radioresistance in cancer therapy [34]. Future studies should prove a possible link between FGF/FGFR system and endoreplication in tumors and promise insight into how to treat therapy resistance cancers.

Fly stocks
Details for all strains genotypes can be obtained from flybase (http://flybase.org) or in references listed here. Conditional activation of either RNAi or gene expression was achieved using the Gal4/Gal80 ts System [35]. To overexpress UAS transgenes either in all tracheal cells or in Spiracular branch cells btlGal4UASGFP; tubGal80 ts or CiGal4; tubGal80 ts was used respectively.

Flip-out clones
Females of the genotype: hsflp 70 ; UASCD8GFP; tub>y + >Gal4 where crossed with the following UAS transgenic males: UAStor-btl, UASbtl DN and UASct and were kept at 25ºC until they reached early third instar stages. After a 30 min heat shock at 37ºC, the larvae where transferred back to 25ºC for 14-16h and dissected.