Characterization of genetic and molecular tools for studying the endogenous expression of Lactate dehydrogenase in Drosophila melanogaster

Drosophila melanogaster larval development relies on a specialized metabolic state that utilizes carbohydrates and other dietary nutrients to promote rapid growth. One unique feature of the larval metabolic program is that Lactate Dehydrogenase (Ldh) activity is highly elevated during this growth phase when compared to other stages of the fly life cycle, indicating that Ldh serves a key role in promoting juvenile development. Previous studies of larval Ldh activity have largely focused on the function of this enzyme at the whole animal level, however, Ldh expression varies significantly among larval tissues, raising the question of how this enzyme promotes tissue-specific growth programs. Here we characterize two transgene reporters and an antibody that can be used to study Ldh expression in vivo. We find that all three tools produce similar Ldh expression patterns. Moreover, these reagents demonstrate that the larval Ldh expression pattern is complex, suggesting the purpose of this enzyme varies across cell types. Overall, our studies validate a series of genetic and molecular reagents that can be used to study glycolytic metabolism in the fly.


ABSTRACT
Drosophila melanogaster larval development relies on a specialized metabolic state that utilizes carbohydrates and other dietary nutrients to promote rapid growth.One unique feature of the larval metabolic program is that Lactate Dehydrogenase (Ldh) activity is highly elevated during this growth phase when compared to other stages of the fly life cycle, indicating that Ldh serves a key role in promoting juvenile development.
Previous studies of larval Ldh activity have largely focused on the function of this enzyme at the whole animal level, however, Ldh expression varies significantly among larval tissues, raising the question of how this enzyme promotes tissue-specific growth programs.Here we characterize two transgene reporters and an antibody that can be used to study Ldh expression in vivo.We find that all three produce similar Ldh expression patterns.Moreover, these reagents demonstrate that the larval Ldh expression pattern is complex, suggesting the purpose of this enzyme varies across cell types.Overall, our studies validate a series of genetic and molecular reagents that can be used to study glycolytic metabolism in the fly.

INTRODUCTION
Lactate dehydrogenase (Ldh) is a highly conserved enzyme that serves a key role in the regulation of cellular redox balance, glycolytic metabolism, and energy production [1,2].Although this enzyme has been studied for over a century [3] [4], we are still discovering new functions for Ldh in metabolism, signal transduction, and even gene expression [1,2,[5][6][7][8].Moreover, due to the central role that Ldh serves in cellular physiology, a wide variety of human diseases are associated with changes in Ldh expression and activity.For example, Ldh is well-known to play a central role in tumor metabolism, and enhanced Ldh serum levels are used as one of the diagnostic parameters in oral, laryngeal, melanoma, renal cell carcinoma and breast cancers, [9][10][11][12].
The role of Ldh in human disease, however, extends far beyond cancer metabolism, as enhanced Ldh activity is also observed in diabetes and hyperglycemia [13][14][15][16], as well as during viral infections [17,18].In this regard, despite the well-documented relationship between elevated Ldh activity and human disease progression, questions remain about the tissue-specific mechanisms that link changes in Ldh expression and activity with the overall disruption of human health.
The fruit fly Drosophila melanogaster has emerged as an ideal system to study the role of Ldh in both healthy and diseased tissues.For example, Ldh activity levels fluctuate during the life-cycle in a predictable manner [19,20].In this regard, Ldh clearly plays a significant role during larval development, when the animal experiences a nearly 200-fold increase in body mass [21].Ldh expression and activity are very high during this growth phase when compared to other phases of fly life-cycle [19], indicating that lactate production is important for larval metabolism.Consistent with this hypothesis, Ldh mutations render larvae unable to maintain a normal redox balance, and when combined with Gpdh1 mutations, induce significant growth defects [22].In this manner, studies of Ldh provide an opportunity to understand how changes in Ldh expression affect Drosophila growth, development, and life-history events.
Similarly, studies of Drosophila disease models have also begun to focus on the link between lactate metabolism activity and tumor growth [for review, see 23], neuronal health and aging [24][25][26][27], as well as during infections and immune challenges [28].
Notable among these findings is that Drosophila tumors up-regulate Ldh in a manner that mimics the elevated Ldh-A activity observed in many human cancer cells [23,[29][30][31][32][33], indicating that Ldh in both flies and humans serves a beneficial role in tumorous growth.
Thus, studies of Drosophila Ldh hold the potential to better understand how mammalian Ldh homologs function in development and disease.

Several genetic and molecular reagents have been used to examine Drosophila
Ldh expression and activity.For example, early studies of Ldh in the fly relied on a biochemical enzymatic assay that generated an easily visualized staining pattern [30,34].These enzymatic assays have been supplemented with transgenes that rely on fluorescent proteins to either directly or indirectly visualize Ldh gene expression [28,30,32,35].However, most published Ldh reporters have not been directly compared, either with each other or with endogenous protein expression, raising the possibility that published genetic reagents might produce artifactual results.To address this issue and thus facilitate more precise studies of Drosophila Ldh function, here we compare the following genetic and molecular reagents: (i) a previously described Ldh genomic rescue construct that consists of a green fluorescent protein (GFP) coding region inserted immediately before the Ldh stop codon (referred to as Ldh-GFP Genomic ) [32], (ii) a previously described Ldh-GFP reporter that consists of EGFP inserted 50 bp upstream of the endogenous start site (referred to as Ldh-GFP enhancer ) [36] [30], and (iii) a commercially available Drosophila Ldh antibody (see methods).As described below, we demonstrate that Ldh-GFP Genomic produces a functional enzyme capable of rescuing the Ldh mutant phenotypes.Moreover, our studies reveal that all three reagents generate similar tissue-specific Ldh expression patterns.Interestingly, our analyses also reveal that Ldh is expressed in a complex manner during larval development, suggesting that this enzyme functions in multiple cell-and tissue-specific roles during larval development.Overall, our study enhances the ability of the Drosophila community to study Ldh within the context of both normal developmental as well as human disease models.

Drosophila melanogaster husbandry and genetic analysis
Fly stocks were maintained at 25ºC on Bloomington Drosophila Stock Center (BDSC) food.Larvae were raised and collected as previously described [37].Briefly, 50 adult virgin females and 25 males were placed into a mating bottle and embryos were collected for 4 hrs on a 35 mm molasses agar plate with a smear of yeast paste on the surface.Collected plates were stored inside an empty 60 mm plastic plate and placed in a 25ºC incubator.
Two transgenes were examined in our study.The p{Ldh-GFP} transgene was generated by inserting GFP immediately upstream of the Ldh stop codon within a previously described Ldh genomic rescue construct [32,38].For our analysis, the p{Ldh-GFP} was placed in the background of Ldh loss-of-function allele Ldh 16 [38].The previously described Ldh-GFP enhancer trap line was a kind gift from Utpal Banerjee's lab [30,36].Finally, the p{Ldh-mCherry} transgene, which was previously described in a study of Drosophila hemocyte metabolism [28], is identical to the p{Ldh-GFP} except that the mCherry coding sequence was inserted immediately upstream of the Ldh stop codon.To distinguish between the two Ldh-GFP and the Ldh-mCherry constructs in the text, we will refer to the p{Ldh-GFP} and p{Ldh-mCherry} rescuing transgenes as Ldh-GFP Genomic and Ldh-mCherry Genomic , respectively, and the Ldh-GFP enhancer trap line will be referred to as Ldh-GFP Enhancer .

Viability assay
Larval viability was measured by placing 20 synchronized embryos of each genotype on molasses agar plates with yeast paste and measuring time until pupariation.Wandering L3 larvae were subsequently transferred into a glass vial containing BDSC food and monitored until eclosion.

Immunofluorescence Assay
Larval tissues were dissected from mid-third instar larvae in 1X phosphate buffer saline (PBS; pH 7.0) and fixed with 4% paraformaldehyde in 1X PBS for 30 minutes at room temperature.Fixed samples were subsequently washed once with 1X PBS and twice with 0.3% PBT (1x PBS with Triton X-100) for 10 mins per wash.
For antibody staining of GFP in larvae expressing either Ldh-GFP genomic or Ldh-GFP Enhancer , tissues were dissected, fixed and incubated with goat serum blocking buffer (4% Goat Serum, 0.3% PBT) for one hour at RT and stained overnight at 4ºC with the primary antibody rabbit anti-GFP diluted 1:500 (#A11122 Thermo Fisher).
Samples were washed three times using 0.3% PBT and stained with secondary antibody Alexa Fluor 488 Goat anti-Rabbit diluted 1:1000 (#R37116; Thermo Fisher) for either 4 hrs at room temperature or overnight at 4ºC.Stained tissues were washed with 0.3% PBT, immersed in DAPI (0.5µg/ml 1X PBS) for 30 mins and then mounted with vector shield with DAPI (Vector Laboratories; H-1200-10).
For larval tissues stained with the anti-Ldh antibody (Bosterbio; DZ41222), fixed tissues were washed three times with 0.03% PBT for 10 minutes and incubated in blocking buffer (3% bovine serum albumin) for 15 mins on a rocking shaker at room temperature.Tissues were then incubated with 200 l of blocking buffer containing a 1:20 dilution of anti-Ldh on a rocking shaker for 12 hrs at room temperature, 12 hrs at 4ºC, and a third incubation of 12 hrs at room temperature.Samples were then washed twice using 200 l of blocking buffer with a 10 min room temperature incubation for each wash.Once the second wash was removed, 200 l of blocking buffer containing a 1:1000 dilution of goat anti-rabbit Alexa 488 (Thermo Fisher; catalog #R37116) was added to the well and samples were incubated overnight on a rocking incubator at 4 ºC.The next day, tissues were washed twice with blocking buffer, once with 0.03% PBT, once with PBS, and subsequently incubated for 15 mins in PBS containing 0.5 µg/ml of DAPI.Stained tissues were then mounted using vector shield containing DAPI (Vector Laboratories; H-1200-10).
For all imaging studies, multiple Z-stacks of individual tissues were acquired using the Leica SP8 confocal microscope in the Light Microscopy Imaging Center at Indiana University, Bloomington.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis
Samples were collected, processed, and analyzed as previously described [39,40].For all experiments, six biological replicates containing 25 mid-L2 larvae were analyzed per genotype.GC-MS data was normalized based on sample mass and internal succinic-d4 acid standard.

Statistical analysis of metabolite data
Statistical analysis was conducted using GraphPad Prism v9.1.Metabolic data are presented as scatter plots, with the error bars representing the standard deviation and the line in the middle representing the mean value.Data were compared using a Kruskal-Walli's test followed by a Dunn's multiple comparison test.

Genetic characterization of the Ldh-GFP Genomic transgene
As a first step towards validating reagents for studying Ldh expression, we initially examined a GFP-tagged genomic rescue construct, referred to here as Ldh-GFP Genomic , which has been previously used to study muscle development and imaginal discs tumors [32].As described above and elsewhere, this transgene consists of a fragment of genomic DNA that contains the entire Ldh locus with GFP inserted at the 3' end of the coding sequence, immediately prior to the stop codon.To determine if the Ldh-GFP Genomic generates a functional GFP-tagged fusion protein, we assayed the ability of this transgene to rescue Ldh mutant phenotypes.Our genetic approach revealed that the resulting fusion protein appears functional, as the Ldh-GFP Genomic transgene rescues both the lethal phenotype and metabolic defects displayed by Ldh 16/17 mutant larvae (Figure 1C-E) [38].We would note, however, that Ldh-GFP Genomic ; Ldh 16/17 mutant larvae exhibited slightly decreased levels of lactate and 2hydroxyglutarate (2HG) as compared with the wild-type control (Figure 1D,E), suggesting the resulting fusion protein harbors lower activity than the endogenous enzyme.Overall, these results indicate that Ldh-GFP Genomic produces a functional Ldh enzyme.
In addition to assessing Ldh-GFP enzymatic function, we also assayed the gross expression pattern of Ldh-GFP Genomic .Previous studies have demonstrated that Ldh is highly active during larval development relative to other developmental stages -peaking during the L3 stage and gradually declining during the wandering stage and early metamorphosis [19].Consistent with previous observations, we noted that Ldh-GFP Genomic is expressed at such a high level during larval development that GFP was apparent in live larvae using a standard dissecting microscope.Overall, these whole animal expression levels mimicked previously reported changes in larval Ldh enzyme expression and activity, with Ldh-GFP levels peaking in the L3 stage and declining thereafter.We would also note that Ldh-GFP Genomic is so highly expressed in the larval central nervous system (CNS) and body wall muscles that GFP fluorescence from these tissues can easily be observed under low magnifications (Figure 1E).Overall, the observed Ldh-GFP Genomic expression pattern is consistent with previous biochemical and genetic studies, as well as a recent manuscript that used this transgene to examine Ldh expression within body wall muscle [32] In addition to the Ldh-GFP Genomic transgene, we have also generated an identical version of this transgene labeled with mCherry, referred to herein as Ldh-mCherry Genomic , which was previously used to study hemocyte metabolism [28].In general, the Ldh-GFP Genomic and Ldh-mCherry Genomic transgenes exhibit similar spatial expression patterns, with notably high expression in the CNS and muscle (Figure S1).
However, the Ldh-mCherry fusion protein persists throughout much of metamorphosis (compare Figure 1E with S1C), suggesting that this fusion protein is either stabilized or accumulates to a higher level than Ldh-GFP.Since the Ldh-GFP Genomic expression pattern more accurately reflects previously reported temporal changes in Ldh expression and activity, we chose to only characterize Ldh-GFP Genomic in our subsequent experiments.

Ldh is expressed in a complex pattern during larval development
Our analysis of the Ldh-GFP Genomic transgene indicates that this genetic reagent can be used to reliably analyze Ldh expression.To further assess this possibility, we compared the tissue-specific larval expression pattern of the Ldh-GFP Genomic transgene with Ldh-GFP enhancer , as well as a previously undescribed Drosophila Ldh antibody (see methods; note that Ldh does not stain Ldh 16/17 mutant tissues; Figure S2).Our comparison demonstrated that all three reagents produced a similar cell-and tissuespecific larval staining pattern.Below we provide a brief description of the Ldh expression pattern in L3 larvae raised under standard growth conditions.
Central Nervous System: The complexity of the Ldh expression pattern is perhaps most apparent in the central nervous system, where Ldh was highly expressed in the central brain and ventral nerve chord but absent from the optic lobe region (medulla, neuroepithelia and lamina; Figure 2A-L).At the level of individual cell types, we observed Ldh expression in a subset of ganglion mother cells (GMCs, Figure 3E-H), neurons (Figure 3I-L) and glia (Figure 3M-P), but not in neuroblasts (Figure 3A-D).We are unsure as to why Ldh is only present within a subset of GMCs, neurons, and glia.
Digestive and Renal Systems: Both the larval gut and Malpighian tubules express Ldh in cell-specific manner.Within the gut, Ldh was expressed at relatively high levels in subset of cells within the AMP clusters but at low or undetectable levels within larger EC cells (Figure 4A-F).We are uncertain as to why only a subset of these cells express such high Ldh levels.In the Malpighian tubules, high Ldh levels were observed only in stellate cells (Figure 4G-L).
Fat Body and Salivary Glands: Previous studies of the Ldh enzyme activity suggest that Ldh expression levels are relatively low in the larval salivary glands and fat body.
Consistent with this observation, Ldh expression was below the level of detection within both the fat body (Figure 5A-F) and salivary glands (Figure 5G-L).
Imaginal discs: Ldh expression has been extensively studied in the larval imaginal discs [23,[29][30][31][32][33].These studies have consistently observed relatively low levels of Ldh expression during normal imaginal disc development.Our findings confirm earlier studies and again demonstrate that Ldh is present at a relatively low level within the leg, wing, and eye-antennal imaginal discs from L3 larvae (Figure 6A-L).However, we found that Ldh is noticeably expressed within a few cells of these tissues.Patches of GFP expression were observed in the leg disc, which based on the similarity of this expression with that of sens [41] [42], we hypothesize to be the sensory organ precursors (SOPs).Also, both transgenic constructs resulted in GFP expression in cells of the eye-antennal disc posterior to the morphogenetic furrow (Figure 6E,F,K,L).
Overall, the similarities in expression between the Ldh-GFP Genomic transgene, the Ldh-GFP enhancer transgene, and the Ldh antibody in the examined larval tissues suggests that the Drosophila metabolism community can use any of these three reagents to study Ldh.However, we would recommend that any future study uses more than one of these reagents to validate observed changes in Ldh expression.

DISCUSSION
Here we demonstrate that two transgenes and a commercially available antibody reveal similar Ldh expression signatures during larval development.While the Ldh-GFP Genomic and Ldh-GFP enhancer transgenes were previously described and used for a variety of studies, our analysis indicates that either reagent can be used to reliably study Ldh expression.Moreover, our characterization of the Ldh antibody provides the first direct visualization of Ldh protein within the fly and will significantly enhance future studies of this enzyme.Overall, our study thus validates use of these reagents for future use by the Drosophila research community.
Beyond our initial validation of these three reagents, our study also reveals that Ldh is expressed in a complex pattern across tissues and cell-types.At the gross tissue level, our analysis agrees with studies dating back to the 1970s [19], as well as modern gene expression analyses that described how Ldh expression varies in intensity across larval tissues [28,30,32,35,43].Consistent with those early studies, we observe very high Ldh expression in muscle and relatively low or undetectable levels in the fat body and salivary glands.All three observations are interesting in regard to the metabolism of those tissues.For example, while high levels of Ldh expression in larval muscle would be expected based on the role of this enzyme in a wide variety of other animals, adults exhibit relatively low levels of Ldh activity [19], indicating that Drosophila muscle has evolved to meet the extreme energetic demands without using this enzyme and raising the question as to why there is such a dramatic difference in Ldh expression levels between larval and adult muscle.One likely explanation is that Ldh in the muscle, as well as other larval tissues, serves to buffer mitochondrial metabolism against the hypoxic environments often encountered by Dipteran larvae -a hypothesis supported by earlier studies [44,45].Future studies should both examine this possibility and investigate why larval salivary glands and fat body exhibit relatively low Ldh activity levels.
In contrast to the muscle, fat body, and salivary glands, other larval tissues display a complex expression pattern.For examples, the AMP clusters of the larval midgut and stellate cells in the Malphigian tubules exhibit notably high levels of Ldh expression relative to other cell types in the surrounding tissue.The larval CNS, however, exhibits the most dramatic example of how Ldh expression can vary across cell types.Notably, there is a striking lack of Ldh expression in neuroblasts relative to other cell-types.Moreover, while we observe Ldh in both neurons and glia -a result consistent with the current hypothesis that lactate functions in a metabolic shuttle between these two cells types [24,46].However, we observed an unexpected heterogeneity in expression levels throughout the brain, with regions of high Ldh expression being observed adjacent to low levels.We are uncertain as to the significance of this observation.
Finally, we would highlight the distinct lack of Ldh in larval imaginal discs at the timepoint examined.Our observation is consistent with several previous studies, which describe how Ldh is expressed at low levels in normal imaginal discs but at dramatically higher levels in tumorous discs [23,[29][30][31][32][33].Together, these suggest that the presence or absence of Ldh within the discs are of importance for cell growth and development.We would also note that much earlier studies supported a model in which Ldh expression is somehow linked to imaginal disc development.After all, the original name for Ldh in Drosophila melanogaster was Imaginal disc membrane protein L3 (ImpL3) [34,47], which was identified as being induced in response to 20hydroxyedcysone signaling.Consistent with this possibility, imaginal discs exposed to 20-hydroxyecdysone in culture exhibit a significant increase in Ldh expression [34] In conclusion, our study validates the use of both genetic and molecular reagents to accurately study Ldh expression during larval development.Moving forward, we would encourage the fly community to use these reagents in combination when conducting studies of Ldh expression.(E-H) Ldh is expressed in ganglion mother cells (GMCs), which are labelled with Prospero (PROS) antibody.Ldh-GFP is also present in both (I-K) neurons stained with Elav and (M-P) and glia stained with Repo.For Ldh-GFP Genomic and Ldh-GFP enhancer expression analysis, the CNS was fixed and stained with an GFP antibody as described in the methods.The scale bar in all images represents 40 M.Scale bar in (A) applies to B-D).Scale bar in (E) applies to (F-H).Scale bar in (I) applies to (J-L).Scale bar in (M) applies to (N-P).and (G,I,K) the stellate cells of the Malpighian tubules.For Ldh-GFP Genomic and Ldh-GFP enhancer expression analysis, tissues were fixed and stained with an GFP antibody as described in the methods.The scale bar in all images represents 40 M.The Ldh-mCherry Genomic spatial expression pattern is consistent with previous studies, with Ldh-mCherry Genomic being expressed at high levels in (A) the body wall muscle.However, unlike Ldh-GFP Genomic , the (A) expression of mCherry Genomic fusion protein persists throughout much of pupal development (compare with Figure 1B).Representative confocal images of the (A-H) central nervous system, (E-H) intestine, (I-L) fat body, and (M-P) salivary gland dissected from third instar w 1118 and Ldh 16 larvae.DAPI is shown in white and Ldh expression is shown in green.Note the brightness in (L) was increased to highlight the lack of staining in small cells within AMP clusters.The scale bar in all images represents 40 M.The scale bar in (A) applies to panels (B-H), the scale bar in (I) applies to panels (J-L), and the scale bar in (M) applies to panels (N-P).

Figure 1 .
Figure 1.Genetic characterization of the p{Ldh-GFP} transgene.(A)A schematic diagram of the Ldh-GFP Genomic transgene illustrating the placement of GFP immediately before the Ldh stop codon.When this transgene is placed in a Ldh16/17 mutant background, the resulting Ldh-GFP fusion protein is capable of not only rescuing (B) the Ldh mutant lethal phenotype, but also restoring steady state levels of (C) lactate and (D) 2-hydroxyglutarate (2HG).(E) The Ldh-GFP Genomic temporal expression pattern is consistent with previous studies, with Ldh-GFP Genomic being expressed at high levels throughout larval development and then declining at the onset of metamorphosis.Data in (B-D) analyzed using one-way ANOVA followed by a post hoc Tukey HSD.***P<0.001.

Figure 2 .
Figure 2. Characterization ofLdh expression in the central nervous system.The Ldh expression pattern in the central nervous system of L3 larvae was examined using Ldh-GFP Genomic , Ldh-GFP enhancer , and an Ldh antibody.(A-L) Representative confocal images of Ldh expression and DAPI staining in the (A-F) ventral and (G-L) dorsal sides of the CNS.For Ldh-GFP Genomic and Ldh-GFP enhancer expression analysis, the CNS was fixed and stained with an GFP antibody as described in the methods.The scale bar in all images represents 40 M.Scale bar in (A) applies to (B,G,H).Scale bar in (C) applies to (D,I,J).Scale bar in (E) applies to (F,K,L).

Figure 3 .
Figure 3. Ldh expression in ganglion mother cells, neurons, and glia.Representative confocal images showing the absence of Ldh in neuroblasts of third instar Ldh-GFP genomic larvae.The first two columns from left to right show DAPI and Ldh.The last column shows the colocalization of Ldh with CNS cell specific markers.(A-D) Ldh is not expressed in neuroblasts, which are labelled with Miranda (MIRA) antibody.In contrast,(E-H) Ldh is expressed in ganglion mother cells (GMCs), which are labelled with Prospero (PROS) antibody.Ldh-GFP is also present in both (I-K) neurons stained with Elav and (M-P) and glia stained with Repo.For Ldh-GFP Genomic and Ldh-GFP enhancer expression analysis, the CNS was fixed and stained with an GFP antibody as described in the methods.The scale bar in all images represents 40 M.Scale bar in (A) applies to B-D).Scale bar in (E) applies to (F-H).Scale bar in (I) applies to (J-L).Scale bar in (M) applies to (N-P).

Figure 4 .
Figure 4. Characterization of Ldh expression in the intestine and Malpighian tubules.The Ldh expression pattern in the intestine and Malphigian tubules of L3 larvae was examined using Ldh-GFP Genomic , Ldh-GFP enhancer , and an Ldh antibody.(A-L) Representative confocal images of Ldh expression and DAPI staining in (A-F) the midgutand (G-L) the Malphigian tubules.Note that Ldh is expressed at significantly higher levels in (A,C,E) a subset of small cells of the AMP clusters (denoted with arrowheads in (A-F))

Figure 5 .
Figure 5. Ldh expression pattern in the fat body and salivary gland.The Ldh expression patterns in the fat body and salivary glands of L3 larvae were examined using Ldh-GFP Genomic and Ldh-GFP enhancer .(A-L) Representative confocal images of Ldh expression and DAPI staining in (A-F) the fat body and (G-L) the salivary glands.For Ldh-GFP Genomic and Ldh-GFP enhancer expression analysis, tissues were fixed and stained with an GFP antibody as described in the methods.The scale bar in all images represents 40 M.

Figure 6 .
Figure 6.Ldh expression pattern in Imaginal discs.Representative confocal images of imaginal discs (top to bottom: wing disc, leg disc and eye disc) dissected from third instar (D5 AEL) Ldh-GFP genomic (A-F) and Ldh-GFP enhancer (G-L) third instar larvae.DAPI is shown in white and Ldh expression is shown in green.Note that the Ldh antibody failed to produce any staining in the imaginal discs and thus was excluded from the figure.The scale bar in all images represents 40 M.

Figure S2 .
Figure S2.Ldh immunostaining in control and Ldh mutant larval tissues.Representative confocal images of the (A-H) central nervous system, (E-H) intestine, (I-L) fat body, and (M-P) salivary gland dissected from third instar w 1118 and Ldh16 larvae.DAPI is shown in white and Ldh expression is shown in green.Note the brightness in (L) was increased to highlight the lack of staining in small cells within AMP clusters.The scale bar in all images represents 40 M.The scale bar in (A) applies to panels (B-H), the scale bar in (I) applies to panels (J-L), and the scale bar in (M) applies to panels (N-P).