Loss of Ubc9 affects gene expression, and size and integrity of third instar lymph gland
Post-embryonic wild type lymph gland development is heterochronic (). From the onset of the third instar, the posterior lobes of wild type lymph glands expand and coalesce so that the initially distinct four to six pairs of cell clusters form two sets of posterior lobes (). The growth of posterior lobes is developmentally synchronous in that the first set expands earlier than the second set (, supplementary material Fig. S1A,C). We call them posterior lobes, first set (PL1), and posterior lobes, second set (PL2) ().
lymph glands are variably overgrown and exhibit aberrant differentiation of hemocytes (Chiu et al., 2005
). Careful analysis of scores of mutant glands revealed differential effects on anterior versus posterior lobes (, supplementary material Fig. S1C,D). In many glands of 6–7-day third instar larvae, the anterior lobes are completely absent or are partially dispersed where peripheral cells in the cortex are lost to the hemolymph (see below). In contrast, most posterior lobes are severely overgrown and either remain tethered to the dorsal vessel or detach (, supplementary material Fig. S1D, and see below). Loss of posterior lobes coincides with the appearance of large compact tumors in the hemolymph. This trend suggests that the lymph gland itself may be the direct source of the microtumors.
To examine whether Ubc9 has one primary function in normal hematopoiesis and probe if all four defects (overgrowth, misdifferentiation, lobe dispersal, and lobe detachment) are triggered from an initial disruption of this primary function, we compared the expression patterns of Dome>GFP
in developing heterozygous and Ubc9
lymph glands. We found no striking difference in late second or even early third instar (day 4 after egg lay) animals (, supplementary material Fig. S1A,B). Most cells of the posterior lobes do not express mature hemocyte markers, but express Dome>GFP,
when the Dome
promoter is active () (Jung et al., 2005
; Krzemien et al., 2007
encodes the receptor for JAK-STAT signaling (Brown et al., 2001
). At mid to late third instar (day 5 to 6), all heterozygous anterior lobes remain relatively small and structurally intact, while anterior lobes of the mutant glands are either larger than control, or they disperse. Mutant posterior lobes expand dramatically, but remain largely intact (, supplementary material Fig. S1C,D, ). We found that the overgrown lobes themselves are displaced and begin to detach from the dorsal vessel (supplementary material Fig. S1D, Fig. S2J,L, ).
Overproliferation of immature cells in Ubc9 lymph gland.
The expression of Dome>GFP in heterozygous lymph glands remains high, while in mutant glands, it gradually decreases during third instar and is virtually absent by late 6 day (). Loss of Dome>GFP expression in mutant lobes does not result from increased apoptosis, as only less than 1% of cells in the lobes of either genetic background are positive for cleaved pro-caspase 3.
Dome>GFP expression is undetectable in circulating hemocytes of both, control and mutant animals. Single Dome>GFP cells in circulation or within microtumors are rare (supplementary material Fig. S2A–D). Surprisingly, while Dome>GFP is expressed weakly in the dorsal vessel of control animals, it is highly upregulated after the onset of anterior lobe dispersal in the mutant background (, ; asterisk). Together, these results suggest that a primary hematopoietic effect of Ubc9 loss is on the cells of the medullary zone. Additionally, Ubc9-dependent gene regulation in the dorsal vessel coincides with loss of lobe integrity.
Dome>Ubc9wt restores Ubc9 lymph gland size and Dome>GFP
The expression of Hml>GFP
is limited largely to the periphery in all 6 day control anterior lobes and in approximately 10% (n
1/8) of the first set of posterior lobes (supplementary material Fig. S1C). In all examined mutant anterior lobes and about 40% (n
3/8) of first posterior lobes, Hml>GFP
cells are scattered throughout the body of the lobe (supplementary material Fig. S1D). The expanded posterior lobes of mutant glands contain more Hml>GFP
-expressing cells than the control posterior lobes (supplementary material Fig. S1D). That both, Dome>GFP
expression becomes more pronounced in the first posterior lobes of control glands at third instar, supports the notion that this capacity to acquire zonation is heterochronic; it emerges only after the anterior lobes have matured. Second, at third instar Dome
expression decreases in Ubc9
lymph gland and Hml
expression increases slightly in posterior lobes compared to controls. These changes in the expression patterns occur simultaneously with lymph gland overgrowth.
The medullary zone exhibits heterogeneity
To understand the effects of the Ubc9
mutation on cells of the medullary zone, we simultaneously expressed Dome>DsRed
(a GFP protein trap) (Morin et al., 2001
) in wild type glands. ZCL2897
is expressed in cells of the medullary zone of control animals () (Jung et al., 2005
). Despite substantial overlap in the expression of Dome>DsRed
, there is significant heterogeneity in gene expression (). At least three cell types are observed: those that express both markers (Domehi
, 1, cells with yellow hue) and those that are strongly positive for one marker (Domehi
, red cells, or Domelo
, green cells, ). Among the doubly-positive cells, there is no apparent correlation in signal intensity of the two markers, suggesting that the medullary zone population consists of distinct cell types.
We next monitored ZCL2897 expression in heterozygous and Ubc9 third instar animals and found that, in contrast to Dome>GFP, loss of Ubc9 activates ZCL2897 expression in anterior and posterior lobes (). Unlike Dome>GFP, high ZCL2897 expression is also found in mutant circulating hemocytes, microtumors and overgrown lobes which are easily spotted through the cuticle (supplementary material Fig. S2E–L). Such overgrown, intact lobes, while still attached to the dorsal vessel (, supplementary material Fig. S2J,L; also see , ), correspond to the freely circulating microtumors in size and shape (supplementary material Fig. S2G,O; also see supplementary material Fig. S5B,H, supplementary material Fig. S6C, supplementary material Fig. S7H). This significant expansion of the ZCL2897hi cell population suggests that Ubc9 restrains division, keeps progenitors from entering an aberrant differentiation program, and maintains organ integrity.
To test if ZCL2897 expression marks lamellocytes, we examined relative expression of either MSNF9mo-mCherry (MSNF9, supplementary material Fig. S3A–D), or Atilla (supplementary material Fig. S3E,F) with ZCL2897. Both methods revealed that while a significant number of mutant ZCL2897-positive cells also express Atilla or MSNF9 (yellow signal, supplementary material Fig. S3C,D,F; white arrowheads in supplementary material Fig. S3G–R'), a number of ZCL2897hi cells do not express either lamellocyte marker (supplementary material Fig. S3G–R', white arrows). We also identified rare cells with low or absent ZCL2897 expression but positive for MSNF9 (supplementary material Fig. S3O,O', blue arrowheads) or Atilla (M.K. and S.G., unpublished data). Thus, expansion of ZCL2897 population in the mutant supports the idea that Ubc9 maintains proliferative quiescence in the progenitor population and prevents their aberrant and lamellocyte differentiation.
Ubc9 affects cells of the transition zone
To probe the properties of the expanded population in mutant glands with a Gal4 driver, whose expression is not downregulated by the effects of the mutation, we examined the expression of the 76B>Gal4
. This driver is expressed in few cells of the lymph gland (Paddibhatla et al., 2010
), although the identity of these cells is not known. We found that at late third instar, many heterozygous 76B>GFP
-expressing cells are located outside the Dome-MESO
boundary (i.e., they are negative for Dome-MESO
; , white arrows) and do not express the Pro-PO (), Nim C (), or MSNF9 (), although rare exceptions are observed (zoomed panels in ; yellow arrows). Thus, 76B>GFP
expression marks the cells that are intermediate to the Dome-MESO
-positive progenitors in the medullary zone and the differentiated cells in the cortex. Because most of the cells expressing 76B>GFP
reside outside the Dome-MESO
boundary, interspersed in the cortex, and the double positives with either Dome-MESO
() or the Pro-PO/Nim C () are rare, they most likely represent the transitional precursors that are derived from the medullary zone progenitors, but have not yet assumed a final differentiated identity. The existence of this transition zone has been suggested in recent studies (Krzemien et al., 2010
76B-Gal4 characterization in control and Ubc9 lymph glands.
population is significantly expanded in Ubc9
mutant glands (). Some mutant 76B>GFP
cells are also positive for either MSNF9 or Nim C (; yellow arrows). [Ubc9
lymph glands have very few crystal cells (Chiu et al., 2005
) and these were therefore not examined in mutant glands.] 76B>GFP
expression is also expanded in single cells in circulation or those in microtumors in the hemolymph (supplementary material Fig. S2M–P). This expanded expression of 76B>GFP
parallels the expression dynamics of ZCL2897
(supplementary material Fig. S2E–L) in the mutants.
Ubc9 is expressed throughout the lymph glands
Ubc9 protein is ubiquitously expressed in the anterior and posterior lobes of the control third instar animals, in both, medulla (nuclear and cytoplasmic) and cortex (mainly nuclear; supplementary material Fig. S4A–D'). In addition to the diffuse nuclear signal (supplementary material Fig. S4B,B',D,D'), speckles are also present (supplementary material Fig. S4B,B'; white arrows). Ubc9 is also expressed in the dorsal vessel (supplementary material Fig. S4A,C,E,G; star). Ubc94-3/5
mutants exhibit significantly lower levels of the protein in the entire organ (supplementary material Fig. S4E–H'). Both hypomorphic alleles have been previously characterized molecularly (Apionishev et al., 2001
SUMO pathway components in hematopoiesis
If changes observed in Ubc9 mutant hematopoietic organ are due to loss of sumoylation, then other enzymes of the sumoylation cascade should be similarly required. To test this idea, we examined larvae carrying loss-of-function mutations in E1 (Aos1c06048) and E3/PIAS (Su(var)2-101/Su(var)2-102) genes. E1 is an activating heterodimer of Aos1 and Uba2 subunits, while PIAS, encoded by Su(var)2-10, serves as the E3 ligase. Like Ubc9 glands, Aos1 and PIAS glands exhibit significant activation of ZCL2897 (). Mutants in each background produce hematopoietic tumors () marked by increased expression of ZCL2897. Numerous lamellocytes appear in dispersing anterior lobes and in circulation (, unpublished data).
Sumoylation enzymes in larval hematopoiesis.
To test if Dome>GFP expression is compromised by loss of sumoylation enzymes, we performed knockdown of E1 subunits via RNAi. Knock-down of either Aos1 or Uba2 led to significant reduction of the Dome>GFP expression, lamellocyte differentiation, anterior lobe dispersal (), and tumorogenesis (). These observations parallel those for Ubc9 mutants and demonstrate that sumoylation is a fundamental mechanism through which cell division and differentiation of hematopoietic progenitors is simultaneously regulated.
Ubc9 microtumors arise from progenitor hyperplasia of anterior and posterior lobes
To more directly study the role of Ubc9 in the cell cycle, we stained lymph glands in late third instar stage (day 6.5–7) for phospho-histone H3 (). At this stage, most control animals pupariated or are about to pupariate; their lymph gland lobes are relatively large and mitotically active (). In mutants, the anterior lobes are dispersed with only few cells remaining (, outline). The enlarged posterior lobes have numerous mitotically-active cells; these lobes show signs of detachment from the dorsal vessel (e.g., in , only single partially detached posterior lobes are visible). Lobes of both PL1 and PL2 are severely affected (, merged confocal Z sections) and the number of phospo-histone H3-positive cells ranges between 200–800 per posterior lobe set, compared to 30–80 phospho-histone H3-positive cells in the corresponding control lobes.
To clarify the identity of mitotic cells and examine their relation to Dome>GFP expression, we stained anterior lobes of slightly younger early 6-day lymph glands (where Dome>GFP is still detectable) and visualized differentiated plasmatocytes (anti-Nimrod C antibody) or lamellocytes (anti-Atilla antibody) with anti-phospho-histone H3 antibody. Most of the Dome>GFP cells in control glands are phospho-histone H3-negative, confirming proliferative quiescence of this cell population (, arrowheads indicate mitotic cells). Not surprisingly, markers for mitosis and Nimrod C rarely colocalized in cells of either genotype (, arrow). None of the lamellocytes were in division (). Notably however, loss of Dome>GFP precedes increase in proliferation, as phospho-histone H3 staining is observed in regions of mutant lobes with low Dome>GFP signal, but only rarely among the Dome>GFP-positive cells ().
Collectively, these observations strongly suggest that the solid compact large Ubc9 microtumors result primarily from the excessive mitoses in the lymph gland lobes. The expanded lobes are severed from the dorsal vessel to become free-floating microtumors. Some small tumors and aggregates are likely derived from clusters of cells dispersed from the anterior lobes. These conclusions are supported by the following: (1) Extensive mitoses and overgrowth in the anterior and posterior mutant lobes of 6 to 7 day old organs and their partial dispersal (, , , , supplementary material Figs S1, S2). (2) Massive overgrowth of the remaining posterior lobes with enhanced expression of ZCL2897 (, supplementary material Fig. S2J,L, supplementary material Fig. S3D,F) or 76B (, supplementary material Fig. S2O,P) in the lobes and microtumors. (3) The morphologies of overgrown ZCL2897hi and 76B>GFPhi lobes match those of the microtumors in the hemolymph. (4) The time of microtumor appearance in the hemolymph correlates with observed detachment of the overgrown lobes from the dorsal vessel.
Ubc9 function is essential in hematopoietic progenitors
To delineate the spatio-temporal requirement of Ubc9 in restraining division and differentiation of hematopoietic progenitors, we provided wild type Ubc9 protein to these populations via Dome-Gal4 and 76B-Gal4. The experimental rescue class (Ubc9; Dome>Ubc9wt) animals exhibit simultaneous and remarkable amelioration from the differential effects of the mutation on the anterior and posterior lobes: (1) The normal temporal and spatial regulation of the Dome promoter is restored in both anterior and posterior lobes and cells of the dorsal vessel (). (2) The normal course of lobe development is restored, i.e., not only are the rescued posterior lobes comparable in size to control posterior lobes, they remain tethered to the dorsal vessel. (3) Even though the cortical zone of some rescue class glands shows differentiating lamellocytes, the overall proportions of the medullary and cortical zones return to normal. Overexpression of Dome>Ubc9wt reduces the number of Dome>GFP cells very slightly (). (4) A stark reduction in tumorogenesis is noted as reduction in the proportion of animals carrying free microtumors (supplementary material Fig. S5D; microtumor penetrance from 75% to 11%) or aggregates (clusters of 15–50 cells; supplementary material Fig. S5E; from 82% to 23%). Other non-hematopoietic defects, i.e., delay in the onset of pupariation and adult lethality, are also rescued. These rescued adults carry no visible microtumors.
Significantly, like Dome>Ubc9wt, 76B>Ubc9wt also rescues Ubc9 defects. Since its expression is high in mutant cells (), it is possible to visualize the remedial effects of 76B>Ubc9wt as it shrinks the GFP-positive cell population, restores coherent lymph gland lobes (I.P. and S.G., unpublished data), prevents posterior lobe detachment, and reduces the tumor burden (supplementary material Fig. S5F–I).
In contrast to the full rescue with the Dome>Ubc9wt
transgenes, we found that large microtumors persisted with Collagen>Ubc9wt
expression (supplementary material Fig. S6A–D; Cg-Gal4
is expressed in the lymph gland cortical zone, circulating hemocytes, and fat body) (Asha et al., 2003
). All together, these observations are consistent with the interpretation that even though Ubc9
influences all hematopoietic compartments and the integrity of the lymph gland, the primary function of the protein is to maintain quiescence in hematopoietic progenitors. Sumoylation appears to serve a critical tumor-suppressive function by regulating the gene expression and the cell cycle of hematopoietic progenitors of the third instar larval lymph gland.
Ubc9 hyperplasia is niche-independent
To examine the requirement for Ubc9
in the niche, we compared niche morphology and size, and the membranous projections emanating from the niche into the medullary zone (Krzemien et al., 2007
; Mandal et al., 2007
) in heterozygous and mutant glands. We found no significant difference in the niche size, measured either as the number of cells expressing Antennapedia protein (supplementary material Fig. S7A–C, 5 day old animals) or Antp>GFP
(supplementary material Fig. S7D–F, 6 day old animals). There was no difference in the niche projections, which were sparse in both backgrounds (supplementary material Fig. S7D,E). Cells of the dorsal vessel immediately adjacent to the niche express Antp
(by both criteria), although we found no difference in its expression between heterozygous and mutant glands (supplementary material Fig. S7A,B,D,E; asterisks). An occasional population of Antp>GFP
cells is found in the posterior lobes of the mutant or in microtumors (M.K. and S.G., unpublished data).
To link Ubc9 function in the niche to overproliferation, we examined Ubc9−
progeny. These rescue class larvae did not experience relief from hematopoietic defects (supplementary material Fig. S7G–K) and died during pupal stages, just like their mutant siblings. Overexpression of Ubc9wt
in the niche (Antp-Gal4
) did not modify the niche or lobe morphology, nor did it induce lamellocytes (M.K. and S.G., unpublished data). Likewise, mutants were not rescued when wild type protein was supplied in the niche by Collier-Gal4
(M.K. and S.G, unpublished data) (Crozatier et al., 2004
). These observations demonstrate that progenitor hyperplasia in mutants is niche-independent and that its function is autonomous with respect to the progenitor pool.
Loss of Ubc9 is linked to reduction of Dacapo levels
Protein interaction data suggested direct association of Ubc9 with Drosophila
CDK inhibitor Dacapo (Dap) (Stanyon et al., 2004
). To test if Dap levels are affected in Ubc9
cells, we stained lymph glands with anti-Dap antibody (described in de Nooij et al.) (de Nooij et al., 1996
). In control glands, levels of Dap protein differ: cytoplasmic Dap is somewhat higher in the compact region of the medullary zone (dotted lines '), than in the cytoplasm of Dome>GFP
-negative cells. This correlation is maintained in Ubc9
glands, where cytoplasmic Dap signal is significantly reduced in cells with lower Dome>GFP
signal and loss of the compact architecture ('). The overall correlation between high Dome>GFP
and high Dap signals suggests that sumoylation maintains quiescence by controlling cell cycle exit by sustaining high levels of Dacapo. While in both, heterozygous and mutant glands, Dacapo levels are lower in cells outside the medulla, in both backgrounds Dap protein is clearly detected ().
Lymph gland cells respond to cell cycle inhibitors Dacapo/p21 and human p21 rescues Ubc9 tumorogenesis.
Expression of human p21 relieves Ubc9 overproliferation
Dacapo shares structural and functional similarity with vertebrate cyclin/cyclin-dependent kinase (CDK) inhibitors, p21/p27 (de Nooij and Hariharan, 1995
; de Nooij et al., 1996
; Lane et al., 1996
). Like overexpression of Ubc9wt
, both Dome>Dap
lead to reduction of the progenitor population (). The effect of Dome>p21
is stronger than that of Dome>Dap
2060±384 cells in control (average ± SD, per both anterior lobes), to n=
1566±449 in Dome>Dap
, and n=
952±301 in Dome>p21
If the primary function of sumoylation is to maintain quiescence in progenitors, expression of p21 in this population may be sufficient to partially restore lymph gland homeostasis. To test this hypothesis, we created Dome>p21; Ubc9 animals. Unlike Dome>Ubc9wt, Dome>p21 resulted in only temporary and weak rescue (supplementary material Fig. S8A) presumably because in Dome>p21; Ubc9 glands, Dome>GFP levels continue to remain low (M.K. and S.G., unpublished data).
In contrast to Dome>p21, both, 76B>Dap and 76B>p21 prevent overgrowth of the progenitor population in mutant glands, restoring their normal compact morphology. There is a decline in the 76B>GFP-positive cells (), the lobes do not disperse or dislocate, and microtumor penetrance is significantly reduced (, supplementary material Fig. S8B). However, when p21 was provided in cells of the cortical zone and circulating hemocytes (with SrpHemo, Hemese, Hml, or Cg), we found no evidence of tumor rescue. Thus, downregulation of Dap expression in Ubc9 mutant lymph gland progenitors and Ubc9 rescue with 76B>Dap/p21 confirm the tumor-suppressive function of Ubc9 in the hematopoietic progenitors and suggest that cell cycle inhibition is likely maintained through sumoylation.