The Drosophila Perlecan gene trol regulates multiple signaling pathways in different developmental contexts
© Lindner et al; licensee BioMed Central Ltd. 2007
Received: 08 March 2007
Accepted: 02 November 2007
Published: 02 November 2007
Heparan sulfate proteoglycans modulate signaling by a variety of growth factors. The mammalian proteoglycan Perlecan binds and regulates signaling by Sonic Hedgehog, Fibroblast Growth Factors (FGFs), Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF), among others, in contexts ranging from angiogenesis and cardiovascular development to cancer progression. The Drosophila Perlecan homolog trol has been shown to regulate the activity of Hedgehog and Branchless (an FGF homolog) to control the onset of stem cell proliferation in the developing brain during first instar. Here we extend analysis of trol mutant phenotypes to show that trol is required for a variety of developmental events and modulates signaling by multiple growth factors in different situations.
Different mutations in trol allow developmental progression to varying extents, suggesting that trol is involved in multiple cell-fate and patterning decisions. Analysis of the initiation of neuroblast proliferation at second instar demonstrated that trol regulates this event by modulating signaling by Hedgehog and Branchless, as it does during first instar. Trol protein is distributed over the surface of the larval brain, near the regulated neuroblasts that reside on the cortical surface. Mutations in trol also decrease the number of circulating plasmatocytes. This is likely to be due to decreased expression of pointed, the response gene for VEGF/PDGF signaling that is required for plasmatocyte proliferation. Trol is found on plasmatocytes, where it could regulate VEGF/PDGF signaling. Finally, we show that in second instar brains but not third instar brain lobes and eye discs, mutations in trol affect signaling by Decapentaplegic (a Transforming Growth Factor family member), Wingless (a Wnt growth factor) and Hedgehog.
These studies extend the known functions of the Drosophila Perlecan homolog trol in both developmental and signaling contexts. These studies also highlight the fact that Trol function is not dedicated to a single molecular mechanism, but is capable of regulating different growth factor pathways depending on the cell-type and event underway.
Heparan sulfate proteoglycans (HSPGs) are a family of cell-surface and extracellular proteins modified by the attachment of glycosaminoglycan chains. The general structure of the protein core determines the family the HSPG belongs to: Syndecans contain a transmembrane domain, Glypicans are tethered to the cell surface via a GPI linkage and Perlecans are secreted components of the extracellular matrix. Both the protein core and glycan chains play important roles in HSPG function through protein-protein and sugar-protein interactions. Genetic studies, first in Drosophila and later in mouse and zebrafish, demonstrated the importance of the heparan sulfate chains on all three types of HSPGs for signaling by multiple growth factors such as the Fibroblast Growth Factors (FGFs), Hedgehogs, Wnts and Transforming Growth Factors (TGFβs) (reviewed in ).
Perlecan is the largest member of the HSPG family with a core protein of approximately 450kD in size. Perlecan has been linked to signaling by the heparan-dependent growth factors FGF2, Vascular Endothelial Growth Factor (VEGF) and Sonic Hedgehog (SHH) in mammalian systems (reviewed in [2, 3]). Studies of Perlecan knock-out mice have demonstrated roles for Perlecan in vascular development and chondrogenesis as well as maintenance of basement membrane integrity [4–7]. Additional mammalian studies have revealed Perlecan's functions in angiogenesis and carcinogenesis ([8–11], reviewed in [2, 12]). Mutation of Perlecan in humans leads to the muscle tone symptoms of Schwartz-Jampel syndrome, possibly through altered excitability of the neuromuscular junction and the skeletal abnormalities of Silver-Handmaker syndrome, presumably through effects on chondrogenesis [13–15].
Studies of Perlecan in invertebrate model systems have led to additional insights into Perlecan function. The single Perlecan gene in C. elegans is encoded by the unc-52 locus . Mutations in unc-52 result in embryonic or adult paralysis due to defects in body wall muscle cells ([16, 17], reviewed in ). Mutations in unc-52 also enhance cell migration defects caused by decreased netrin, FGF, TGFβ or Wnt signaling. In Drosophila, Perlecan is encoded by the trol gene on the X chromosome [19, 20], which was initially implicated in the control of stem cell division in the developing larval brain [21, 22]. In the larval brain, trol promotes the cell cycle progression of mitotically arrested neuroblasts [23, 24] through modulation of FGF and Hedgehog signaling . These Drosophila studies were the first to link Perlecan to Hedgehog signaling. More recently, studies of oogenesis in Drosophila have uncovered a role for Perlecan in the maintenance of epithelial cell polarity through interactions with the extracellular matrix receptor Dystroglycan .
The many signaling pathways associated with HSPGs in general and Perlecan in particular led us to ask what other biological processes may require Perlecan function. We used a series of trol mutants to investigate several phenotypes ranging from overall developmental progress to specific alterations of stem cell division and hemocyte production. Furthermore, analysis of signaling pathway response genes revealed that while mutations in Perlecan decrease signaling in multiple pathways, at least some of these effects are tissue specific.
Results and discussion
Development and lethal phase
Why would animals mutant for trol7 (that has a strong effect on neuroblast proliferation) be able to progress further in development than animals mutant for trol4 which causes a weaker neuroblast proliferation phenotype? One possibility is that trol modulates the activity of different signaling pathways in different tissues. For example, a mutation that affects the ability of Trol to function in the Hh pathway would have a severe effect on developmental decisions that require Hh activity and very little effect on decisions that do not require Hh signaling. To address this possibility we investigated the impact of trol mutations on two distinct developmental events and several signaling pathways.
Effects of trol mutations on TNb proliferation
trol was initially identified as a mutation on the X chromosome that affected the proliferation pattern of neuroblasts in the brain lobes and ventral ganglion [21, 22]. Since neuroblasts in the thoracic region of the ventral ganglion begin proliferation in early second instar [21, 26, 27], we evaluated the ability of thoracic neuroblasts (TNbs) to enter S phase in trol mutant animals. We adapted the idea of phenotypic classes to produce a scale for the extent of TNb proliferation at four hours post molt (Fig. 1C–G). Five TNb classes were defined as follows: Class 1, no neuroblasts labeled; Class 2, a small number of labeled neuroblasts with no distinct segmental pattern; Class 3, labeled neuroblasts in a segmentally repeated lines with very few labeled neuroblasts in between the lines or in the medial region of the ventral ganglion; Class 4, labeled neuroblasts in a segmentally repeated line with some labeled neuroblasts in between the lines or in the medial region of the ventral ganglion; and Class 5, labeled neuroblasts in heavily populated segmental pattern with many labeled neuroblasts in the medial portion of the ventral ganglion. When both sides of a ventral ganglion did not conform to a single class, the sample was scored as the higher class. This will have a conservative effect of scoring a partial loss-of-proliferation TNb phenotype as more wild-type. Thus we can have greater confidence in the significance of TNb proliferation phenotypes observed compared to controls.
TNb BrdU incorporation phenotype of trol mutants at 4–5 hours pm.
trol affects Bnl and Hh signaling in the ventral ganglia
Trol localization in the larval brain
Effects of trol mutations on larval hemocyte number
Trol localization and function in hemocytes
The decrease in the number of circulating plasmatocytes in trol mutants versus controls suggested that trol might indeed function to promote Ras-MAPK signaling by PDGF/VEGF in circulating plasmatocytes. This predicts that Trol protein would be localized on these plasmatocytes. We used Trol-GFP protein trap to examine the plasmatocytes for the presence or absence of Trol protein. Fluorescence microscopy revealed that Trol-GFP is indeed found on circulating plasmatocytes in third instar larvae (Fig. 5B,C), but not in the lymph gland (data not shown).
This result is consistent with the requirement for Ras-MAPK activation in plasmatocytes for plasmatocyte proliferation and for PVR in plasmatocytes to avert apoptosis, and supports the hypothesis that Trol modulates PVR-Ras-MAPK signaling in plasmatocytes. The ETS-transcription factor pnt is a MAPK-response gene and will drive plasmatocyte proliferation . Therefore we asked if trol mutant plasmatocytes show decreased levels of pnt compared to controls. Plasmatocytes were collected by bleeding third instar trolb22 and trol7 mutant larvae and sibling controls, RNA was extracted and amplified, and subjected to qRT-PCR analysis. qRT-PCR studies demonstrated that plasmatocytes isolated from either trolb22 or trol7 mutants show decreased expression of pnt compared to controls, further evidence that trol modulates Ras-MAPK signaling in plasmatocytes (Fig. 5D).
Trol and other growth factor signaling pathways
To determine if Trol is necessary for growth factor signaling in other tissues at other stages we assayed for dpp, wg and hh expression and activity in trolb22 and trol sd third instar brain lobes and eye discs. No significant changes in either growth factor expression or signaling were observed in trolb22 samples (data not shown). In trol sd samples, expression of all three growth factors decreased by 65–85%, as did the expression of their response genes (Fig. 6B). The sole exception is wg/slp, where wg expression decreased about 65% and slp expression decreased only about 50%. These data indicate that mutations in trol do not dramatically decrease the signaling efficiency of Dpp, Wg or Hh in third instar brain lobes and eye discs, unlike the effect of those same trol mutations in second instar.
trol and Drosophila development
We have previously demonstrated that mutations in trol prevent the onset of neuroblast division in the first instar brain and that most trol mutations are lethal. Mutations in a second gene, anachronism, also affect the onset of neuroblast proliferation but in the opposite manner: in anachronism mutants, mitotically regulated neuroblasts begin cell division too early . However, when a lethal trol mutation was combined with a viable allele of anachronism, the lack of neuroblast division was rescued (double mutants exhibited the anachronism phenotype of premature neuroblast division) but lethality was not . This outcome suggested that trol function is required for other developmental events necessary for survival. Further analyses revealed that trol modulates Hh and Bnl signaling in the first instar brain . Here we have demonstrated that trol function is required for developmental progression to third instar and for pupariation. Analogous to its function in the first instar brain, trol is required to initiate the division of a second, independent and spatially distinct population of neuroblasts in the second instar brain (Table 1, Fig. 1). This initiation of division is also dependent on Bnl and Hh signaling (Fig. 2). We have also demonstrated that the Trol protein is localized to the surface of the brain at all larval stages, which places it in close proximity to the regulated neuroblasts. This localization is consistent with our model where Trol regulates Bnl and Hh signaling to cells adjacent to the regulated neuroblasts by binding the growth factors directly . Trol protein localization to the basal lamina is not limited to the larval brain, as Trol-GFP studies also showed Trol protein in the basal lamina surrounding the salivary glands (Fig. 4G). trol function is not limited to the nervous system, as mutations in trol also diminish the number of circulating plasmatocytes by decreasing expression of pnt, a PVR response gene in plasmatocytes (Fig. 5). We speculate that trol may be necessary for signaling by the Drosophila PDGF and/or VEGF growth factor, just as mammalian Perlecan has been shown to function during angiogenesis . Our studies of Dpp and Wg indicate a positive feedback between dpp expression and Dpp signaling and wg expression and Wg signaling in the second instar ventral ganglion. Signaling by Dpp and Wg is also dependent on trol in the second instar brain, but not (or very little) in the third instar brain lobes and eye discs (Fig. 6), despite the fact that Dpp and Wg signaling are taking place in those tissues. In fact, even Hh signaling appears to be independent of trol in this context. These results highlight an important concept in trol, and indeed, in proteoglycan function: that the Trol protein will be used at different times and places to regulate the signaling of different growth factors. Deciphering the role of trol in different developmental decisions will require that we examine each event individually, as trol will not necessarily mediate the same molecular mechanism each time.
Involvement of HSPGs in growth factor signaling
The requirement for heparan sulfate proteoglycans in signaling by different families of growth factors is well established , but what is not yet clear is why different organs and tissue types use different HSPGs to modulate these signaling pathways. One possibility is that the specific mechanism(s) through which these molecules modulate signaling activity allows for site-specific variations in the regulation of signaling activity. HSPGs with varied amino acid sequence can act in the same signaling pathway, such as Syndecan-4 and Perlecan for FGF2 [28, 44] or Glypicans, Syndecan-3 and Perlecan for Hh [19, 43, 45]. Mutations that affect heparan sulfate synthesis or modification strongly affect FGF2 and Hh signaling . Furthermore, Perlecan isolated from various endothelial cell sources has different binding affinities for FGF2 . These data initially suggested that the protein core of the HSPG might have little to do with signaling specificity and that the main functional domain of HSPGs is concentrated in the sequence of the heparan sulfate chains.
The carbohydrate-centric view is being challenged by studies that indicate a role for the protein-protein interactions of HSPGs with growth factors and other signaling molecules. For example, expression of chimeric molecules has shown that the cytoplasmic tail of Syndecan is specifically required for FGF2 signaling in addition to its heparan sulfate chains . Perlecan protein-protein interactions include the ability of Perlecan to bind growth factors and extracellular matrix molecules at various sites on its protein core. Further mechanisms that allow for differential regulation include processing of HSPGs. These studies suggest a reason for the use of a particular HSPG during an individual developmental decision – the flexibility of combining both carbohydrate-based regulation and protein-based regulation of cell-cell signaling may make a specific HSPG uniquely suited for a given situation.
In the context of combined carbohydrate and protein inputs into HSPG function, it becomes clear that a given HSPG may be expressed and function in very specific contexts that take advantage of its unique regulatory abilities. It is interesting to note that we have connected Perlecan with FGF and Hh signaling in the developing fly brain while mouse studies have shown that Perlecan knock-out mice have cerebral cortex abnormalities [6, 19, 21]. trol mutant larvae have decreased numbers of circulating hemocytes that are likely due to decreased Ras-MAPK signaling by VEGF/PDGF. Perlecan knock-out mice also have defects in chondrogenesis and cardiovascular development and mammalian studies have demonstrated a role for Perlecan in angiogenesis driven by FGFs, VEGF and PDGF . Finally, we have shown that Perlecan is required for SHH signaling during human prostate cancer growth , which reveals a new system for the investigation of the mechanism of Perlecan action. Further analysis of the ability of HSPGs to substitute for each other in cell fate decisions and the means by which they individually regulate cell-cell communication will lead to a clearer understanding of the inputs necessary for cells to carry out a developmental or disease progression.
Stocks of the viable trolb22 allele and the lethal trol4, trol7, trol8 and trol sd alleles have been described previously [19, 21, 22, 29]. All trol mutant stocks with the exception of trolb22 are y trol x w/Binsn where the chromosome carrying the trol mutation is marked with y to facilitate identification of y trol mutant versus y+control larvae. The trol-GFP protein trap was obtained from Dr. Stephane Noselli. The bnl06916 and hh AC stocks were obtained from the Bloomington stock center and used to construct y ; bnl06916/TM3y+ and y ; hh AC /TM3y+ stocks for genetic studies.
Early first instar larvae were collected and placed on apple juice plates with yeast. Each plate initially had 50 mutant or control animals per plate, segregated to prevent competition between mutant and wildtype siblings. Two plates of each genotype were examined. The number and stage of larvae still present on each plate were assayed every 24 hours and the survivors transferred to a fresh plate. Since none of the trol mutants with the exception of trolb22 produce viable adults, individual animals were followed only until pupariation.
Developmental synchronization was carried out as previously described [19, 21, 23, 48]. Flies were allowed to lay eggs on apple juice agar plates with fresh yeast overnight or for about 24 hours. For staging of synchronized first instar larvae, the plate was first cleared of any larvae and newly hatched larvae collected in one hour windows and placed on new apple juice plates with yeast at 25°C for aging. For staging of second instar larvae, late first instar larvae were placed on fresh apple juice plates with yeast. Newly molted second instars were collected in one hour windows and placed on apple juice plates with yeast at 25°C for aging or dissected immediately.
BrdU assays were carried out as previously described [19, 21, 23, 48]. Briefly, animals were fed BrdU-containing artificial medium for one hour, dissected in PBST and fixed with Histochoice (Amresco) for 10 minutes. Brain samples were denatured in PBST-HCl for 30 minutes, washed and blocked in PBNT for one hour. Primary anti-BrdU antibody (Becton-Dickinson) was added at 1:200 overnight at 4°C. Samples were washed and incubated with HRP-conjugated secondary antibody at 1:400 for 2–4 hours at room temperature. Signal was developed using a DAB substrate (Sigma).
Larval hemocyte assay
Hemocytes from three third instar larvae were harvested using a Pasteur pipette pulled to generate a capillary end, pooled and counted on a standard hemacytometer slide. Five 16-square regions were counted for each pooled sample. Three replicates were assayed for each genotype.
Quantitative RealTime PCR
Whole first instar brains or ventral ganglia dissected from the brains of second instar larvae were used for RNA isolation. For first instar brain samples, total RNA was isolated using Trizol (Invitrogen) following manufacturer's directions. Samples were DNAsed and reverse transcribed using oligo dT primers. The resulting cDNA was used to perform quantitative Real Time PCR with SYBR Green dye. For ventral ganglia isolated during second instar RNA was extracted and the sequences amplified as described in [49, 50]. Hemocyte studies were carried out on pooled hemolymph from three third instar larvae per sample. RNA was extracted and amplified as for ventral ganglia. All qRT-PCR reactions were carried out in triplicate at three different template concentrations to ensure that we were within linear template range. Primer sequences are available upon request. β-actin expression was used as an internal control. Data were analyzed using the delta-delta calculation method to yield fold change compared to controls.
Determination of significance was accomplished by use of Student's t test or ANOVA, depending on the design of the study.
List of Abbreviations
Fibroblast Growth Factor
Green Fluorescent Protein
Heparan Sulfate Proteoglycan
Platelet Derived Growth Factor
PDGF- and VEGF-Receptor Related
quantitative Real Time PCR
Transforming Growth Factors
Vascular Endothelial Growth Factor
The authors would like to thank past and present members of the Datta lab, anonymous reviewers and Drs. Subhabrata Sanyal and Vlad Panin for helpful discussions, the Bloomington stock center and Dr. Stephane Noselli for fly stocks, Dr. Larry Ringer for help with statistical analyses and Dr. Shubha Govind for advice on hemocyte counting. Alyssa Rosenbloom, Bryan Tackett, Rachel Miller, Megan Carrier and Allegra Lamison provided technical support. This work was supported by NIH grant NS 036737-04 to S. D.
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