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Dual role for Jumu in the control of hematopoietic progenitors in the Drosophila lymph gland

  1. Yangguang Hao
  2. Li Hua Jin  Is a corresponding author
  1. College of Life Sciences, Northeast Forestry University, China
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Cite this article as: eLife 2017;6:e25094 doi: 10.7554/eLife.25094

Abstract

The Drosophila lymph gland is a hematopoietic organ in which the maintenance of hematopoietic progenitor cell fate relies on intrinsic factors and extensive interaction with cells within a microenvironment. The posterior signaling center (PSC) is required for maintaining the balance between progenitors and their differentiation into mature hemocytes. Moreover, some factors from the progenitors cell-autonomously control blood cell differentiation. Here, we show that Jumeau (Jumu), a member of the forkhead (Fkh) transcription factor family, controls hemocyte differentiation of lymph gland through multiple regulatory mechanisms. Jumu maintains the proper differentiation of prohemocytes by cell-autonomously regulating the expression of Col in medullary zone and by non-cell-autonomously preventing the generation of expanded PSC cells. Jumu can also cell-autonomously control the proliferation of PSC cells through positive regulation of dMyc expression. We also show that a deficiency of jumu throughout the lymph gland can induce the differentiation of lamellocytes via activating Toll signaling.

https://doi.org/10.7554/eLife.25094.001

Introduction

The development and specification of blood cells in humans and Drosophila are controlled by conserved transcriptional regulators and signaling pathways. Drosophila has become a suitable model for investigating the genetic and molecular mechanisms of hematopoiesis and associated leukemias (Crozatier and Vincent, 2011). Similarly to the process in vertebrates, hematopoiesis in Drosophila occurs in two waves during development. The first population of hemocytes is derived from the embryonic head mesoderm and provides two types of circulating blood cells, specifically plasmatocytes and crystal cells (Holz et al., 2003). The second wave of hematopoiesis occurs during the larval stage in a specialized hematopoietic organ known as the lymph gland (Lanot et al., 2001). At the onset of metamorphosis, the lymph gland breaks down, releasing plasmatocytes and crystal cells into the circulating hemolymph (Crozatier and Vincent, 2011). In response to an immune challenge, the lymph gland can also give rise to a third cell type, the lamellocytes, which are produced in response to wasp infestation (Lanot et al., 2001; Crozatier and Meister, 2007). The third-instar larval lymph gland contains a pair of primary lobes and a variable number of secondary lobes posterior to the primary lobes. The primary lobes are organized in three zones: the cortical zone (CZ) contains differentiated hemocytes, the medullary zone (MZ) is composed of prohemocytes, and the posterior signaling center (PSC) functions as a hematopoietic niche for maintaining a population of blood cell precursors (Jung et al., 2005). Similar to the hematopoietic stem cell (HSC) niche in the mammalian bone marrow, the PSC plays a key role in supporting Drosophila blood cell homeostasis (Krzemień et al., 2007; Mandal et al., 2007). PSC cells are specified by the expression of two transcription factors, the homeotic protein Antennapedia (Antp) and Collier (Col), which is the Drosophila early B-cell factor (EBF) ortholog (Crozatier et al., 2004; Mandal et al., 2007). In addition, PSC cells also selectively express the secreted factor Hedgehog (Hh) and the Serrate (Ser) signaling molecules (Lebestky et al., 2003; Krzemień et al., 2007; Mandal et al., 2007). In the col mutant, all Antp-positive PSC cells are missing, and the population of MZ hematopoietic precursors is lost (Mandal et al., 2007). Conversely, increasing the PSC size via Antp overexpression can increase the size of the MZ at the expense of the CZ during late stages of the third instar (Mandal et al., 2007). Moreover, the decapentaplegic/bone morphogenetic protein (Dpp/BMP), Wingless (Wg) and insulin/TOR signaling pathways cell-autonomously control the size of the PSC by regulating the expression of dMyc (Pennetier et al., 2012; Sinenko et al., 2009; Benmimoun et al., 2012; Tokusumi et al., 2015). A recent study suggested that Slit/Robo signaling from the cardiac tube also controls PSC morphology (Morin-Poulard et al., 2016). Prohemocytes within the MZ express the Hh receptor Patched (Ptc), the Jak/Stat signaling pathway receptor Domeless (Dome), and DE-cadherin (Shg), which is a target of Wg function in prohemocytes (Mandal et al., 2007; Sinenko et al., 2009). Furthermore, a significant up-regulation of ROS levels occurs during the third larval instar, and this up-regulation primes hematopoietic progenitors for differentiation (Owusu-Ansah and Banerjee, 2009). It has been shown that the Hh signal from the PSC contributes to the maintenance of prohemocytes within the MZ (Mandal et al., 2007). In addition, Wingless and insulin/TOR signaling can autonomously maintain hemocyte progenitors in the lymph gland (Sinenko et al., 2009; Benmimoun et al., 2012).

Jumeau (Jumu) is a member of the winged-helix/forkhead (Fkh) transcription factor family in Drosophila. Previous studies have shown that Jumu is a suppressor of position-effect variegation and is involved in neurogenesis and in eye, wing and bristle development (Strödicke et al., 2000; Cheah et al., 2000). A recent study showed that Jumu and its homolog, Checkpoint suppressor homologue (CHES-1-like), regulate the division of cardiac progenitor cells through a Polo-dependent pathway (Ahmad et al., 2012). Our previous study demonstrated that the loss of jumu affects the number and phagocytosis of circulating hemocytes (Jin et al., 2008). Moreover, the overexpression of jumu induces melanotic nodules by activating Toll signaling (Zhang et al., 2016). In this paper, we show that Jumu is expressed in the entire lymph gland and plays crucial roles during Drosophila hematopoiesis. Jumu protein within the entire lymph gland suppresses the activation of Toll signaling by down-regulating Col expression and thereby prevents lamellocyte differentiation. Jumu protein within the MZ maintains the proper differentiation of prohemocytes by cell-autonomously preventing the local overexpression of Col in the MZ and by non-cell-autonomously inhibiting the ectopic and expanded Col+ PSC cells. Moreover, our results also demonstrate that Jumu cell-autonomously controls PSC cell proliferation by regulating the expression of dMyc.

Results

Jumu is involved in the differentiation of hemocyte precursors

To further analyze the function of Jumu in larval hematopoietic homeostasis, we first detected the expression of the plasmatocyte marker P1, the crystal cell marker Hindsight (Hnt) and the lamellocyte marker L1 in the lymph glands of jumu mutants. Because all jumu homozygous null mutants died during embryogenesis, to further reduce the expression of Jumu, we utilized the double heterozygotes generated by crosses between different jumu heterozygote mutants. Only a few jumu double heterozygotes mutants survived until adulthood, and these were found to exhibit poor viability and female sterility. The jumu heterozygote mutant lymph gland showed obvious reductions in plasmatocyte and crystal cell differentiation (Figure 1A–J). Moreover, the number of crystal cells in the jumu double heterozygotes mutants was clearly less than that found in heterozygotes (Figure 1I,J). However, plasmatocyte differentiation in the jumu double heterozygotes mutants was not clearly reduced compared with that found in w1118 (Figure 1D,E). In addition, only very few lamellocytes could be detected in less than 10% of the lymph glands (n = 30 lobes) of jumu heterozygotes, whereas more than 90% of the lymph glands in the jumu double heterozygotes mutants (n = 40 lobes) showed large numbers of lamellocytes in the primary and secondary lobes (Figure 1K–N). Moreover, a reduced primary lobe size and an enormous overgrowth of secondary lobes were features of jumu double heterozygotes lymph glands (Figure 2E,L). To further determine whether the change in hematopoietic homeostasis observed in the jumu double heterozygotes mutants depends on the expression levels of jumu, the transcription levels of jumu in heterozygotes and double heterozygotes third-instar larvae were quantified. The jumu transcription levels were reduced by approximately half and 40-fold in the heterozygotes and the double heterozygotes mutants, respectively (Figure 1EE). In summary, these results indicate that a change in jumu expression substantially disturbs blood cell homeostasis and the growth and development of the lymph gland.

Loss of jumu affects prohemocyte differentiation in the lymph gland.

(A–D) Immunostaining against the plasmatocyte marker P1 (red) in the lymph glands of third-instar larvae. The numbers of plasmatocytes are significantly reduced in jumu heterozygotes but not obviously reduced in double heterozygotes mutants. (F–I) Immunostaining against the crystal cell marker Hnt (red) in the lymph glands of third-instar larvae. The numbers of crystal cells are significantly reduced in the mutant flies. (E, J) Quantification of plasmatocyte and crystal cell indexes in primary lobes. (K–N) Immunostaining against the lamellocyte marker L1 (red) shows few lamellocytes in jumu heterozygotes (K, L) and substantial increases in the lamellocyte number in jumu double heterozygotes mutants (M, N). (O–Q) The lymph glands of jumuDf2.12and jumuDf3.4 third-instar larvae display expansion of the medullary zone (MZ) as marked by dome>GFP (green) and a reduced plasmatocyte signal (red). (R–X) Immunostaining against Ptc (red) (R–T) and Shg (green) (U–X) shows MZ expansion in mutant third-instar larvae. (Y) Quantification of the proportions of the primary lobes occupied by prohemocytes (Shg+ MZ area/primary lobe area). (Z–CC) The ROS levels (red) are increased in the MZ of w1118third-instar larvae, but similar increases are rarely observed in mutant third-instar larvae. (DD) Quantification of the ROS+ cell index in primary lobes. (EE) Real-time PCR analysis of the jumu level in the entire third-instar larvae. The data are presented as box-and-whisker representations in E, J, Y and DD and as column-bar-graph representations (in which the error bars represent ± S.E.M.) in EE. For all quantifications: NS, not significant; *p<0.1; **p<0.01; ***p<0.001 (Student’s t test). Dashed white and yellow lines outline the edges of the primary and secondary lobes, respectively, in all of the figures. Scale bars: 50 μm.

https://doi.org/10.7554/eLife.25094.002
Loss of jumu causes expansion of the populations of Col+ PSC cells in the lymph gland.

(A–E) Immunostaining against the PSC cell marker Antp (green) shows an increased number and ectopic expansion of PSC cells in jumu-mutant lymph glands. (F–G’) Increased hhF4f-GFP-positive PSC cells (green) are located throughout the lymph glands of jumuDf2.12 third-instar larvae, and these cells are co-localized with Antp (red). (H–I’) Antp immunostaining (red) of the lymph glands of third-instar larvae expressing GFP under the control of col-Gal4 (green) shows col>GFP ectopic expression and co-localization with Antp in jumuDf2.12. (J–L) Immunostaining against Col (green) shows ectopic expansion of Col+ PSC cells in jumu-mutant lymph glands. (M–P) Deficiency in the col levels (col/+) reduces the numbers of both Col+ and Antp+ PSC cells in jumuDf2.12. (Q–T) Deficiency in the col levels (col/+) reduces Shg expression (green) (Q, R) and increases the differentiation of plasmatocytes (red) (S, T) in jumuDf2.12. (U) Quantification of the number of PSC cells (Antp+ cells) per lymph gland lobe. The data are presented through a box-and-whisker representation. **p<0.01 and ***p<0.001 (one-way ANOVA). Dashed white and yellow lines outline the edges of the primary and secondary lobes, respectively, in E and L. Scale bars: 100 μm.

https://doi.org/10.7554/eLife.25094.003

We subsequently analyzed the expression pattern of the MZ-specific markers domeless-Gal4,UAS-2×EGFP (dome>GFP), E-cadherin (Shg) and Patched (Ptc), which is a receptor in the Hh pathway. In contrast to wild-type larvae, the lymph glands of jumu heterozygotes third-instar larvae strongly expressed prohemocyte markers and presented a greatly enlarged MZ (Figure 1O–Y). However, the lymph glands of jumu double heterozygotes mutants did not show an obviously enlarged MZ compared with the wild-type (Figure 1X,Y). In addition, we also assessed ROS as a specific marker for MZ. ROS appear by the third instar, and increased levels of ROS can prime progenitor cells for differentiation (Owusu-Ansah and Banerjee, 2009). Consistent with the results of a previous study, ROS appeared in the MZ of the lymph gland of w1118 mid-third-instar larvae and remained increased until late stages (Figure 1Z); however, ROS were rarely observed in jumu mutants during the entire third instar stage (Figure 1AA-DD). These results indicate that the loss of jumu prevents the differentiation of hematopoietic progenitors into plasmatocytes and crystal cells.

Jumu negatively regulates the proliferation and ectopic expansion of Col+ PSC cells

Previous studies have indicated that an increased PSC domain size can cause an increase in the MZ and a reduction of hemocyte differentiation (Mandal et al., 2007; Pennetier et al., 2012). To investigate whether an increase in the number of PSC cells could be observed in jumu mutants, we detected the expression of Antp, a specific marker of the PSC in the lymph gland. As predicted, the loss of jumu indeed led to substantial increases in the PSC size and cell number to different degrees (Figure 2A–E,U). In particular, the deletion mutants jumuDf2.12 and jumuDf3.4 showed strong increases in PSC cells and ectopic expansion of Antp; these ectopic PSC cells were scattered and often formed two or more cell clusters with higher accumulation in the MZ and CZ (Figure 2C,D). Moreover, the jumu double heterozygotes mutants showed more severe defects in PSC morphology, specifically the existence of PSC cells mostly in the secondary lobes (Figure 2E). In addition, the Hh signal from the PSC is required for the maintenance of prohemocytes (Mandal et al., 2007); thus, we subsequently detected Hh expression using the hhF4-GFP reporter gene. Similarly to the expression of Antp, jumu mutants also showed increased numbers of Hh+ cells, and these were highly co-localized with Antp+ PSC cells (Figure 2F–G’). It has been indicated that Col is also a PSC-specific marker and regulates Antp and Hh expression in the PSC in third-instar larvae (Mandal et al., 2007; Pennetier et al., 2012). Thus, we tested whether jumu affects the PSC by controlling the level of Col. Similar to Antp and Hh, increased Col was also observed in jumu mutant lymph glands, and increased numbers of Col>GFP+ PSC cells were colocalized with Antp+ cells (Figure 2H–L). Moreover, a reduced expression of Col can inhibit the phenotypes associated with jumu mutants: specifically clearly reduced numbers of both Col+ and Antp+ PSC cells accompanied by a reduction in the MZ size and an increased differentiation of plasmatocytes (Figure 2M–U).

To determine whether the ectopic PSC cells in jumu mutant lymph glands are migrated from the PSC or are de novo arising in the MZ and CZ, we first detected the morphology of the PSC at different larval stages. Expanded PSC cells were observed in jumu mutant L1 larvae and were amplified in L2 and L3 larval stages (Fgure 3A-L’). In addition, we observed that the ectopically expanded Antp+ PSC cells did not co-localize with any dome-GFP+ cells or Hml-GFP+ cells in the MZ and CZ domains, particularly among the ectopic PSC cells adjacent to MZ or CZ cells during the early larval stage; in fact, we did not detect any intermediate cells co-expressing both Antp and GFP appeared (Figure 3E–L’). In summary, these results indicate that the ectopic PSC cells in jumu mutants are derived from the PSC domain, and Jumu controls hematopoietic homeostasis by serving as a crucial negative regulator of Col+ PSC cell proliferation and ectopic expansion.

jumu is required for the prevention of ectopic PSC cells derived from the PSC domain during larval development.

(A–D) Immunostaining against the PSC cell marker Antp (green) in L1 larvae (A, B) and L2 larvae (C, D). (E–J’) The expanded Antp+ PSC cells (red) in jumuDf2.12 lymph glands show no co-localization with dome-GFP+ cells during the L1 larvae (E–F’), L2 larvae (G–H’) and early L3 (I–J’) stages. (K–L’) The expanded Antp+ PSC cells (red) in jumuDf2.12 lymph glands show no co-localization with Hml-GFP+ cells during the early L3 (K, K’) and L3 (L, L’) stages. Scale bars: 50 μm.

https://doi.org/10.7554/eLife.25094.004

Jumu expression in prohemocytes is required for preventing the ectopic expansion of PSC cells

To further investigate where and how Jumu regulates the number and organization of PSC cells, we first identified the expression pattern of Jumu protein in the lymph gland at different larval stages through anti-Jumu antibody staining. In wild-type larvae, Jumu is expressed in the entire lymph gland and mainly located in the nuclei and presents markedly higher expression in the PSC than in the other domains during the L1 to L3 larval stage (Figure 4A–F”); however, the signal was nearly abolished in jumu double heterozygotes (Figure 4E). Moreover, we found that Jumu is also expressed in cardiomyocytes and is colocalized with Antp in some cells (Figure 4A–A’’). A recent study suggested that Slit/Robo signaling from the cardiac tube is required to preserve the morphology and function of the PSC (Morin-Poulard et al., 2016). To determine whether the expansion of the PSC cells in jumu mutants is caused by the loss of jumu in cardiomyocytes, we used a heart-specific Gal4 driver (Hand-Gal4) to express jumu in jumuDf2.12. However, the PSC morphology defects detected in jumuDf2.12 were not rescued by the overexpression of jumu (Figure 4G,H and S). We then used tissue-specific Gal4 lines to achieve a forced expression of jumu in the jumuDf2.12 PSC niche (Antp-Gal4), MZ (dome-Gal4, TepIV-Gal4) and CZ (Hml-Gal4) and found that the overexpression of jumu in the PSC or CZ domain cannot rescue the ectopic expansion of PSC cells in jumuDf2.12 (Figure 4I,J,M,N and S). However, the overexpression of jumu using dome-Gal4 and TepIV-Gal4 effectively inhibited the generation of expanded PSC cells in the MZ and CZ (Figure 4K,L and S; Figure 5A,B and R). Moreover, the overexpression of jumu in the entire lymph gland using the pan-lymph gland specific driver Srp-Gal4 or the ubiquitous driver Tub-Gal4ts also rescued the PSC morphology defects detected in jumuDf2.12 (Figure 4O–S). Thus, these results indicate that Jumu protein in prohemocytes might prevent the ectopic expansion of PSC cells through a non-cell-autonomous mechanism.

Expression of Jumu in prohemocytes is required for preventing the expansion of PSC cells.

(A–D) Immunostaining against Jumu protein shows that Jumu (green) is expressed throughout the w1118 lymph gland and shows co-localization with Antp (red) during the entire larval development process (A–D). Jumu is also expressed in cardiomyocytes and colocalizes with Antp in some cells (arrow) (A’’). (E) The expression of Jumu (green) is obviously decreased in jumu double heterozygotes mutants. (F–F’’) Jumu (green) is expressed at high levels in Antp+ PSC cells (red). (G–R) The overexpression of jumu using Hand-Gal4 (heart specific), Antp-Gal4 (PSC niche specific) and Hml-Gal4 (CZ specific) in jumuDf2.12 lymph glands cannot inhibit the expansion of PSC cells (red) (H, J, N); however, the use of dome-Gal4 (MZ specific), Srp-Gal4 (entire lymph gland) or Tub-Gal4ts (entire lymph gland) to overexpress jumu in jumuDf2.12 lymph glands effectively prevents the expansion of PSC cells (L, P, R). (S) Quantification of the number of PSC cells (Antp+ cells). The data are presented in a box-and-whisker representation. NS, not significant, ***p<0.001 (Student’s t test). Scale bars: 50 μm (A–E), 20 μm (F–F’’), and 100 μm (G–R).

https://doi.org/10.7554/eLife.25094.005
Figure 5 with 1 supplement see all
Jumu in prohemocytes non-cell-autonomously regulates the expression of dMyc in the PSC.

(A–I) The knockdown of jumu in prohemocytes (TepIV>jumu RNAi) (C) or in the entire lymph gland (Srp>jumu RNAi, e33C>jumu RNAi) (F, I) causes the expansion of Antp+ PSC cells (red), and the overexpression of jumu in prohemocytes (TepIV>UAS-jumu) or in the entire lymph gland (Srp>UAS-jumu) does not affect the number of PSC cells (D, G). (J–L’) The knockdown of jumu in prohemocytes (TepIV>jumu RNAi) causes increased levels of dMyc (red) in PSC cells (green) (K, K’), but the dMyc level is not changed in TepIV>UAS-jumu (L, L’). (M–Q’) The expression of dMyc (red) is substantially increased in PSC cells (green) of the e33C>jumu RNAi (N, N’), jumuGE27806 (P, P’) and jumuDf2.12 mutants (Q, Q’). (R) Quantification of the number of PSC cells (Antp+ cells). (S) Quantification of the pixel intensity of dMyc (integrated density of the dMyc signal in the PSC divided by the PSC area). (T–V) The use of col-Gal4 to knock down dmyc can rescue the expanded PSC cells in jumuDf2.12. All of the quantification data are presented as box-and-whisker representations. NS, not significant, ***p<0.001 (Student’s t test). Dashed white lines outline the PSC in J-Q’. Scale bars: 50 μm.

https://doi.org/10.7554/eLife.25094.006

The loss of jumu in the MZ non-cell-autonomously induces increases in dMyc in the PSC

To further determine whether jumu expression in the MZ non-cell-autonomously prevents the expansion of PSC cells, we knocked down or overexpressed jumu using the MZ-specific drivers TepIV-Gal4 and dome-Gal4 and assessed the resulting PSC morphology. Similar to jumu mutants, the TepIV>jumu RNAi and dome>jumu RNAi mutants also showed scattered and increased numbers of PSC cells; however, the overexpression of jumu in MZ did not cause any defects in PSC morphology (Figure 5A–D,R; Figure 6A–C,E). We then used the pan-lymph gland specific driver Srp-Gal4 and e33C-Gal4 to knock down or overexpress jumu in the entire lymph gland and both Srp-Gal4>jumu RNAi and e33C>jumu RNAi displayed the scattered and increased numbers of PSC cells (Figure 5E–I,R). Because e33C>UAS-jumu was lethal during embryogenesis, we only detected the PSC of Srp>UAS-jumu and did not observe any changes in the morphology and cell number of the PSC domain (Figure 5G,R). These results strengthen the notion that Jumu protein located in the MZ mainly prevents the expansion and scattering of PSC cells toward the MZ and indirectly maintains the normal MZ development, but is not sufficient to initiate the inhibition of the proliferation of PSC.

Figure 6 with 3 supplements see all
Jumu cell-autonomously regulates the differentiation of blood cell progenitors by maintaining low expression levels of Col of the MZ.

(A–D) Immunostaining against Antp shows that the knockdown of jumu in the MZ causes ectopic and increased PSC cells (B), but the overexpression of jumu or col in the MZ does not change the number of PSC cells (C, D). (E) Quantification of the number of PSC cells (Antp+ cells). The data are shown through a box-and-whisker representation. NS, not significant, ***p<0.001 (Student’s t test). (F–I, K–N) The knockdown of jumu in prohemocytes does not cause an obvious reduction in plasmatocyte and crystal cell differentiation compared with the control (G, L), whereas the forced expression of jumu in prohemocytes causes increases in plasmatocyte and crystal cell differentiation (H, M). The overexpression of col suppresses the increases in plasmatocyte and crystal cell numbers in dome>GFP>UAS-jumu (I, N). (J, O) Quantification of plasmatocyte and crystal cell indexes in primary lobes. The data are presented in a box-and-whisker representation. NS, not significant, ***p<0.001 (one-way ANOVA). (P–U) High-exposure visualization of Col immunostaining (red) shows that compared with the controls, the Col levels of the MZ are increased in dome>GFP>jumu RNAi (Q, Q’) and in heterozygous jumu mutants (T, U) but are reduced in dome>GFP>UAS-jumu (R, R’). (W–Z’) Dlp (red) is expressed in the MZ of dome>GFP and is increased in dome>GFP>UAS-col (X, X’) and dome>GFP>jumu RNAi (Y, Y’); however, Dlp expression is reduced in dome>GFP>UAS-jumu (Z, Z’). (V, AA) Quantification of the pixel intensity of Col or Dlp (integrated density of Col or Dlp signal in the MZ divided by the area of the primary lobe). The data are presented in a box-and-whisker representation. **p<0.01, ***p<0.001 (Student’s t test). Scale bars: 50 μm.

https://doi.org/10.7554/eLife.25094.008

Slit/Robo signaling controls the clustering and number of PSC cells by regulating DE-cadherin (Shg) and Cdc42 expression and dMyc activity, respectively (Morin-Poulard et al., 2016). Notably, a deficiency in Slit/Robo signaling resulted in defects in the PSC similar to those obtained in jumu mutants, and both Slit, which is the canonical ligand of Drosophila Robo, and Jumu are expressed in the dorsal vessel (Morin-Poulard et al., 2016). Thus, we first asked whether Jumu protein in cardiomyocytes controls Slit/Robo signaling and, in turn, regulates both proliferation and clustering of the PSC. However, the knockdown or overexpression of jumu under the control of hand-Gal4 did not cause the abnormal PSC morphology (Figure 6—figure supplement 1A–D). This result suggests that Jumu does not directly regulate Slit/Robo signaling in the dorsal vessel. Moreover, the loss of Shg or the activation of Cdc42 in the PSC can induce the clustering of PSC cells (Morin-Poulard et al., 2016). Thus, we questioned whether Jumu protein in the MZ controls the PSC morphology by regulating the expression of Shg or the activation of Cdc42. Therefore, we first analyzed Shg expression in the PSC and found that the expression levels of Shg were not reduced in the PSC of jumuDf2.12 compared with the wild-type (Figure 5—figure supplement 1A–B’’), suggesting that the role of Jumu in the PSC morphology is independent of Shg. Moreover, the overexpression of the dominant negative form of Cdc42 (cdc42-DN) in the PSC of jumuDf2.12 did not rescue the increased number and clustering of the PSC cells (Figure 5—figure supplement 1C–E). Slit/Robo signaling controls PSC cell proliferation via dmyc, and promotes dmyc transcription through the negative regulation of the HSPG (heparin sulfate proteoglycan) Dally-like (Dlp) and BMP/Dpp signaling pathways (Morin-Poulard et al., 2016). We subsequently asked whether Jumu also controls the PSC cell number by regulating dMyc expression. The use of TepIV-Gal4 to knock down or overexpress jumu did not change the expression levels of dMyc in the MZ and CZ; however, we observed increased dMyc expression in the PSC cells of TepIV>jumu RNAi mutants (Figure 5J–L’, S). In addition, we detected dMyc in the lymph gland of e33C>jumu RNAi and jumu mutant larvae. The loss of jumu throughout the lymph gland did not cause an obvious reduction of dMyc in the MZ and CZ but caused an observable increase in the dMyc levels in PSC cells (Figure 5M–Q’, S). Moreover, the knockdown of dmyc in the PSC of jumuDf2.12 effectively suppressed the scattering of and the increase in the number of PSC cells (Figure 5T–V). These results indicate that Jumu protein in prohemocytes might be required to prevent the overexpression of dMyc in the neighboring PSC cells in a non-cell-autonomous manner and that it accordingly inhibits the overproliferation and ectopic presence of PSC cells in an indirect manner. The loss of BMP/Dpp activity leads to increased numbers of PSC cells through the up-regulation of the expression of dMyc, and Dpp signaling is regulated directly by Dlp (Pennetier et al., 2012; Morin-Poulard et al., 2016). Thus, we subsequently detected whether Jumu controls dMyc expression by regulating Dlp and Dpp signaling. However, the normal expression of Dlp and phosphorylated Mad (P-Mad), which targets the activity of the BMP/Dpp signaling pathway, were detected in the PSC cells of jumuDf2.12 (Figure 5—figure supplement 1F–I’’). Taken together, these results suggest that Jumu in MZ might control the morphology of the PSC by indirectly regulating dMyc expression in PSC cells through a Slit/Robo and BMP/Dpp-independent mechanism.

Jumu cell-autonomously controls the maintenance of blood cell progenitors by regulating the low levels of Col in the MZ

Because Jumu is expressed throughout the lymph gland, we investigated the role of jumu in the various domains of the lymph gland. We knocked down or over expressed jumu under the control of a driver specific to differentiating cells (Hml-Gal4), but this had no effect on the size of the PSC niche or on prohemocyte differentiation (Figure 6—figure supplement 1E–L). To determine whether Jumu is involved in prohemocyte differentiation in a cell-autonomous manner in addition to its non-cell-autonomous role in the regulation of PSC morphology, we used the dome-Gal4 driver to knock down or overexpress jumu in the MZ. Although an expansion of the neighboring PSC population was observed (Figure 6A,B and E), the knockdown of jumu in prohemocytes did not obviously reduce plasmatocyte or crystal cell differentiation (Figure 6F,G,J,K,L and O). However, the overexpression of jumu in the MZ resulted in massive differentiation of plasmatocytes and crystal cells and a reduction in hematopoietic progenitors (Figure 6H,J,M and O). Because dome-Gal4 is also expressed in the brain, and some signaling pathways derived from the brain also control the differentiation of prohemocytes (Shim et al., 2013), we examined the possible involvement of the brain by overexpressing or knocking down jumu using the brain-specific driver elav-Gal4, but this did not result in an abnormal differentiation of prohemocytes (Figure 6—figure supplement 1M–P). Because the forced expression of jumu in the MZ was not sufficient to reduce the number of PSC cells (Figure 6C,E), the overdifferentiation of prohemocytes could not be indirectly caused by the reduction in the number of neighboring PSC cells. Hh signaling from the PSC is required for the maintenance of hematopoietic progenitors (Mandal et al., 2007). Thus, we subsequently detected Hh expression in the PSC using the HhF4-GFP reporter gene. However, the overexpression of jumu in the MZ did not reduce the HhF4-GFP expression levels in the PSC, and the knockdown of jumu in the MZ also did not increase the HhF4-GFP expression levels in the PSC (Figure 6—figure supplement 2). Based on these results, jumu might control the differentiation of prohemocytes through other cell-autonomous mechanisms.

To further confirm whether Jumu cell-autonomously affects prohemocyte differentiation, we knocked down or overexpressed jumu in lymph gland-specific clonal populations of hemocytes using HHLT-Gal4 (Hand-Hml lineage-traced Gal4). A proportion of P1+ differentiated plasmatocytes were colocalized with the wild-type clones (GFP+) generated by HHLT-Gal4 (Figure 6—figure supplement 3A–A’’). However, most P1+ differentiated plasmatocytes did not overlap with clonal cells lacking jumu (GFP+) (Figure 6—figure supplement 3B–B’’), suggesting that the loss of jumu in clonal cells can inhibit hemocyte differentiation cell-autonomously. Conversely, most P1+ cells were colocalized with clonal cells overexpressing jumu (GFP+) (Figure 6—figure supplement 3C–C’’), suggesting that the overexpression of jumu in clonal cells can promote hemocyte differentiation in a cell-autonomous manner. Altogether, the HHLT clonal analyses indicate the cell-autonomous requirement of Jumu in controlling lymph gland hemocyte differentiation.

A recent study suggested that in addition to its expression in the PSC, Col is also expressed at low levels in prohemocytes and is required for preventing the differentiation of prohemocytes (Benmimoun et al., 2015). The above-described results show that the loss of col in the lymph gland of the jumu mutants also rescued the increased MZ size and the reduced differentiation of plasmatocyte phenotypes in addition to inhibiting the expansion of PSC cells (Figure 2M–T). Thus, we evaluated whether Jumu could cell-autonomously inhibit the expression of Col in prohemocytes in addition to preventing the ectopic expansion of Col+ PSC cells. The intensity of the Col signal in the MZ was quantified. As predicted, the expression of Col was clearly increased in the dome>jumu RNAi lymph gland and significantly reduced in the dome>UAS-jumu lymph gland (Figure 6P–R’, V). Similarly, jumu mutants also displayed an increased Col expression signal in the MZ (Figure 6S–V). A previous study suggested that Col regulates the expression of (Dlp) in the PSC (Pennetier et al., 2012). We found that Dlp is also expressed in the MZ (Figure 6W,W’), and the overexpression of col in the MZ enhanced the production of Dlp protein, an effect that was accompanied by the expansion of the MZ (Figure 6X,X ’and AA). Consistent with this phenotype, we found increased and reduced levels of Dlp expression in the MZ of dome>jumu RNAi and dome>UAS-jumu mutants, respectively (Figure 6Y–AA). In addition, the dome-driven expression of col was sufficient to suppress the plasmatocyte and crystal cell differentiation phenotypes associated with jumu overexpression in prohemocytes (Figure 6I,J,N and O). These results demonstrate that Jumu cell-autonomously promotes the differentiation of prohemocytes by inhibiting the expression of Col in the MZ. Moreover, the overexpression of Col in prohemocytes did not induce the ectopic expansion of PSC cells (Figure 6D,E); thus, the expanded PSC cell phenotype caused by jumu loss-of-function is independent of the increased expression of Col in the MZ.

Jumu cell-autonomously controls the size of the PSC by regulating the expression of dMyc

To test whether jumu is also cell-autonomously required in the PSC, we used a col-Gal4 driver to knock down and overexpress jumu in the PSC. The expression of dome-MESO, a reporter of JAK-STAT signaling, was used to identify prohemocytes. In contrast to knockdown in the MZ or the entire lymph gland, the knockdown of jumu in the PSC (col>jumu RNAi) led to a reduction in the number of PSC cells and the overdifferentiation of plasmatocytes at the expense of the MZ; consistently, the overexpression of jumu in the PSC (col>UAS-jumu, Antp>UAS-jumu) substantially increased the number of PSC cells and resulted in an expansion of the MZ at the expense of hemocyte differentiation (Figure 7A–L; Figure 7—figure supplement 1A–C). Moreover, we also observed the similar phenotypes by clonally knocking down or overexpressing jumu in the PSC using HHLT-Gal4 (Figure 6—figure supplement 3D–F). These results suggest that Jumu cell-autonomously controlsthe size and cell number of the PSC. A previous study suggested that a reduction in the Col levels in the PSC can cause an increase in the number of PSC cells by down-regulating Dlp and Hh expression (Pennetier et al., 2012). The above-described results indicate that jumu negatively regulates the expression of Col in prohemocytes. Thus, we first asked whether the overexpression of jumu also reduces the expression of Col in the PSC, thereby indirectly increasing the number of PSC cells. However, although the number of Col+ PSC cells was significantly increased, the average pixel intensity of the Col signal per PSC cell was not reduced in the Antp>UAS-jumu mutant (Figure 7—figure supplement 1D–F). In addition, the overexpression of jumu in the PSC did not result in a loss of Hh and Dlp expression, which are directly regulated by Col (Figure 7—figure supplement 1G–K’). Dpp signaling is regulated directly by Dlp and indirectly by Col (Pennetier et al., 2012). We subsequently detected whether Jumu protein in the PSC controls the proliferation of PSC cells by suppressing the activity of the Dpp signaling pathway. The normal expression level of P-Mad could be detected in the Antp>UAS-jumu mutant (Figure 7—figure supplement 1L–M”). These results indicate that jumu cell-autonomously controls the size of the PSC niche through a Col-independent and Dpp-independent mechanism.

Figure 7 with 3 supplements see all
Jumu cell-autonomously controls the size of the PSC by regulating the expression of dMyc and indirectly affects prohemocyte differentiation.

(A–C) Immunostaining against Antp (red) shows that jumu RNAi in the PSC (col>GFP>jumu RNAi) causes a reduction in the number of PSC cells (B), but the overexpression of jumu in the PSC (col>GFP>UAS-jumu) can substantially increase the size and cell number of the PSC niche (anti-Antp, red) (C). (D–F) The use of dome-MESO (LacZ) (red), which serves as a marker of prohemocytes, shows a reduction of the MZ in col>GFP>jumu RNAi (E) and an expansion of the MZ in col>GFP>UAS-jumu (F). (G–I) Plasmatocyte differentiation (anti-P1, red) is increased in col>GFP>jumu RNAi and reduced in col>GFP>UAS-jumu. (J) Quantification of the number of PSC cells (Antp+ cells). (K) Quantification of the proportion of the primary lobes occupied by prohemocytes (dome-MESO+ MZ area divided by the primary lobe area). (L) Quantification of the plasmatocyte index in primary lobes. (M–O’) Compared with the control (col>GFP), the expression of dMyc (red) is reduced in the PSC of col>GFP>jumu RNAi (N, N’) and significantly increased in col>GFP>UAS-jumu (O, O’). Dashed white lines outline the PSC. (P–Q’’) The levels of Jumu (green) are not decreased in the PSC cells (red) of Antp>dmyc RNAi. (R, S) Quantification of the pixel intensity of dMyc or Jumu (integrated density of dMyc or Jumu signal in the PSC divided by the PSC area). (T, U) The knockdown of dmyc in the PSC (col>GFP>dmyc RNAi) reduces the number of PSC cells (anti-Antp, red) (T) and suppresses the increase in PSC cells observed in col>GFP>UAS-jumu (U). (V) Quantification of the number of PSC cells (Antp+ cells). The quantification data are presented as box-and-whisker representations. NS, not significant. **p<0.01 and ***p<0.001 (Student’s t test) in J-L, R and S; ***p<0.001 (one-way ANOVA) in V. Scale bars: 20 μm (A–C, M’–O’) and 50 μm (D–I, M, P–Q’’, T, U).

https://doi.org/10.7554/eLife.25094.012

Recent studies have suggested that some genes and signaling pathways control the PSC cell number and size by regulating dMyc expression in the PSC (Pennetier et al., 2012; Sinenko et al., 2009; Benmimoun et al., 2012; Tokusumi et al., 2015). Moreover, the above results suggest that Jumu protein in the MZ non-cell-autonomously prevents an increase in dMyc expression in the PSC. In addition, the knockdown of dmyc or jumu in the PSC resulted in a similar phenotype of fewer PSC cells, and correspondingly, the overexpression of dmyc or jumu in the PSC also caused increased numbers of PSC cells. Thus, we hypothesized that the existence of a regulatory relationship between Jumu and dMyc. Therefore, we first detected the expression of dMyc in jumu loss- and gain-of-function lymph glands. Surprisingly, a reduced level of dMyc was observed in the PSC of col>jumu RNAi lymph glands, whereas an increased level of dMyc was observed in col>UAS-jumu lymph glands (Figure 7M–O’, R). However, the knockdown of dmyc in the PSC did not result in an obvious absence of Jumu expression in the PSC (Figure 7P–Q”, S). Additionally, the knockdown of dmyc in PSC cells can suppress the increase in PSC cells caused by the overexpression of jumu (Figure 7T–V). These results indicate that Jumu is a direct or indirect positive regulator of dMyc expression in the PSC and promotes the overproliferation of PSC cells.

The above results show that Jumu in the MZ does not cell-autonomously regulate dMyc expression in the MZ (Figure 5J–L’). Similarly, the loss or overexpression of jumu in the CZ did not cause obvious changes in the dMyc levels (data not shown). These results suggest that the role of Jumu in the regulation of dMyc expression is specifically restricted to the PSC of the lymph gland. To further address whether Jumu can directly regulate dmyc transcription, we subsequently detected the protein and transcription levels of the dmyc gene in circulating hemocytes and found that the overexpression of jumu in circulating hemocytes led to increases in the dMyc protein level and the transcription of the dmyc gene (Figure 8A,B). We then analyzed the 2000 bp promoter sequence of the dmyc gene using Ensembl, a web system for predicting promoters, and found 23 putative FKH transcription factor- binding sites on the promoter sequence using JASPAR, a database of transcription factor binding profiles (Figure 8C,D). Because Jumu contains a conserved FKH DNA-binding domain, we then examined whether Jumu regulates the expression of dmyc at the transcriptional level by directly binding to its promoter via the FKH domain in vivo through a chromatin immunoprecipitation (ChIP) assay. We designed specific primers for possible FKH domain-binding sites in the dmyc promoter (Figure 8D). Total lysates from S2 cells stably expressing Flag-Jumu were used as an input positive control, along with immunoprecipitation with IgG (as a negative control) or antibodies against Flag, and the immunoprecipitated chromatin fragments were detected using PCR. As shown in Figure 8E, using anti-Flag antibodies compared with IgG control, there was no obvious positive signal at the dmyc promoter region of −1993 bp to −105 bp, but a specific enrichment of the dmyc gene region of −46 bp to +235 bp was observed. This fragment contains three putative FKH-binding sites, one inside the promoter region and two inside the 5’UTR (Figure 8F). To further determine the binding site recognized by Jumu in the dmyc promoter, we mutated each of the three putative FKH-binding sites at two positions crucial for the efficiency of FKH binding (Figure 8G). The Fragment from −643 to +467 of the dmyc promoter was cloned into the luciferase reporter vector pGL3, and a reporter assay showed that the co-transfection of S2 cells with a jumu expression construct and pGL3-dmyc (WT-643/+467) resulted in a nearly two-fold induction of reporter gene activity compared with the control. The disruption of the Site 1 (Mut1) led to an almost three-fold reduction of reporter gene activity compared with WT-643/+467, whereas the mutation of the Site 2 (Mut2) and Site 3 (Mut3) did not affect the reporter gene activity. Taken together, these results suggest that Jumu can be endogenously recruited to the conserved FKH-binding sites located in −27/–17 of the dmyc promoter via its FKH domain, and further strengthen the possibility that Jumu positively up-regulates dmyc expression by directly binding to the dmyc promoter.

Jumu regulates the expression of dmyc by binding to its FKH-binding site.

(A, B) The overexpression of jumu in circulating hemocytes (Hml>GFP>UAS-jumu) up-regulates dmyc expression at the translation (A) and transcription levels (B). ***p<0.001 (Student’s t test). Scale bars: 20 μm in A. (C) Optimal FKH DNA-binding consensus motif obtained from the JASPAR database. (D) Schematic of the dmyc gene promoter locus showing putative FKH-binding sites (red lines) and DNA fragments from ChIP PCR amplification (black lines). (E) S2 cells stably expressing Flag-jumu were used for ChIP experiments to screen for the presence of endogenous Jumu on the dmyc promoter region. The enrichment of target DNA fragments relative to IgG is shown on an agarose gel. ChIP-PCR demonstrates that Jumu can bind to the dmyc gene in the region from −46 bp to +235 bp. (F) The analysis of the dmyc gene region from −46 bp to +235 bp contains one putative FKH site (red) inside dmyc promoter region and two putative FKH sites (green) inside the 5’UTR of dmyc. (G) Three putative FKH-binding sites (in −46 bp to +235 bp of the dmyc gene region) were mutagenized. The quantification of the results from a dual-luciferase reporter assay of S2 cells transfected with pMK33-jumu (white bars) or empty pMK33 vector (red bars) shows the activation of the wild-type (WT) dmyc promoter and that the mutagenesis of the Mut1 site, but not of the Mut2 or Mut3 sites, prevents activation of the dmyc promoter. The normalized luciferase values are the ratios of firefly-to-Renilla luciferase. The data are presented as the means ± S.E.M. NS, not significant, *p<0.1, **p<0.01, ***p<0.001 (Student’s t test).

https://doi.org/10.7554/eLife.25094.016

The above results show that the knockdown of jumu in the MZ and PSC has oppositing effects on the expression of dMyc and PSC morphology. We subsequently analyzed the spatial-temporal regulation of Jumu on PSC development during larval development. First, the expression pattern of dMyc during larval development was detected. dMyc is expressed at high levels in MZ and CZ cells but at low levels in PSC cells, and its expression pattern remains the same at all stages of larval development (Figure 7—figure supplement 2). Similarly, the expression pattern of Jumu does not change during larval development (Figure 4A–F’’), suggesting that Jumu protein in the PSC might be responsible for the maintenance of dMyc expression. We then used a temporal and regional gene expression targeting system (Gal80ts/Gal4 expression system) (McGuire et al., 2003) to knock down jumu in the PSC at different larval stages (Figure 7—figure supplement 3M). Similar to the phenotype observed after the knockdown of jumu in the PSC from embryogenesis, the knockdown of jumu in the PSC from L1 larval stage or L2 stage also caused the reductions in the PSC cell number to different degrees; in fact, the earlier knock down of jumu resulted in a greater reduction in the PSC cell number (Figure 7—figure supplement 3A–F,N). This result indicates a continuous role for Jumu in PSC cell proliferation during larval development. The above results show that the loss of jumu in the MZ is responsible for the ectopic PSC cells detected in the early larval stages (Figure 3A–D). We then used the Gal80ts/Gal4 expression system to knock down jumu in the MZ at different larval stages (Figure 7—figure supplement 3M). The loss of jumu in the MZ from L1 larval stage or L2 stage also caused the ectopic expansion of PSC cells, and an earlier knockdown of jumu resulted in an increased ectopic expansion of PSC cells (Figure 7—figure supplement 3G–L,O). This result further confirms that the expression of Jumu in the MZ prevents the expansion of PSC cell toward the MZ throughout larval development. In conclusion, the expression of Jumu in the MZ and PSC simultaneously maintains the normal development of the PSC. Moreover, changes in the jumu expression levels in the entire lymph gland result in defects in PSC morphology similar to those detected in response to changes in the jumu levels in the MZ but opposite to those caused by changes in the jumu levels in the PSC, indicating that the expression of Jumu in the MZ might play a more prominent role in regulating PSC morphology when jumu is lost or overexpressed in both the PSC and the MZ.

The loss of jumu in the entire lymph gland leads to the generation of lamellocytes through the activation of Toll signaling

The above-described results suggest that the severe loss of jumu can induce an increase in lamellocytes (Figure 1K–N). We subsequently investigated where and how Jumu controls the generation of lamellocytes. The knockdown of jumu in different zones of the lymph gland did not cause obvious increases in the lamellocytes; in fact, only less than 20% of the lymph gland showed a few lamellocytes (n > 30) (Figure 9—figure supplement 1A–C). However, using e33C-Gal4 to knock down jumu in the entire lymph gland led to the generation of numerous lamellocytes in more than 50% of the lymph gland (n = 32), and similar to jumu mutant, e33C>jumu RNAi also showed reduced numbers of crystal cells (Figure 9A–E). Similarly, more than 60% of lymph gland of the Srp> jumu RNAi lymph gland also showed obvious increased numbers of lamellocytes (n = 36) (Figure 9F,G). Our previous study demonstrated that the overexpression of jumu in both hemocytes and the fat body induces the deposition of hemocytes and melanotic nodules through the activation of Toll signaling (Zhang et al., 2016). The constitutive activation of Toll signaling in hemocytes or the fat body can lead to the differentiation of lamellocytes (Schmid et al., 2014). Moreover, a recent study show that the Toll signaling transcription factors Dorsal and Dorsal-related immunity factor (Dif) are expressed in the lymph gland and that the ectopic expression of both factors in the PSC can cause the generation of lamellocytes in the lymph gland (Gueguen et al., 2013). Thus, we asked whether the lamellocytes caused by the loss of jumu in the lymph gland are linked to Toll signaling. The expression and localization of Dorsal and Dif were detected via antibody staining, and interestingly, we found that Dorsal was enriched in nuclei throughout the lymph glands in the control animals (Srp-Gal4), whereas Dif was located in the cytoplasm and showed high expression levels in the PSC and lower signals in the MZ and CZ (Figure 9L,R). To rule out the effect of the transgenes background of the Srp-Gal4 line on the localization of Dorsal and Dif, we also detected the expression of Dorsal and Dif in the wild-type (w1118) lymph gland and observed the same results (data not shown). The knockdown of jumu in the entire lymph gland did not affect the expression and localization of Dorsal but caused an activation of Dif, mainly in the CZ and partly in the MZ but not in the PSC; in fact, most cells in the CZ showed obvious nuclei enrichment of Dif (Figure 9M,S; Figure 9—figure supplement 2E–F’). In addition, Dorsal and Dif were not enriched in the nuclei of circulating cells of the Srp>jumu RNAi mutant (Figure 9—figure supplement 2A–D), suggesting that the knockdown of jumu might activate the Toll signaling specifically in the lymph gland. We found that the activation of the Toll pathway throughout the through the overexpression of Toll10b under the control of Srp-Gal4 also caused a robust increase in the lamellocyte in more than 80% of the lymph glands (n = 30), and also led to enrichment of Dif in the nuclear in the MZ and CZ without affecting the Dorsal expression and localization (Figure 9N,T; Figure 9—figure supplement 2G,G’). To further confirm whether Toll signaling is involved in the generation of lamellocytes in the Srp>jumu RNAi mutant, we performed rescue experiment with a dif mutant. The loss of dif effectively reduced the lamellocytes in the Srp>jumu RNAi mutant; specifically, only less than 10% of the lymph gland generated a few lamellocytes, and the nuclear enrichment of Dif was abolished (n = 36) (Figure 9J,V). Taken together, these results suggest that the activation of Dif in the lymph gland can spontaneously induce lamellocyte differentiation and that Jumu can prevent the activation of Dif and consequently maintain the normal hemocyte differentiation in the lymph gland.

Figure 9 with 2 supplements see all
Loss of jumu in the entire lymph gland induces the generation of lamellocytes through the activation of Toll signaling.

(A–D) Immunostaining for L1 (red) and Hnt (red) shows that the knock down of jumu in the entire lymph gland increases the number of lamellocytes (B) and reduces the number of crystal cells (D). (E) Quantification of the number of PSC cells (Antp+ cells). The data are presented in a box-and-whisker representation. ***p<0.001 (Student’s t test). (F–K) Immunostaining for L1 (red) shows that only less than 20% of lymph glands of the Srp-Gal4 control display a few lamellocytes (n = 24) (F), but numerous lamellocytes are generated in more than 60% of the lymph glands of Srp>jumu RNAi (n = 36) (G) and in more than 80% of the lymph glands of Srp>Toll10b (n = 30) (H) and Srp>UAS-col (n = 26) (I). The deficiency of dif and col in Srp>jumu RNAi clearly reduces the number of lamellocytes, and only less than 10% (n = 36) (J) and 20% of the lymph glands (n = 32) (K) generated a few lamellocytes, respectively. (L–Q) Immunostaining for Dorsal (green) shows that Dorsal is enriched in nuclei throughout the lymph glands of the control animals (L), and the expression and localization of Dorsal is not changed in the crosses (M–Q). (R–W) Immunostaining for Dif (green) shows that Dif is expressed in the cytoplasm of cells throughout the lymph glands of the control animals (R) but shows nuclear enrichment mainly in the CZ and partly in the MZ of Srp>jumu RNAi (S), Srp>Toll10b (T) and Srp>UAS-col (U). The deficiency of dif (V) and col (W) in Srp>jumu RNAi clearly reduces the nuclear enrichment of Dif. The regions indicated by the arrows are magnified. Dashed white lines outline the edges of the primary lobes. Scale bars: 100 μm.

https://doi.org/10.7554/eLife.25094.017

Similar to the phenotype caused by the loss of jumu in the lymph gland, a previous study indicated that the ectopic expression of col in the entire lymph gland also spontaneously triggers lamellocyte differentiation at the expense of crystal cells spontaneously (Figure 9I) (Crozatier et al., 2004). Moreover, we found that the forced expression of col in different zones of the lymph gland also did not cause an obvious increase in the lamellocytes, with less than 10% of the lymph gland showing a few lamellocytes (n > 30) (Figure 9—figure supplement 1D–F). The above results suggest that Jumu negatively regulates Col expression; thus, we asked whether Col is also involved in the activation of Toll signaling. As expected, col overexpression in the entire lymph gland also resulted in Dif enrichment, mainly in the nuclei of CZ cells, but did not affect Dorsal expression (Figure 9O,U; Figure 9—figure supplement 2H,H’). Moreover, the knockdown of col in Srp>jumu RNAi mutants also inhibited lamellocyte differentiation and nuclear Dif enrichment (Figure 9K,Q and W). Altogether, these results demonstrate that the loss of jumu in the entire lymph gland might indirectly activate Toll signaling by up-regulating the expression of Col, consequently inducing lamellocyte differentiation.

Discussion

In recent years, the Drosophila lymph gland has been extensively studied as a model for investigating the genetic control of hematopoiesis in flies and humans. The more recent results suggest that the development and maintenance of Drosophila hematopoietic progenitors are associated with niche interactions and systemic interactions (Krzemień et al., 2007; Mandal et al., 2007; Mercier et al., 2012; Shim et al., 2013). Moreover, some conserved molecules and regulatory networks controlling lymph gland homeostasis have been gradually elucidated.

In this study, we showed that Jumu is expressed in the entire lymph gland and regulates the differentiation of the lymph gland via multiple regulatory mechanisms. It has been demonstrated that Jumu regulates nucleolar morphology and function and chromatin organization and that the correct genetic dose is required for nucleolar integrity and correct nucleolar function (Hofmann et al., 2010). Here, we found that Jumu also regulates the hemocyte differentiation of the lymph gland in a dose-dependent manner. The change in the level of jumu expression in the entire lymph gland substantially alters the composition of the hemocyte population and the morphology of the lymph gland. The loss of one copy of jumu leads to the expansion of Col+ PSC cells and to a reduced differentiation of plasmatocytes and crystal cells (Figure 10A). The loss of two copies of jumu induces a more severe phenotype of ectopic Col+ PSC cells throughout the primary and secondary lobes of the lymph gland, favors lamellocyte differentiation at the expense of crystal cells, and causes obvious overgrowth of the secondary lobes (Figure 10A). Notably, the obvious lamellocyte differentiation phenotype was only observed in larvae in which jumu was knocked in the entire lymph gland and not in the larvae in which jumu was knocked down in different specific zones. This phenomenon suggests that the generation of lamellocytes might require a systemic deficiency of jumu in the entire lymph gland and that local jumu expression might be sufficient to prevent lamellocyte differentiation. Our results suggest that the loss of jumu in the entire lymph gland induces lamellocyte generation through the activation of Toll signaling (Figure 10B). Our previous study has showed that the overexpression of jumu in both circulating hemocytes and the fat body activates the Toll pathway in hemocytes that were deposited on the fat body, and subsequently leads to the formation of melanotic nodules (Zhang et al., 2016). These results suggest that the correct genetic dose of jumu is important for the activation of Toll-dependent immune response and chronic inflammation. Moreover, several pathways, such as the JAK/STAT, JNK, Insulin/TOR and Spi/EGFR signaling pathways, have been reported to be directly involved in the differentiation of lamellocytes in different zones of the lymph gland (Krzemień et al., 2007; Owusu-Ansah and Banerjee, 2009; Sinenko et al., 2011). However, although the role of Toll signaling in the immune response and larval hematopoiesis has been investigated widely, most studies focused on the fat body and hemolymph, and few studies investigated the lymph gland. Here, we found that in contrast to its expression in circulating hemocytes, Dorsal is highly expressed in nuclei in the lymph gland, suggesting that it might be involved in lymph gland development and homeostasis in addition to participating in the Toll-dependent immune response. Different from Dorsal, Dif is expressed in the cytoplasm of cells in the lymph gland and becomes enriched in nuclei upon activation of the Toll pathway. This result suggests that similar to its role in the fat body and circulating hemocytes, Dif might also mainly participate in the Toll-mediated immune response. Toll signaling in the PSC also non- cell-autonomously influences fate choice in basal and parasite-activated hematopoiesis (Gueguen et al., 2013). In addition, the Toll ligand, Spz protein and SPE transcripts are expressed in cells of the lymph gland lobes (Paddibhatla et al., 2010; Mulinari et al., 2006; Jang et al., 2006). These findings indicate that Toll signaling might play an important role in the regulation of lymph gland hematogenesis. However, the regulatory mechanism of Toll signaling on the development and immune response of the lymph gland remain to be addressed. Moreover, we found that ectopic expression of col throughout the lymph gland also leads to the activation of Toll signaling and that Jumu might control the Toll signaling by regulating col expression (Figure 10B). Some elements, such as fly IkB, cact, the SUMO conjugase and Ubc9, can balance the activation of immune signaling, the loss of function of these factors result in constitutive activation of Toll signaling in the fat body and circulating hemocytes and indirectly causes chronic inflammation (Paddibhatla et al., 2010). Similarly, the correct genetic dose of Jumu and Col might maintain the normal immune homeostasis of the lymph gland and indirectly prevent the development of chronic inflammation. Future studies will address the mechanism through which Jumu and Col regulate the activation of Toll signaling in the lymph gland.

Model of the role of Jumu in lymph gland hematopoiesis.

(A) Schematic representation showing that the fate of hemocyte differentiation is determined by the level of jumu expression in the entire lymph gland. The loss of one copy leads to the overproliferation of lymph gland hemocytes, the expansion of PSC cells and a reduction in the differentiation of plasmatocytes and crystal cells. The loss of two copies induces a more severe phenotype of PSC cell expansion throughout the primary and secondary lobes of the lymph gland and favors lamellocyte differentiation. Furthermore, the loss of two copies also leads to the overgrowth of the secondary lobes of the lymph gland. (B) Jumu protein in the entire lymph gland prevents the activation of Toll signaling by down-regulating the expression of Col, consequently preventing the differentiation of lamellocytes. (C) Schematic representation showing the mechanisms through which Jumu regulates PSC homeostasis and the maintenance of prohemocytes. In the PSC, Jumu controls the proper PSC cell proliferation by regulating the expression of dMyc as a positive transcription factor. In the MZ, Jumu cell-autonomously maintains the differentiation of prohemocytes via the repression of Col levels. Moreover, Jumu protein in the MZ prevents the overexpression of dMyc in the PSC in a non-cell-autonomous manner and consequently suppresses the expansion of PSC cells.

https://doi.org/10.7554/eLife.25094.020

The role of the PSC in prohemocyte maintenance remains controversial. Previous studies suggest that the PSC functions as a hematopoietic niche and is essential for the maintenance of prohemocytes (Krzemień et al., 2007; Mandal et al., 2007). Conversely, recent studies indicate that ablation of the PSC does not affect prohemocyte maintenance and that the PSC is dispensable for the maintenance of prohemocytes under laboratory breeding conditions (Benmimoun et al., 2015; Oyallon et al., 2016). However, changes in the levels of certain proteins or signal elements from the PSC indeed affect prohemocyte differentiation. It has been suggested that dMyc is expressed at low levels in PSC cells and that the forced expression of dMyc selectively in the PSC leads to an increased number of PSC cells and further results in an expansion of the prohemocyte pool at the expense of hemocyte differentiation (Pennetier et al., 2012). In addition, Slit/Robo signaling, Dpp/Wg signaling and the genetic regulators InR, Bam, and Rbf control the number of PSC cells by regulating the expression of dMyc (Morin-Poulard et al., 2016; Pennetier et al., 2012; Sinenko et al., 2009; Tokusumi et al., 2015). Our results indicate that Jumu cell-autonomously ensures proper PSC cell proliferation by positively regulating dMyc expression in the PSC and that it further affects the maintenance of prohemocytes in an indirect manner (Figure 10C). In addition, through an in vitro experiment, we also confirmed that Jumu promotes the transcription of the dmyc gene via directly binding to its promoter. Moreover, Jumu shows no obvious regulatory effect on the dMyc level in the MZ and CZ, which suggests that the role of Jumu in the regulation of dMyc expression is zone-specific. In addition to the cell-autonomous mechanism, Jumu also controls the numbers of PSC cells in a non-cell-autonomous manner. The loss of jumu in the entire lymph gland or the MZ can lead to the ectopic expansion of PSC cells, and this effect is accompanied by the overexpression of dMyc in these cells. This result suggests that Jumu might regulate the expression of some regulatory factors in the MZ that can deliver signals to neighboring PSC cells and indirectly prevent the overexpression of dMyc, thereby maintaining the proper PSC cell number and location (Figure 10C). However, overexpression of jumu in the MZ alone is not sufficient to reduce dMyc expression and the number of PSC cells. In fact, this result indicates that Jumu protein in the MZ might mainly function in the maintenance of the normal development margin between the MZ and the PSC and further maintains the homeostasis of each zone of the lymph gland but does not directly regulate proliferation in the PSC in a spontaneous manner. It has been suggested that Slit from the cardiac tube controls PSC morphology and that inter-organ crosstalk between the cardiac tube and the PSC is required to preserve the normal PSC morphology (Morin-Poulard et al., 2016). Similarly, Jumu might also participate in the inter-zone communication between the MZ and the PSC and thereby preserve the normal development of each zone. Notably, different from the PSC phenotypes caused by the loss of jumu in the MZ or in the entire lymph gland, the additional PSC cells induced by the overexpression of jumu or dmyc in the PSC are all located in the original PSC domain rather than being scattered through the MZ or CZ, further strengthening the notion that Jumu protein in the MZ and the PSC regulate the distribution of PSC cells through different mechanisms. The relationship between Jumu and the known signaling pathways associated with PSC development remains to be addressed.

In addition to regulating the homeostasis of the PSC, Jumu also maintains prohemocyte differentiation in a cell-autonomous manner by preventing the overexpression of Col in the MZ (Figure 10C). More recently, it was shown that the EBF transcription factor Col is also expressed at low levels in prohemocytes as well as in the PSC and that Col directly promotes the maintenance of Drosophila blood cell progenitors independently of the niche (Benmimoun et al., 2015; Oyallon et al., 2016). The overexpression of jumu in the MZ is sufficient to reduce the Col level and induce an increased differentiation of prohemocytes. However, we found that although the knockdown of jumu in the MZ causes an increase in the Col level, the differentiation of plasmatocytes and crystal cells is not markedly reduced. We speculate that this phenomenon might be caused by the dose-dependent function of jumu and that the RNAi-induced decrease in the jumu expression level in the MZ is insufficient to obtain a sufficient expression of Col to inhibit the differentiation of prohemocytes. Moreover, it has been suggested that Col protein in the PSC controls the number of PSC cells by activating the expression of Dally-like (Dlp) (Pennetier et al., 2012). In this study, we showed that Col also positively regulates the expression of Dlp in the MZ (Figure 10C). Different from its role in the MZ, ectopic expression of col throughout the lymph gland leads to the generation of lamellocytes and reduced crystal cells but does not affect plasmatocyte differentiation (Crozatier et al., 2004); interestingly, these phenotypes were also observed in jumu double heterozygotes but not in jumu heterozygotes. These results suggest that the correct expression levels and regions of jumu or col are required for the normal lymph gland development. Further work is needed to decipher the regulation and the mechanisms of action of Col in the maintenance of lymph gland homeostasis. Moreover, the signals associated with jumu and other transcription factors that regulate col expression remain to be addressed. A recent study showed that Ebf2, the mammalian ortholog of Col, can promote hematopoietic stem cell (HSC) maintenance by controlling the development of osteoblastic cells (Kieslinger et al., 2010). Moreover, it has been shown that the members of the forkhead-box (FOX) transcription factor family in human and mouse play crucial roles in various aspects of immune regulation, from lymphocyte survival to thymic and embryonic development and cell cycle regulation (Coffer and Burgering, 2004; Lehmann et al., 2003; von Both et al., 2004; Martínez-Gac et al., 2004). Foxn1, the mammalian ortholog of Jumu, has been shown to regulate the growth and differentiation of thymic epithelial cells in mouse. Foxn1-knockout mice show congenital hairlessness, severe immunodeficiency and a rudimentary thymus with a lack of T-cell development (Nehls et al., 1994; Balciunaite et al., 2002; Su et al., 2003). Therefore, our present results provide a better understanding of the regulation of Drosophila hematopoiesis and important insights into the regulatory mechanisms of the EBF and FOX families of transcription factors in the mammalian hematopoietic niche.

Materials and methods

Drosophila strains

The following mutant alleles, deficiencies, and transgenes were used: jumuGE27806 and shgGE13814were purchased from GenExel (Daejeon, South Korea); jumuDf2.12, jumuDf3.4 and UAS-jumu were gifts from Alan M. Michelson (Ahmad et al., 2012; Zhu et al., 2012); Hml-Gal4 UAS-2xEGFP, domeless-Gal4 UAS-2xEGFP, col1/CyO;P(col5-cDNA)/TM6B, Tb, Antp-Gal4/TM3, Sb and Srp-Gal4 were gifts from Utpal Banerjee (Mandal et al., 2007); col-Gal4 UAS-mCD8GFP, domeMESO, hhF4-GFP, UAS-col and UAS-col RNAi were gifts from Lucas Waltzera (Benmimoun et al., 2015); P{en2.4-GAL4}e33C (e33C-Gal4) was a gift from Dominique Ferrandon; Tep-IV-Gal4 was a gift from the Kyoto Stock Center (DGRC); UAS-jumu RNAi (jumuGD4099) was obtained from the Vienna Drosophila RNAi Center (VDRC); Tub-Gal4, Tub-Gal80ts (Tub-Gal4ts), UAS-dmyc RNAi (JF01761) and elav-Gal4 were obtained from the Tsinghua Fly Center; Hand-Gal4 was a gift from Wuzhou Yuan; cdc42-DN was obtained from Bloomington; HHLT-Gal4 was a gift from Jiwon Shim; and UAS-Toll10b and dif were gifts from Bruno Lemaitre. The w1118 line or the respective UAS or Gal4 parent lines were used as controls when required. The crosses involving RNAi lines or Tub-Gal4ts were reared at 29°C, and the other strains and crosses were reared at 25°C. All strains and crosses were cultured on standard cornmeal-yeast medium under a 12 hr light/12 hr dark cycle.

Antibody production

A fusion protein containing a nonconserved portion of the deduced Jumu protein (residues 141–330) was produced in E. coli using the pRSETA (6x-His) vector from Qiagen. The fusion protein was purified using Ni-NTA resin and used to immunize rats.

Immunohistochemistry and ROS detection

For antibody staining, the lymph glands were dissected in PBS, fixed with 3.7% formaldehyde in PBS for 20 min, pre-incubated in blocking solution (PBS with 0.1% Tween-20% and 5% goat serum) and then incubated with primary antiserum diluted in blocking solution. The following primary antibodies were used: mouse anti-P1, mouse-anti-L1 (gifts from I. Ando), mouse anti-Hnt (AB_528278), mouse anti-Ptc (AB_528441), Rat anti-Shg (AB_528120), mouse anti-Antp (AB_528082), mouse anti-Dlp (AB_528191) (Developmental Studies Hybridoma Bank), mouse anti-Col (gift from M. Crozatier) (Pennetier et al., 2012), mouse anti-β-galactosidase (Sigma), rat anti-Jumu, rabbit anti-dMyc (Santa Cruz), and mouse anti-P-Mad (gift from Ed Laufer, USA). Alexa Fluor 488-, Alexa Fluor 568- and Alexa Fluor 594-conjugated secondary antibodies (Molecular Probes) were used. ROS detection was performed as previously described (Evans et al., 2014) using dihydroethidium (Invitrogen). Images were obtained using a Zeiss LSM510 confocal microscope or a Zeiss Axioplan 2 microscope equipped with fluorescence optics. All staining was performed in samples from at least three independent experiments.

Image analyses

For quantifications, the lymph glands were scanned at an optimized number of slices (aiming to acquire all staining signals throughout the tissue) using a Zeiss Axioplan 2 microscope, usually less than three optical sections from the Z stack can contain all signal from one tissue. To quantify the crystal cell and ROS indexes and the PSC cell number, the consecutive slices from the same primary lobe were assembled in Photoshop CS6, and the merged images were used for the quantifications. The crystal cell and ROS indexes were calculated as the total number of Hnt+ or ROS+ cells (quantified using ImageJ, NIH), respectively, in each primary lobe divided by the relative area of the primary lobe. The total PSC cell (Antp+ cell) number in each primary lobe was also quantified using ImageJ. At least 30 lymph glands were scored for each genotype. For the quantification of P1+ cells and the Shg+ and domeMESO+ cell indexes, the fluorescent images collected by Zeiss Axioplan 2 microscope were converted to 8-bit images and then recalibrated to obtain an identical threshold using ImageJ. The area with the identical threshold was captured and measured with ImageJ. To measure the total size of the primary lobes, the DAPI-expressing area was captured using freehand selection tool of ImageJ and measured. The plasmatocyte differentiation index was assessed by determining the average area of P1+ cells from all optical sections of a primary lobe compared with the area of the primary lobe. The proportion of prohemocytes in the primary lobes (MZ/lobe 1) was defined as the average area of Shg+ or domeMESO+ cells in all optical sections of a primary lobe compared with the area of the primary lobe. At least 30 lymph glands were analyzed for each genotype. For quantification of the Col, Dlp, dMyc and Jumu fluorescence signal intensities in the MZ or PSC, images were captured with a Zeiss LSM510 confocal microscope at the same exposure times, and at least three confocal slides (4 μm) per stack were analyzed. The total intensity values for Col, Dlp, dMyc and Jumu in each ROI (region of interest) were measured using ImageJ. For the analysis of Col and Dlp staining in the MZ, dome-GFP was used to define the ROIs. For the analysis of dMyc and Jumu staining in the PSC, Antp labeling was used for to define the ROIs. The ROIs in the fluorescent images were captured using the freehand tool and then converted to 8-bit images. The total intensity value of the ROIs with an identical threshold was captured and measured with ImageJ. The average pixel intensity values of dMyc or Jumu in PSC cells (integrated intensity/area of Antp-labeled cells) were calculated. The pixel intensity values of Col or Dlp in the MZ were assessed as the integrated intensity in the ROIs compared with the area of the primary lobe. For each fluorescence quantification experiment, at least 20 lymph glands were analyzed.

Chromatin immunoprecipitation (ChIP) assays

ChIP assays were performed as described previously (Kim et al., 2005). In brief, S2 cells (CVCL_Z232, Invitrogen, Cat#R690-07) were transfected with the pMK33-Flag-jumu full CDS construct using the Effectene Transfection kit (Qiagen). S2 cells stably expressing Flag-jumu were fixed in 1% formaldehyde at room temperature. The samples were then sonicated to obtain DNA fragments between 200 and 800 bp in length. The fragmented chromatin was incubated with the anti-Flag antibody (Sigma) or IgG (Santa Cruz) bound to Salmon Sperm DNA/Protein A Agarose Slurry (Upstate) overnight at 4°C. The immunoprecipitated DNA fragments were recovered using a MiniElute purification kit (Qiagen) and analyzed by PCR amplification. Similar results were obtained in three independent ChIP experiments. The primer sequences are shown in Supplementary file 1.

Construction of dmyc promoter reporter plasmid and site-directed mutagenesis 

The dmyc promoter −643 to +467 was cloned into the pGL3 basic vector (firefly luciferase reporter plasmid, Promega) using KpnI and XhoI sites. The two base mutations of FKH-binding sites in the dmyc promoter were generated by the overlapping PCR method (Vallejo et al., 2008). The recombinant pGL3 plasmid with the dmyc promoter −643 to +467 was used as a template. The first and second rounds of PCR were performed using a primer pair composed of the forward primer amplifying dmyc promoter −643 to +467 (F) and the reverse mutated primer containing the two base exchanges in the FKH-binding sites (M1R, M2R or M3R) of the dmyc promoter and a second primer pair corresponding to the complementary forward mutated primer (M1F, M2F, or M3F) and the reverse primer amplifying dmyc promoter −643 to +467 (R), respectively. The two amplified overlapping fragments were used as templates for a subsequent overlap extension PCR with the primer pair amplifying the dmyc promoter −643 to +467 (F+R). The primer sequences used are shown in Supplementary file 1. The PCR fragment containing the mutated FKH-binding site was then cloned into the pGL3 basic vector. The introduction of the mutations was verified by DNA sequencing.

Transfection and luciferase reporter gene assay

A total of 5 × 105 S2 cells (CVCL_Z232, Invitrogen, Cat#R690-07) stably expressing the pMK33-Flag-jumu full CDS construct or empty pMK33 vector were plated per well in a 24-well plate and then co-transfected with 200 ng of dmyc promoter firefly luciferase reporter plasmid and 20 ng of Renilla luciferase pRL-TK reporter vector. The cells were harvested after 48 hr of incubation. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega), and the normalized luciferase values were calculated as the ratio of the firefly to the Renilla luciferase activities. Plasmid transfection was performed in at least triplicate, and luciferase activity was measured in duplicate or triplicate.

Quantitative real-time PCR

The total RNA from the entire third-instar larvae or the dissected hemocytes (from 300 to 500 third-instar larvae) was isolated with TRIzol (Invitrogen). The obtained total RNA was used to generate cDNA with M-MLV Reverse Transcriptase (Promega). The real-time PCR amplification was performed using SYBRR Select Master Mix (Applied Biosystems) on a Roche 4500 real-time PCR system. The results were normalized to the level of RpL32 mRNA in each sample. Three experiments per genotype were averaged. A biological replicate was performed, and the same results were obtained. The primer sequences used are shown in Supplementary file 1.

Statistical analysis

The statistical analyses were performed through two-tailed unpaired Student’s t-tests or one-way ANOVAs using GraphPad Prism software. The threshold for statistical significance was established as *p<0.1, **p<0.01 and ***p<0.001.

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Decision letter

  1. Utpal Banerjee
    Reviewing Editor; University of California, Los Angeles, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Dual role for Jumu in the control of hematopoietic progenitors in the Drosophila lymph gland" for consideration by eLife. Your article has been favorably evaluated by K VijayRaghavan (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Jiwon Shim (Reviewer #2).

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

The exact verbatim reviewer's comments are attached below. As you will see, the reviewer's initial reaction to the paper is upbeat and they suggest many individual experiments that we hope will be useful to you to enhance the manuscript. But in the follow up discussion, it became clear that there are too many questions raised by the reviewers, and particularly given that much of the criticism is over whether a proper mechanistic basis has been established, we do not feel that a revision within the eLife timing and standards can be reached. You do have the option of resubmission, but that will be evaluated as a fresh manuscript on the basis of relevance, advancement and in light of the reviews for this version.

Reviewer #1:

I was excited about this paper when it was first sent to me. It was particularly intriguing to read about the autonomous and non-autonomous signals that the authors mention in the Abstract. Upon further scrutiny, I am not as enthusiastic as I was before in terms of depth and novelty of this work and although I can be convinced otherwise by other expert reviewers, I do not see this manuscript moving the field much farther.

Jumu/Domina was identified as a position effect variegation causing mutation a couple of decades ago. The authors are the first to investigate its role in hematopoiesis. In their previous paper, the emphasis was on producing melanotic tumors and through unknown mechanisms activating Toll signaling. These results are not properly described here. Activation of Toll should have a big effect on the lymph gland as well. Perhaps the extra lamellocytes in the homozygote are related to this effect?

Given what is known about this gene, it is expected to participate in the regulation of large numbers of genes and the zone-specific phenotypes coupled with ubiquitous expression pattern would suggest that the developmental context is provided by signals, partners, regulators and epigenetic status and not by the Jumu protein itself. Understanding the hierarchies and interactions between the various pathway members generates mechanisms. Unfortunately, other than myc regulation (already shown by Crozatier's laboratory to control PSC size; Mourin-Pollard et al., Nature communication), the paper is to be viewed as a very preliminary phenotypic analysis mostly without mechanisms. The is not even an attempt to identify the non-autonomous MZ to PSC signal which is what got me really interested after reading the abstract. As shown, the authors imply a morphogen involved in the process. A handful has been described and could easily have been tested with available tools.

1) The data suggest Jumu functions downstream of Antp but upstream of Col and Hh. Does this allow some ordering of events? When does Jumu expression begin? Is it first expressed in the PSC, later expanding to the rest of the gland? Richard Mann had shown fkh is controlled by the Hox gene Scr and Exd. In other contexts, Antp can do that as well. An interesting interplay between Antp and Homothorax has been reported in the lymph gland. Is that related to Jumu expression?

2) Why were only the heterozygotes used in Figure 1? It is understandable that homozygous null is lethal, but the transheterozygote has to show these phenotypes as well.

3) Why does ROS go down as the MZ expands?

4) I have a great deal of difficulty understanding the quantification data throughout the paper. It seems that what is being quantitated is different in each figure such as number of cells, pixel density or staining index. There is no clear mention of how much of the tissue is quantitated. The methods describe that an optimal number of sections were used. What does this mean? Was the number of optical sections kept constant in each case? Indeed, are these all confocal images? An LSM510 is mentioned but most of the analysis was done on an Axioplan2 microscope. If the entire depth of the tissue is imaged, there will be a great deal of variability. How does pixel intensity converted to number of cells when the intensity per cell could vary and how were the thresholds set? Why are the quantitations in the later figures represented as histograms? Ideally, the entire paper should have a uniform standard in how images are quantified so that the readers can get a clear comparison between all of the data presented. Perhaps that is what was done, but it does not come through that way.

5) In Figure 2., the data look very nice, but a developmental series will be useful in knowing whether the cells are migrating or arising in the wrong site. Overexpression of Antp causes an expansion of the PSC, the reason has never been clear. This again brings up the question of whether ANTP controls JUMU.

6) Figure 3. It would be good to see the genotype in panel B stained for MZ and CZ markers.

7) Figure 8., how does panel 1 match with the results in the authors' previous paper?

8) Also Figure 8, the binding assay needs a critical control with mutated binding sites.

Reviewer #2:

Jin et al. describes dual functions of Jumu in the regulation of blood progenitors in both the MZ and in the PSC. Authors have shown that one copy loss of Jumu inhibits the blood progenitor differentiation due to the expansion of PSC cells. These phenotypes are restored by expression of Jumu specifically in the blood progenitors, while the other cell-type specific drivers do not result in the rescue phenotype. Authors have also shown that overexpression of Jumu in the blood progenitors induces differentiation of mature blood cells, and confirmed that expression of Col and Dlp are regulated by levels of Jumu in the MZ. In addition to the function of Jumu in the MZ, authors have identified that Jumu in the PSC controls the proliferation of PSC cells and thereby indirectly affects the progenitor maintenance. One possible downstream of Jumu identified in this paper is dMyc, of which expression can be directly controlled by Jumu. Overall, these data suggest two independent functions of Jumu in the regulation of progenitors by inhibition of Col in the MZ or by activating dMyc expression in the PSC.

1) One of the main findings of Jin and Hao is that one-copy loss of Jumu induces expansion of the PSC cells in the lymph gland. However, the phenotype shown by Jumu heterozygote (Figure 2F,F' or H) includes both proliferation and clustering defects of the PSC. Previous study has shown that these two phenomena are regulated by two independent pathways in the PSC; proliferation is regulated by Dpp/dMyc and clustering, by DE-cad/Cdc42 (Morin-Poulard et al., 2015). Authors have demonstrated that expression of Jumu in the MZ rescues both proliferation and clustering phenotypes of the PSC. To support the authors' claim, it will be important to identify genetic correlations in between Jumu in the MZ and two other pathways in the PSC in detail.

2) Along the same lines; if Jumu is widely expressed in other tissues including the dorsal vessel, it is possible that Jumu regulates Slit/Robo that in turn regulates both proliferation and clustering of the PSC. To exclude the involvement of Slit/Robo pathway and its indirect regulation, experiments such as Hand-gal4 driving Jumu RNAi or overexpression of Jumu will simply solve this concern.

3) One-copy loss of Jumu induces expansion of PSC cells in the lymph gland (Figure 2), and this phenotype is recapitulated by Dome-gal4 driving Jumu RNAi (Figure 4L). However, downregulation of Jumu in the PSC further reduces the number of PSC cells, while overexpression of Jumu leads to the proliferation of PSC cells. These data indicate that Jumu regulates dMyc in opposite ways in two zones; Jumu in the MZ non-autonomously inhibits dMyc whereas Jumu in the PSC autonomously activates dMyc. However, in the development of the PSC, PSC cells require proliferation at earlier stages while Jumu needs to inhibit the proliferation in later instars. Developmental relevance of Jumu would be also important for understanding the gene function. Supplementing strong evidence that there are changes in the expression levels of Jumu/dMyc throughout larval development, or different phenotypes led by early vs late knockdown of Jumu in different zones would strengthen the developmental function of Jumu/dMyc in the PSC.

Reviewer #3:

In this study, Hao and Lin explore the role of Jumu in controlling blood progenitor function in Drosophila, specifically in the larval lymph gland. Their findings demonstrate that Jumu maintains prohemocytes by cell-autonomously preventing the overexpression of Col in the MZ and by non-cell-autonomously inhibiting dMyc in the PSC to regulate its size. Their study also studies reveals a requirement for Jumu in cell-autonomously controlling PSC cell proliferation via dMyc. Their findings provide novel insight into the molecular regulation of progenitor maintenance, in particular a novel mechanism in which the progenitor population provides feedback to the cells of the niche/PSC for their own maintenance. The quality of the data is excellent and consistent with established standards for this field. However, there are a few concerns that need to be addressed:

Expression of Hedgehog in the PSC should be assessed upon overexpression of Jumu in the MZ to determine if the increased differentiation observed is due to loss of this maintenance signal.

It cannot be assumed that lamellocytes present in the lymph gland of trans-heterozygote Jumu mutants is due to loss of collier (Discussion, second paragraph). Staining of lymph glands for lamellocytes in which Jumu is removed in specific zones would allow insight into the process.

Dome-gal4 is expressed outside of the MZ in many other organs and the authors cannot rule an effect external to the signal responsible for the observed effect of inhibiting PSC expansion in Jumu mutants. This should be stated.

It would be useful to have clones of Jumu made in which the cell autonomous effects observed can be established, particularly given that the levels of Jumu may have different effects on cell maintenance and differentiation.

In addition, the following need to be corrected prior to publication:

Figure 1S-U: the ROS staining looks poorly done.

Figure 2M and N: these experiments need labeling with a MZ marker like shg or ptc.

Figure 2C and D and H: it is not clear if the ectopic Antp cells are derived from the PSC or the result of a homeotic transformation in which an ectopic PSC is generated. The fact that Dome>UAS-Jumu rescues the phenotype of ectopic PSC may have to do with Jumu preventing the PSC fate in the MZ (domeless is expressed very early in lymph gland development) rather than the PSC increasing in size.

Figure 2O: The quantification panel can go in supplementary to maintain symmetry for the figure.

Figure 3A-C: the Jumu staining looks cytoplasmic while clearly nuclear with some cytoplasmic in the PSC. Have the authors looked at the developmental pattern of expression of Jumu to determine if it is ever nuclear in the lymph gland outside of the PSC? The developmental pattern of Jumu expression is essential to understand its function.

Figure 3M and O: images too blurry.

Figure 3P: can go in supplementary.

Figure 4N: the figure is missing dome-gal4, UAS-Jumu, UAS-col with Antp immunostaining.

Figure 9: the schematic model is misleading since the trans heterozygote must include a hypomorphic mutation and not a complete loss of function to allow survival into adulthood. Please modify the description to trans heterozygote.

https://doi.org/10.7554/eLife.25094.024

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

I was excited about this paper when it was first sent to me. It was particularly intriguing to read about the autonomous and non-autonomous signals that the authors mention in the Abstract. Upon further scrutiny, I am not as enthusiastic as I was before in terms of depth and novelty of this work and although I can be convinced otherwise by other expert reviewers, I do not see this manuscript moving the field much farther.

We thank the reviewer for these constructive comments. We have performed an in-depth analysis of the regulation of PSC morphology by Jumu in the MZ and the mechanism of lamellocyte differentiation in the subsections “The loss of jumu in the MZ non-cell-autonomously induces increases in dMyc in the PSC” and “The loss of jumu in the entire lymph gland leads to the generation of lamellocytes through the activation of Toll signaling”, respectively. We believe that this additional information will improve the interest of this manuscript.

Jumu/Domina was identified as a position effect variegation causing mutation a couple of decades ago. The authors are the first to investigate its role in hematopoiesis. In their previous paper, the emphasis was on producing melanotic tumors and through unknown mechanisms activating Toll signaling. These results are not properly described here. Activation of Toll should have a big effect on the lymph gland as well. Perhaps the extra lamellocytes in the homozygote are related to this effect?

Thank you for the advice. Our previous paper indicated that the overexpression of jumu simultaneously in both hemocytes and the fat body leads to an enrichment of Dorsal and Dif in the nuclei of aggregated circulating hemocytes and deposited hemocytes on the fat body. However, the Dif/Dorsal levels did not increase in the lymph gland. Here, we used the lymph gland-specific driver Srp-Gal4 to knock down jumu and detected the activation of Toll signaling in the lymph gland using Dorsal and Dif antibodies. Surprisingly, we found that Dorsal is enriched in nuclei throughout the lymph glands in the control, whereas Dif is located in the cytoplasm and showed high expression levels in the PSC and lower signals in the MZ and CZ. Moreover, Dif was found to be activated and mainly enriched in nuclei in the MZ and CZ of the lymph gland in Srp>jumu RNAi and Srp> Toll10b(Figures 9L-9N and 9R-9T). In addition, the loss of dif effectively reduced the lamellocytes in Srp>jumu RNAi (Figure 9J). These results suggest that the loss of jumu in the lymph gland activates Toll signaling and thereby induces the generation of lamellocytes. We have described these results in the subsection “The loss of jumu in the entire lymph gland leads to the generation of lamellocytes through the activation of Toll signaling”.

Given what is known about this gene, it is expected to participate in the regulation of large numbers of genes and the zone-specific phenotypes coupled with ubiquitous expression pattern would suggest that the developmental context is provided by signals, partners, regulators and epigenetic status and not by the Jumu protein itself. Understanding the hierarchies and interactions between the various pathway members generates mechanisms. Unfortunately, other than myc regulation (already shown by Crozatier's laboratory to control PSC size; Mourin-Pollard et al., Nature communication), the paper is to be viewed as a very preliminary phenotypic analysis mostly without mechanisms. The is not even an attempt to identify the non-autonomous MZ to PSC signal which is what got me really interested after reading the abstract. As shown, the authors imply a morphogen involved in the process. A handful has been described and could easily have been tested with available tools.

Thank you for the advice, We have performed an in-depth analysis of the relationship between jumu and Slit/Rob signaling in the control of the PSC morphology and found that Jumu in the MZ controls PSC morphology by indirectly regulating dMyc expression in PSC cells through a Slit/Robo and BMP/Dpp-independent mechanism in an non-autonomous manner (Figure 5 and Figure 5—figure supplement 1; subsection “The loss of jumu in the MZ non-cell-autonomously induces increases in dMyc in the PSC”).

1) The data suggest Jumu functions downstream of Antp but upstream of Col and Hh. Does this allow some ordering of events? When does Jumu expression begin? Is it first expressed in the PSC, later expanding to the rest of the gland? Richard Mann had shown fkh is controlled by the Hox gene Scr and Exd. In other contexts, Antp can do that as well. An interesting interplay between Antp and Homothorax has been reported in the lymph gland. Is that related to Jumu expression?

Thank you for the advice. We did not mention that jumu functions downstream of Antp but rather suggested that jumu affects the proliferation and expansion of Col+, Antp+ and HH+ PSC cells and that the loss of col can inhibit the phenotype caused by jumu deficiency.

With respect to jumu expression, previous studies have shown that jumu beings to be expressed at the embryo stage. (“Cheah PY, Chia W, Yang X. 2000. Jumeaux, a novel Drosophila winged-helix family protein, is required for generating asymmetric sibling neuronalcell fates. Development 127: 3325–3335.” and “Strödicke M, Karberg S, Korge G. 2000. Domina (Dom), a new Drosophila member of the FKH/WH gene family, affects morphogenesis and is a suppressor of position-effect variegation. Mech Dev 96: 67–78.”). In addition, we detected the expression pattern of Jumu protein in the lymph gland at different larval stages through staining with anti-Jumu antibody and observed that Jumu is expressed in the entire lymph gland and mainly localized in the nuclei (Figures 4A-4F”; subsection “Jumu expression in prohemocytes is required for preventing the ectopic expansion of PSC cells”).

With respect to the known role of Hox gene Homothorax (Hth) in Antp expression is restricted to embryo, Antp+ cells were decreased in the embryo of the Homothorax (Hth) mutant (Mandal L, Martinez-Agosto JA, Evans CJ, Hartenstein V, Banerjee U. 2007. A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors. Nature 446: 320–324.); however, Mandal et al.did not investigate the larvae stage. Therefore, we knocked down hth using the PSC-specific driver col-Gal and found that Antp+ cells presented no difference between the col>hth RNAi and control PSCs (the data was shown in Author response image 1). These results suggest that different from the function of Jumu, Hth might not directly participate in PSC development during the larval stage.

2) Why were only the heterozygotes used in Figure 1? It is understandable that homozygous null is lethal, but the transheterozygote has to show these phenotypes as well.

We added the trans-heterozygote detection data to Figures 1 and 2, which reveal a significant phenotype different compared with heterozygotes.

3) Why does ROS go down as the MZ expands?

ROS is different from other MZ markers, such as dome, Shg and Ptc, which prevent prohemocyte differentiation. ROS only appear in the MZ until the middle- third larval stage and can primes hematopoietic progenitors for differentiation (Owusu-Ansah E, Banerjee U. 2009. Reactive oxygen species prime Drosophila hematopoietic progenitors for differentiation. Nature461: 537–541”). We observed low ROS levels in jumu mutant third-instar larva; therefore, the progenitor cells are unable to differentiate into mature hemocytes. This result is also consistent with the reduced CZ detected in the jumu mutants.

4) I have a great deal of difficulty understanding the quantification data throughout the paper. It seems that what is being quantitated is different in each figure such as number of cells, pixel density or staining index. There is no clear mention of how much of the tissue is quantitated. The methods describe that an optimal number of sections were used. What does this mean? Was the number of optical sections kept constant in each case? Indeed, are these all confocal images? An LSM510 is mentioned but most of the analysis was done on an Axioplan2 microscope. If the entire depth of the tissue is imaged, there will be a great deal of variability. How does pixel intensity converted to number of cells when the intensity per cell could vary and how were the thresholds set? Why are the quantitations in the later figures represented as histograms? Ideally, the entire paper should have a uniform standard in how images are quantified so that the readers can get a clear comparison between all of the data presented. Perhaps that is what was done, but it does not come through that way.

We apologize for the unclear description of the quantifications. We have described the quantification methods in detail in the Materials and methods section(subsection “Image analyses”). With respect to the quantification method and standards, we mainly refer to some articles: “Shim J, Mukherjee T, Banerjee U. 2012. Direct sensing of systemic and nutritional signals by hematopoietic progenitors in Drosophila. Nat Cell Biol14: 394-400.”, “Benmimoun B, Polesello C, Waltzer L, Haenlin M. 2012. Dual role for Insulin/TOR signaling in the control of hematopoietic progenitor maintenance in Drosophila. Development139: 1713–1717.”, “Gueguen G, Kalamarz ME, Ramroop J, Uribe J, Govind S. 2013. Polydnaviral ankyrin proteins aid parasitic wasp survival by coordinate and selective inhibition of hematopoietic and immune NF-kappa B signaling in insect hosts. PLoS Pathog9:e1003580.” and “Benmimoun B, Polesello C, Haenlin M, Waltzer L. 2015. The EBF transcription factor Collier directly promotes Drosophila blood cell progenitor maintenance independently of the niche. Proc Natl Acad Sci U S A112: 9052–9057.”.

For the quantifications of the numbers of crystal cells and PSC cells and ROS indexes, at least 30 lymph glands were scored per genotype. For the quantifications of the areas of P1+ cells and the Shg+ and domeMESO+ cell indexes, at least 30 lymph glands were scored per genotype. For the quantifications of the Col, Dlp, dMyc and Jumu fluorescence signal intensities in the MZ or PSC, at least 20 lymph glands were analyzed. We provide a detailed description about the collection and analysis of images in the Materials and methods section(subsection “Image analyses”).

In this study, the Antp, Hnt and ROS signals were converted to the number of cells, because these three markers are all located in nuclei and show a clearer signal. Moreover, the signal intensity in each cell was similar, and these signal points are relatively scattered and can thus be counted easily. The image acquisition settings were adjusted to avoid over-exposure, but allow observation of a clear scattered signal.

All quantification graphs are presented as box-and-whiskers with the exceptions of Figures 1EE and 9B, which show the quantification of mRNA levels.

5) In Figure 2., the data look very nice, but a developmental series will be useful in knowing whether the cells are migrating or arising in the wrong site. Overexpression of Antp causes an expansion of the PSC, the reason has never been clear. This again brings up the question of whether ANTP controls JUMU.

We have added a description of the morphology of the PSC at different larval stages and the colocalization of Antp+ PSC cells and dome-GFP+cells or Hml-GFP+cells, as shown in Figure 3(subsection “Jumu negatively regulates the proliferation and ectopic expansion of Col+ PSC cells”). We observed that the ectopically expanded Antp+ PSC cells did not co-localize with any dome-GFP+ or Hml-GFP+ cells in the MZ and CZ domains. In particular, the ectopic PSC cells adjacent to MZ cells or CZ cells in the early larval stage showed no intermediate cells co-expressing both Antp and GFP. The results indicate that the ectopic PSC cells in the jumu mutants are derived from the PSC domain. In addition, previous studies have shown that Antp is only located in the PSC of the lymph gland (Mandal L, Martinez-Agosto JA, Evans CJ, Hartenstein V, Banerjee U. 2007. A Hedgehog- and Antennapedia-dependent niche maintains Drosophila hematopoietic precursors. Nature 446: 320–324.), and we also did not find the expression of Antp in MZ using anti-Antp antibody; however, ectopic expansion of the PSC is caused by the loss of jumu in the MZ, suggesting that Antp cannot directly control jumu in the regulation of PSC morphology.

6) Figure 3. It would be good to see the genotype in panel B stained for MZ and CZ markers.

We have added the results of P1, Hnt, Shg and ROS staining of the jumuGE27806/ jumuDf2.12mutant to Figure 1.

7) Figure 8., how does panel 1 match with the results in the authors' previous paper?

Thank you for the advice. dMyc expression is up-regulated in circulating hemocytes of Hml>UAS-jumu. Our previous studies showed increased circulating hemocytes and lamellocyte differentiation in Hml>UAS-jumu. To further analyze whether these phenomena are related to dMyc, we overexpressed dmyc under the control of Hml-Gal4. Hml>UAS-dmyc did not present lamellocytes in the lymph glands (the data was shown in Author response image 2A). This result suggests that the overexpression of dmyc in CZ do not affect lamellocyte differentiation in the lymph gland. However, we found that similar to Hml>UAS-jumu, Hml>UAS-dmyc also showed increased circulating hemocytes (the data was shown in Author response image 2B), suggesting dMyc regulates the proliferation of circulating hemocytes. Although dMyc might be one of the factors responsible for the increase in circulating hemocytes in Hml>UAS-jumu, some other signaling pathways and elements might also be involved in the proliferation of circulating hemocytes. The role of Jumu in hemocyte proliferation remains to be further investigated.

8) Also Figure 8, the binding assay needs a critical control with mutated binding sites.

We mutated two positions in each of the three putative FKH-binding sites, the mutated promoter was cloned to the pGL3 vector, and a luciferase reporter gene assay was used to detect their activation. The results suggest that Jumu can endogenously be recruited to the -27/-17 site of the dmyc promoter and further strengthen the hypothesis that Jumu positively up-regulates dmyc expression by directly binding to the dmyc promoter (Figure 8G). The main results are described in the third paragraph of the subsection “Jumu cell-autonomously controls the size of the PSC by regulating the expression of dMyc”, and the methods used in the transfection and luciferase reporter gene assay are detailed in subsections “Construction of dmyc promoter reporter plasmid and site-directed mutagenesis” and “Transfection and luciferase reporter gene assay”.

Reviewer #2:

[…] 1) One of the main findings of Jin and Hao is that one-copy loss of Jumu induces expansion of the PSC cells in the lymph gland. However, the phenotype shown by Jumu heterozygote (Figure 2F,F' or H) includes both proliferation and clustering defects of the PSC. Previous study has shown that these two phenomena are regulated by two independent pathways in the PSC; proliferation is regulated by Dpp/dMyc and clustering, by DE-cad/Cdc42 (Morin-Poulard et al., 2015). Authors have demonstrated that expression of Jumu in the MZ rescues both proliferation and clustering phenotypes of the PSC. To support the authors' claim, it will be important to identify genetic correlations in between Jumu in the MZ and two other pathways in the PSC in detail.

We have analyzed the correlations between Jumu and DE-cad/Cdc42 or Dpp/dMyc in the MZ in the regulation of PSC morphology. The expression levels of Shg (DE-cad) were not reduced in the PSC of jumuDf2.12compared with the wild-type (Figure 5—figure supplement 1A-1B’’). We then performed a rescue experiment to identify the correlation between Jumu and Cdc42. The overexpression of the dominant negative form of Cdc42 (cdc42-DN) in the PSC of jumu Df2.12did not rescue the increased number and clustering defect of the PSC cell (Figure 5—figure supplement 1C-1E). These results suggest that Jumu protein in the MZ controls the clustering morphology of the PSC in a DE-cad/Cdc42-independent manner. In addition, we also found that the expression of Dlp and P-Mad was not reduced in the PSC of jumuDf2.12compared with the wild-type, suggesting that Jumu does not regulate Dpp signaling in the PSC (Figure 5—figure supplement 1F-1I’’, subsection “The loss of jumu in the MZ non-cell-autonomously induces increases in dMyc in the PSC”).

2) Along the same lines; if Jumu is widely expressed in other tissues including the dorsal vessel, it is possible that Jumu regulates Slit/Robo that in turn regulates both proliferation and clustering of the PSC. To exclude the involvement of Slit/Robo pathway and its indirect regulation, experiments such as Hand-gal4 driving Jumu RNAi or overexpression of Jumu will simply solve this concern.

We knocked down or overexpressed jumu using the Hand-Gal4 driver to detect PSC cells but did not observe defects in either proliferation or clustering in the PSC (Figure 6—figure supplement 1A-1D). Moreover, using Hand-Gal4 driver to overexpress jumu in the jumuDf2.12mutant did not rescue the PSC phenotype (Figure 4G and 4H; subsection “The loss of jumu in the MZ non-cell-autonomously induces increases in dMyc in the PSC”).

3) One-copy loss of Jumu induces expansion of PSC cells in the lymph gland (Figure 2), and this phenotype is recapitulated by Dome-gal4 driving Jumu RNAi (Figure 4L). However, downregulation of Jumu in the PSC further reduces the number of PSC cells, while overexpression of Jumu leads to the proliferation of PSC cells. These data indicate that Jumu regulates dMyc in opposite ways in two zones; Jumu in the MZ non-autonomously inhibits dMyc whereas Jumu in the PSC autonomously activates dMyc. However, in the development of the PSC, PSC cells require proliferation at earlier stages while Jumu needs to inhibit the proliferation in later instars. Developmental relevance of Jumu would be also important for understanding the gene function. Supplementing strong evidence that there are changes in the expression levels of Jumu/dMyc throughout larval development, or different phenotypes led by early vs late knockdown of Jumu in different zones would strengthen the developmental function of Jumu/dMyc in the PSC.

First, we detected the expression pattern of Jumu and dMyc in the lymph gland during larval development. We observed that Jumu and dMyc are detected throughout larval developmental stage and that is expression pattern is similar to that detected in the third-instar lymph gland (Figures 4B-4D and Figure 7—figure supplement 2; subsection “Jumu cell-autonomously controls the size of the PSC by regulating the expression of dMyc”, last paragraph). This result suggests that Jumu protein in the PSC and MZ might consistently balance the dMyc expression levels.

We then used a temporal and regional gene expression targeting system to knock down jumu in the PSC or MZ at different developmental stages and detect the PSC numbers. The results show that the PSC cells were reduced or expanded to different degrees at each developmental stage from first to third instar (Figure 7—figure supplement 3, subsection “Jumu cell-autonomously controls the size of the PSC by regulating the expression of dMyc”, last paragraph). These results suggest that Jumu expression in both the MZ and the PSC ensures maintenance of normal PSC development.

Moreover, we hypothesize that Jumu protein located in the MZ mainly prevents the expansion and scattering of PSC cells toward the MZ and indirectly maintains normal MZ development during lymph gland development but is not sufficient to initiate the inhibition of PSC cell proliferation. We also found that the loss of jumu in the MZ causes an expansion of the PSC and an increase in dMyc expression and that the overexpression of jumu in the MZ did not spontaneously lead to the reduction in the PSC cell number and dMyc expression (Figure 5D, 5R, 5L, 5L’ and 5S). In other words, the main function of Jumu in the MZ may be maintenance of the normal development zone margin between the MZ and the PSC as well as maintenance of the homeostasis of each zone of the lymph gland. A detailed discussion is provided in the third paragraph of the Discussion section.

Reviewer #3:

In this study, Hao and Lin explore the role of Jumu in controlling blood progenitor function in Drosophila, specifically in the larval lymph gland. Their findings demonstrate that Jumu maintains prohemocytes by cell-autonomously preventing the overexpression of Col in the MZ and by non-cell-autonomously inhibiting dMyc in the PSC to regulate its size. Their study also studies reveals a requirement for Jumu in cell-autonomously controlling PSC cell proliferation via dMyc. Their findings provide novel insight into the molecular regulation of progenitor maintenance, in particular a novel mechanism in which the progenitor population provides feedback to the cells of the niche/PSC for their own maintenance. The quality of the data is excellent and consistent with established standards for this field. However, there are a few concerns that need to be addressed:

Expression of Hedgehog in the PSC should be assessed upon overexpression of Jumu in the MZ to determine if the increased differentiation observed is due to loss of this maintenance signal.

We overexpressed or knocked down jumu in the MZ and detected the Hh-GFP expression levels. The results showed that the Hh-GFP signal was not changed compared with the control (Figure 6—figure supplement 2; subsection “Jumu cell-autonomously controls the maintenance of blood cell progenitors by regulating the low levels of Col in the MZ”, first paragraph). This result suggests the prohemocyte overdifferentiation due to the overexpression of jumu is not related to Hh signaling.

It cannot be assumed that lamellocytes present in the lymph gland of trans-heterozygote Jumu mutants is due to loss of collier (Discussion, second paragraph). Staining of lymph glands for lamellocytes in which Jumu is removed in specific zones would allow insight into the process.

The previous description was “Previous studies have reported that the overexpression of collier in the entire lymph gland causes lamellocyte differentiation and reduces the number of crystal cells (Crozatier M, Ubeda JM, Vincent A, Meister M. 2004. Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier. PLoS Biol2: E196). Thus, Jumu might control the differentiation of lamellocytes by negatively regulating col expression in the entire lymph gland”.

According to the suggestion, we have knocked down jumu or overexpressed col in different zones of the lymph gland (CZ, MZ and PSC) and did not observe obvious increases in the lamellocyte numbers in the lymph gland; in fact, less than 20% and 10% of the lymph glands showed a few lamellocytes ((Figure 9—figure supplement 1A-1F). However, jumu knockdown and col overexpression in the entire lymph gland caused obvious increases in lamellocytes and reductions in crystal cells. Moreover, the knockdown of col in Srp>jumu RNAi can inhibit lamellocyte differentiation (Figure 9B, 9D, 9E, 9G, 9I and 9K; subsection “The loss of jumu in the entire lymph gland leads to the generation of lamellocytes through the activation of Toll signaling”, last paragraph). These results indicate that the loss of jumu in the entire lymph gland might induce lamellocyte differentiation by up-regulating the expression of Col.

Dome-gal4 is expressed outside of the MZ in many other organs and the authors cannot rule an effect external to the signal responsible for the observed effect of inhibiting PSC expansion in Jumu mutants. This should be stated.

With the exception of the cardiac tube, the other organs involved in PSC morphology have not been reported. However, we knocked down or overexpressed jumu using the Hand-Gal4 driver (cardiac tube) to detect the PSC cells and did not neither proliferation nor clustering in the PSC (Figure 6—figure supplement 1 A-1C).

To further confirm our conclusion, we used a pan lymph gland-specific Srp-Gal4 or MZ-driver TeplV-Gal4 to over express jumu in the jumu mutant and found that this overexpression can rescue the PSC defect (Figure 4P and Figure 5B). However, the overexpression of jumu using Antp-Gal4 (PSC driver) and Hml-Gal4 (CZ driver) did not rescue the PSC defect in the jumu mutant (Figures 4J and 4N). Therefore, the results indicate that Jumu protein in the MZ inhibits PSC expansion in the lymph gland.

It would be useful to have clones of Jumu made in which the cell autonomous effects observed can be established, particularly given that the levels of Jumu may have different effects on cell maintenance and differentiation.

Thank you for this suggestion. We have added the results of a clonal analysis using HHLT-Gal4 to Figure 6—figure supplement 3in the second paragraph of the subsection “Jumu cell-autonomously controls the maintenance of blood cell progenitors by regulating the low levels of Col in the MZ” and in the first paragraph of the subsection “Jumu cell-autonomously controls the size of the PSC by regulating the expression of dMyc”.

We previously indicated two cell-autonomous functions of Jumu in lymph gland development: First, Jumu protein in the MZ can cell-autonomously promote prohemocyte differentiation (Figures 6H and 6M), and second, Jumu protein in the PSC can cell-autonomously promote PSC cell proliferation (Figures 7A-7C). The clonal analysis confirmed these conclusions. First, clonal cells in which jumu was knocked down rarely overlapped with P1+ cells (Figure 6—figure supplement 3B-B’’), but most P1+ cells overlapped with clonal cells overexpressing jumu (Figure 6—figure supplement 3C-3C’’), suggesting that Jumu autonomously promotes hemocyte differentiation. Second, the clonal knock down of jumu in PSC cells can reduce the number of PSC cells (Figure 6—figure supplement 3E); however, the clonal cells overexpressing jumu in the PSC tended to increase the PSC cell number (Figure 6—figure supplement 3F). Altogether, these results further confirm that Jumu cell-autonomously promotes PSC cell proliferation.

In addition, the following need to be corrected prior to publication:

Figure 1S-U: the ROS staining looks poorly done.

Fluorescence staining for ROS levels is typically performed using DHE (dihydroethidium), which is a redox-sensitive dye that intercalates into cellular DNA when oxidized. Thus, the fluorescence signal does not look as clear as that obtained through antibody staining. Mondal et al.provided a description of a DHE staining protocol for the lymph gland, which is similar to that used in the present study.

Figure 2M and N: these experiments need labeling with a MZ marker like shg or ptc.

We have added Shg staining to Figures 2Q and 2Rand describe these results in the subsection “Jumu negatively regulates the proliferation and ectopic expansion of Col+ PSC cells”.

Figure 2C and D and H: it is not clear if the ectopic Antp cells are derived from the PSC or the result of a homeotic transformation in which an ectopic PSC is generated. The fact that Dome>UAS-Jumu rescues the phenotype of ectopic PSC may have to do with Jumu preventing the PSC fate in the MZ (domeless is expressed very early in lymph gland development) rather than the PSC increasing in size.

We thank the reviewer for these constructive comments. We analyzed PSC cells at different larval stages but did not observe the colocalization of Antp+ PSC cells with dome-GFP+cells or Hml-GFP+cells (Figure 3, subsection “Jumu negatively regulates the proliferation and ectopic expansion of Col+ PSC cells”, last paragraph). These results indicate that the ectopic PSC cells in the jumu mutants are derived from the PSC domain.

Figure 2O: The quantification panel can go in supplementary to maintain symmetry for the figure.

The construction and order of images in this figure have been adjusted (Figure 2U).

Figure 3A-C: the Jumu staining looks cytoplasmic while clearly nuclear with some cytoplasmic in the PSC. Have the authors looked at the developmental pattern of expression of Jumu to determine if it is ever nuclear in the lymph gland outside of the PSC? The developmental pattern of Jumu expression is essential to understand its function.

We have detected the expression pattern of Jumu in the lymph gland throughout larval development and found that Jumu is always expressed in the entire lymph gland, with markedly higher expression in the PSC, and that it is mainly localized in the nuclei (Figures 4A-4F’). Previous studies have shown that Jumu is located in the nuclei of syncytial embryos, is present in all nuclei of the cellular blastoderm, and is predominantly expressed in the brain lobes, in the segmented CNS and in elements of the PNS. In third-instar larvae, the most prominent expression of Jumu is in imaginal discs, the CNS, the ring gland and the hindgut (Cheah, P.Y., Chia, W., Yang, X., 2000. Jumeau, a novel Drosophila winged-helix family protein, is required for generating asymmetric sibling neuronal cell fates. Development. 127, 3325-3335; Sugimura, I., Adachi-Yamada, T., Nishi, Y., Nishida, Y., 2000. A Drosophila Winged-helix nude (Whn)-like transcription factor with essential functions throughout development. Dev Growth Differ.42, 237-248). We successfully prepared anti-jumu polyclonal antibodies, and the results were published in the Immunological Journal of China (2011, 27:1038-1042). The specificity and titer of the antibodies were detected by Western blotting and immunostaining. The subcellular localization and tissue-specific expression of Jumu protein were analyzed by immunostaining. The results showed endogenous Jumu protein in the nuclei of blood cells and the fat body, and this signal was not be detected in jumu mutants flies. In this study, we show that Jumu is localized in the nuclei of lymph gland cells and that the subcellular localization of Jumu does not change during larval development.

Figure 3M and O: images too blurry.

The images in Figure 3E-Lhave been replaced’

Figure 3P: can go in supplementary.

This figure has been renumbered as Figure 4S.

Figure 4N: the figure is missing dome-gal4, UAS-Jumu, UAS-col with Antp immunostaining.

Both dome>UAS-jumu and dome>UAS-col did not cause the PSC defect; thus, we do not think that it is necessary to overexpress col in dome>UAS-jumu to rescue the PSC cell numbers.

Figure 9: the schematic model is misleading since the trans heterozygote must include a hypomorphic mutation and not a complete loss of function to allow survival into adulthood. Please modify the description to trans heterozygote.

We apologize for the incorrect description. We used double heterozygotes instead of trans-heterozygotes.

https://doi.org/10.7554/eLife.25094.025

Article and author information

Author details

  1. Yangguang Hao

    Department of Genetics, College of Life Sciences, Northeast Forestry University, Harbin, China
    Contribution
    YH, Investigation, Visualization, Writing—original draft
    Competing interests
    The authors declare that no competing interests exist.
  2. Li Hua Jin

    Department of Genetics, College of Life Sciences, Northeast Forestry University, Harbin, China
    Contribution
    LHJ, Supervision, Funding acquisition, Project administration, Writing—review and editing
    For correspondence
    lhjin2000@hotmail.com
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5912-9800

Funding

National Natural Science Foundation of China (General Program 31270923)

  • Li Hua Jin

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Alan M Michelson, Utpal Banerjee, Lucas Waltzera, Dominique Ferrandon, Jiwon Shim, Wuzhou Yuan and Bruno Lemaitre for providing the numerous fly strains used in this study. We acknowledge István Andó, Michèle Crozatier, and Ed Laufer for providing the anti-P1, anti-L1, anti-Col, and anti-P-Mad antibodies. We thank the GenExel Drosophila Stock Center, Kyoto Drosophila Genetic Resource Center, Vienna Drosophila RNAi Center, and Tsinghua Fly Center for sharing the numerous fly stocks utilized in this research. We also thank Michèle Crozatier for the valuable suggestions provided regarding this work. This work was supported by the National Natural Science Foundation of China (31270923).

Reviewing Editor

  1. Utpal Banerjee, University of California, Los Angeles, United States

Publication history

  1. Received: January 13, 2017
  2. Accepted: March 20, 2017
  3. Accepted Manuscript published: March 28, 2017 (version 1)
  4. Accepted Manuscript updated: April 3, 2017 (version 2)
  5. Version of Record published: April 13, 2017 (version 3)

Copyright

© 2017, Hao et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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