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

Madhulika Rai; Sarah M Carter; Shefali A Shefali; Geetanjali Chawla; Jason M Tennessen

doi:10.1371/journal.pone.0287865

. 2024 Jan 3;19(1):e0287865. doi: 10.1371/journal.pone.0287865

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

Madhulika Rai ¹, Sarah M Carter ¹, Shefali A Shefali ¹, Geetanjali Chawla ^2,^*, Jason M Tennessen ^1,^*

Editor: Jyotshna Kanungo³

PMCID: PMC10763966 PMID: 38170735

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 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.

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 both Ldh and lactate in metabolism, signal transduction, the cell cycle, and even gene expression [1, 2, 5–9]. 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 [10–13]. The role of Ldh in human disease, however, extends far beyond cancer metabolism, as enhanced Ldh activity is also observed in diabetes and hyperglycemia [14–17], as well as during viral infections [18, 19]. 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 [20, 21]. In this regard, Ldh clearly plays a significant role during larval development, when the animal experiences a nearly 200-fold increase in body mass [22]. Ldh expression and activity are very high during this growth period when compared to other phases of fly life-cycle [20, 23, 24], 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 [25]. 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 26], neuronal health and aging [27–34], as well as during infections and immune challenges [35]. 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 [26, 36–40], 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 [24, 37]. These enzymatic assays have been supplemented with transgenes that rely on fluorescent proteins to either directly or indirectly visualize Ldh gene expression[35, 37, 39, 41]. 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) [39], (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) [37, 42], and (iii) a commercially available Drosophila Ldh antibody (Bosterbio; DZ41222). 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.

Methods

Drosophila melanogaster husbandry and genetic analysis

Fly stocks were maintained at 25°C on Bloomington Drosophila Stock Center (BDSC) food. Larvae were raised and collected as previously described [43]. 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 25°C incubator.

Ldh mutations were maintained in trans to the balancer chromosome TM3, p{Dfd-GMR-nvYFP}, Sb¹ (BDSC Stock 23231). Unless noted, Ldh mutant larvae harbored a trans-heterozygous combination of Ldh¹⁶ and Ldh¹⁷ as described in previous studies [44].

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 [39, 44]. For our analysis, the p{Ldh-GFP} was placed in the background of Ldh loss-of-function allele Ldh¹⁶ [44]. The previously described Ldh-GFP enhancer trap line was a kind gift from Utpal Banerjee’s lab [37, 42]. Finally, the p{Ldh-mCherry} transgene, which was previously described in a study of Drosophila hemocyte metabolism [35], 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.

Whole-larvae imaging

Expression of the LDH-GFP^Genomic and LDH-mCherry^Genomic transgenes in intact larvae and pupae was visualized without fixation using a MZ10F microscope with a EL6000 light source. GFP was visualized with the Leica ET GFP filter set (470 nm excitation filter; 525 emission filter). mCherry was visualized using the Leica ET mCherry filter set (560 nm excitation filter; 630 nm emission filter). Images were taken using a Leica MC170 HD microscope camera.

Immunofluorescence

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 4°C 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 4°C. Stained tissues were washed with 0.3% PBT, immersed in DAPI (0.5μg/μl 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 μl of blocking buffer containing a 1:20 dilution of anti-Ldh on a rocking shaker for 12 hrs at room temperature, 12 hrs at 4°C, and a third incubation of 12 hrs at room temperature. Samples were then washed twice using 200 μl of blocking buffer with a 10 min room temperature incubation for each wash. Once the second wash was removed, 200 μl 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°C. 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).

The following primary antibodies from the Developmental Studies Hybridoma Bank (DSHB) were used to in our study: mouse anti-Prospero (MR1A-S, 1:500 dilution), rat anti-Elav (7E8A10, 1:500 dilution), mouse anti-Repo (8D12, 1:20 dilution). In addition, the mouse anti-Miranda antibody (1:20 dilution, [45]) was a kind gift from Alex Gould. Secondary antibodies used herein were goat anti-mouse Alexa Fluor 568 (Thermo Fisher, A11004; 1:1000 dilution) and goat anti-rat Alexa Fluor 568 (Thermo Fisher, A11077; 1:1000 dilution).

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 and a representative section was used for the figures. The third instar larval CNS was imaged using a 20X objective, while all other tissues were imaged with a 40X objective. Excitation/emission max for Alexa Fluor 488 was 499/520 nm and for Alexa Fluor 568 was 579/603 nm.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis

Samples were collected, processed, and analyzed as previously described [46, 47]. 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 Kruskal–Wallis test followed by a Dunn’s multiple comparison test.

Results

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 [39]. 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 (Fig 1A). 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 (Fig 1B and S1 Table) and metabolic defects displayed by Ldh^16/17 mutant larvae (Fig 1C and 1D) [44]. We would note that Ldh-GFP^Genomic; Ldh^16/17 mutant larvae exhibited slightly decreased levels of lactate and 2-hydroxyglutarate (2HG) as compared with the wild-type control (Fig 1C and 1D and S1 Table), however, the observed differences were not significant. Regardless, the general trend of decreased lactate and 2HG in Ldh-GFP^Genomic; Ldh^16/17 mutant larvae suggests that the resulting fusion protein harbors lower activity than the endogenous enzyme. While there are a number of factors that could result in the Ldh-GFP enzyme exhibiting reduced activity relative to endogenous Ldh, a reasonable hypothesis stems from the fact that, in general, Ldh functions as a tetramer [48–50], thus the GFP tag could potentially disrupt the assembly or function of the higher order complex. Overall, our findings indicate that Ldh-GFP^Genomic produces a functional Ldh enzyme.

Fig 1 — (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 *Ldh*^16/17 mutant background, the resulting Ldh-GFP fusion protein is capable of rescuing (B) the *Ldh* mutant lethal phenotype and partially restoring (C) lactate and (D) 2-hydroxyglutarate (2HG) accumulation. (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 Kruskal–Wallis test followed by a Dunn’s multiple comparison test. ***P<0.001.

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 metamorphosis [20, 51]. 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 (Fig 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 [39].

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 [35]. 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 (S1 Fig). However, the Ldh-mCherry fusion protein persists throughout much of metamorphosis (compare Fig 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 aLdh does not stain Ldh^16/17 mutant tissues; S2 Fig). Our comparison demonstrated that all three reagents produced a similar cell- and tissue-specific 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; Fig 2A–2L). At the level of individual cell types, we observed identical expression patterns using both GFP transgenes as well as the Ldh antibody—Ldh is highly expressed in a subset of neurons and glia (Fig 3I–3P), as determined by co-staining with antibodies that recognize the neuronal protein Elav and the glial protein Repo [52, 53]. We would also note that Ldh expression was absent in neuroblasts that co-stain with a Miranda antibody [54, 55], but present in ganglion mother cells (GMCs) co-stained with Prospero antibody [54, 55]. Thus, our observations reveal that Ldh is expressed in a subset of GMCs (Fig 3E–3H), neurons (Fig 3I–3L) and glia (Fig 3M–3P), but not in neuroblasts (Fig 3A–3D). We are unsure as to the significance of this Ldh expression profile.

Fig 2 — The *Ldh* expression pattern in the central nervous system of L3 larvae was examined using *Ldh-GFP*^Genomic, *Ldh-GFP*^enhancer, and an aLdh 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. The scale bar in all images represents 40 μM. 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).

Fig 3 — 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 aMiranda (MIRA) antibody. In contrast, (E-H) *Ldh* is expressed in ganglion mother cells (GMCs), which are labelled with aProspero (PROS) antibody. Ldh-GFP is also present in both (I-L) neurons stained with aElav and (M-P) and glia stained with aRepo. The scale bar in all images represents 40 μM. 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). The scale bar in all images represents 40 μM.

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 adult midgut progenitors (AMPs) clusters, but at low or undetectable levels within larger EC cells (Fig 4A–4F). Considering that AMPs are small clusters of proliferating cells found within the larval gut epithelium [56], future studies should examine how Ldh influences the identify and proliferative capacity of these cells.

In the Malpighian tubules, Ldh levels were notably high in stellate cells as compared with principal cells and other renal cell types (Fig 4G–4L). This result is notable because stellate cells possess fewer mitochondria than principal cells [57]. Thus, our observation supports previous studies and suggest that stellate cells are more reliant on glycolysis than principal cells.

Fat body and salivary glands

Previous studies indicate that Ldh gene expression as well as Ldh enzymatic activity are relatively low in the larval salivary glands and fat body [20, 58]. Consistent with this observation, Ldh expression was below the level of detection within both the fat body (Fig 5A–5F) and salivary glands (Fig 5G–5L).

Fig 5 — 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. The scale bar in all images represents 40 μM.

Imaginal discs

Ldh expression has been extensively studied in the larval imaginal discs [26, 36–40]. 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 (Fig 6A–6L). 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 [59, 60], 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 (Fig 6E, 6F, 6K and 6L).

Fig 6 — 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 aLdh 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 μM.

Overall, the similarities in expression between the Ldh-GFP^Genomic transgene, the Ldh-GFP^enhancer transgene, and the aLdh 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. We would also note that while we did not fully characterize the Ldh-mCherry transgene in this study, the use of the mCherry fluorophore provides some flexibility in systems that are using GFP transgenes for other purposes. However, care should be used when using this reagent, as the resulting fusion protein appears to be stabilized relative to the endogenous 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 [20], as well as modern gene expression analyses that described how Ldh expression varies in intensity across larval tissues [35, 37, 39, 41, 61]. 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 [20], 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 [62, 63]. 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 example, 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 [27, 29]. 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 [26, 36–40]. 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) [24, 64], which was identified as being induced in response to 20-hydroxyedcysone signaling. Consistent with this possibility, imaginal discs exposed to 20-hydroxyecdysone in culture exhibit a significant increase in Ldh expression [24].

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.

Supporting information

S1 Fig. Expression of Ldh-mCherry^Genomic during larval development.

The Ldh-mCherry^Genomic spatial expression pattern is consistent with previous studies, with Ldh-mCherry^Genomic being expressed at high levels in the body wall muscle. However, unlike Ldh-GFP^Genomic, the expression of mCherry^Genomic fusion protein persists throughout much of pupal development (compare with Fig 1B).

(PDF)

Click here for additional data file.^{(7.5MB, pdf)}

S2 Fig. aLdh 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 w¹¹¹⁸ and Ldh¹⁶ larvae. DAPI is shown in white and Ldh expression is shown in green. Note the brightness in (L) was increased by 30% to highlight the lack of staining in small cells within AMP clusters. The scale bar in all images represents 40 μM. 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).

(PDF)

Click here for additional data file.^{(9.4MB, pdf)}

S1 Table. Data for Fig 1B–1D.

Data and statistical analysis used to generated figure panels 1B-1D.

(XLSX)

Click here for additional data file.^{(19.9KB, xlsx)}

Acknowledgments

We thank Alex Gould for generously sharing the Miranda antibody. The antibodies recognizing Elav, Repo, and Prospero were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

G.C is supported by the DBT/Wellcome Trust India Alliance Fellowship/Grant [grant number IA/I(S)/17/1/503085]. https://www.indiaalliance.org/fellowshiptype/basic-biomedical-research-fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. J.M.T. is supported by the National Institute of General Medical Sciences of the National Institutes of Health under a R35 Maximizing Investigators’ Research Award (MIRA; 1R35GM119557). https://nigms.nih.gov. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0287865.r001

Decision Letter 0

Jyotshna Kanungo

4 Sep 2023

PONE-D-23-18419Characterization of genetic and molecular tools for studying the endogenous expression of Lactate dehydrogenase in Drosophila melanogasterPLOS ONE

Dear Dr. Tennessen,

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Reviewer #1: In this manuscript Rai et. al. demonstrate where Ldh is localized in Drosophila larva across developmental stages and tissue types. The authors provide validation for valuable tools to examine expression of this enzyme that is highly relevant to development and many other physiological processes. Moreover, their discussion is clear and concise. The primary strengths of this study are the number of tissues measured and the direct comparison of similar tools in equivalent contexts. The weaknesses of this study are a lack of quantification for the fluorescence images across larval development, lack of any comment on individual variation, and absence of raw values for quantitative data with statistical analyses. Nonetheless, this study provides insightful findings that are suitable for publication in this journal. I recommend the editors accept this manuscript with revisions.

Here are my recommended revisions in order of importance:

1. Quantitative data provided in this manuscript is presented exclusively in Figure 1B-D. In the figure legend of Figure 1 the authors state that this data was analyzed using “one-way ANOVA followed by a post hoc Tukey HSD.” On page 9 the authors write in the methods section that for metabolite “Data were compared using a Kruskal–Walli’s test followed by a Dunn’s multiple comparison test.” The raw values and statistical analyses for all quantitative data (including the viability assay) were not provided. I recommend that the authors provide the raw values and statistical analyses (including test statistics and p value for all tests) for all quantitative data in the manuscript and clarify in the methods section which data were analyzed by ANOVA and Tukey or Kruskal-Wallis and Dunn’s.

2. In Figure 1 and S1, the authors provide qualitative data depicting detection of change in Ldh level across larval development using two different lines, Ldh-GFPGenomic and Ldh-mCherryGenomic. To strengthen these findings, I recommend the authors provide a quantification of the fluorescence depicted in these images in order to highlight the magnitude of changes that were detected and to compare more clearly to previous findings.

3. Throughout the manuscript, the authors provide images from a single representative individual animal fore every result. Although this may be sufficient for a binary measure whereby the outcome is only either positive or negative, instances whereby patterns are evaluated should be examined in more than a single animal. I recommend the authors provide supplementary images from additional animals for at least Figure 1, S1, and 2. At a minimum, even if additional images are not provided, I recommend that the authors discuss in the body text whether they observed any individual variation in their results from each tool they used, including Ldh-mCherry.

4. The authors highlight the discovery of new functions of Ldh on page 3. This description of Ldh seems to conflate the function of lactate to the function of Ldh. I recommend clarification of the distinction between functions attributed to Ldh and the substrates of Ldh, lactate and pyruvate, of which Ldh reversibly interconverts. Furthermore, a particularly important newly discovered function of lactate which warrants citing because it may have a significant impact on development is the ability of lactate to regulate cell cycle through zinc chelation (Liu, W., Wang, Y., Bozi, L.H.M., Fischer, P., Jedrychowski, M.P., Xiao, H., Wu, T., Darabedian, N., He, X., Mills, E.L., et al. (2023). Lactate regulates cell cycle by remodeling the anaphase promoting complex. Nature 616, 1–2. DOI:10.1038/s41586-023-05939-3 PMID:36921622.)

5. The authors state on page 3 “Ldh expression and activity are very high during this growth phase when compared to other phases of fly life-cycle [19]”. This citation only measures Ldh activity and not expression. I recommend adding a citation that provides evidence supporting the claimed changes in expression, for example (Graveley, B., Brooks, A., Carlson, J. et al. The developmental transcriptome of Drosophila melanogaster. Nature 471, 473–479 (2011). DOI: 10.1038/nature09715)

6. The authors cite three studies related to the connection between lactate metabolism activity and “neuronal health and aging [24-27]”. I recommend four additional critical citations (1. Hunt, L.C., and Demontis, F. (2021). Age-Related Increase in Lactate Dehydrogenase Activity in Skeletal Muscle Reduces Lifespan in Drosophila. J. Gerontol. A. Biol. Sci. Med. Sci. XX, 1–32. DOI:10.1093/gerona/glab260 PMID:34477202. 2. Frame, A.K., Robinson, J.W., Mahmoudzadeh, N.H., Tennessen, J.M., Simon, A.F., and Cumming, R.C. (2023). Aging and memory are altered by genetically manipulating lactate dehydrogenase in the neurons or glia of flies. Aging (Albany. NY). 10, 1–53. DOI:10.18632/aging.204565. 3. Long, D.M., Frame, A.K., Reardon, P.N., Cumming, R.C., Hendrix, D.A., Kretzschmar, D., and Giebultowicz, J.M. (2020). Lactate dehydrogenase expression modulates longevity and neurodegeneration in Drosophila melanogaster. Aging (Albany. NY). 12, 10041–10058. DOI:10.18632/aging.103373. 4. Lee, J.E., Oney, M., Frizzell, K., Phadnis, N., and Hollien, J. (2015). Drosophila melanogaster Activating Transcription Factor 4 Regulates Glycolysis During Endoplasmic Reticulum Stress. G3 Genes|Genomes|Genetics 5, 667–675. DOI:10.1534/g3.115.017269 PMID:25681259.

7. On page 6 the authors describe the production of Ldh-mCherryGenomic with reference to a past study which has used this resource and having produced this resource in a similar manner to Ldh-GFPGenomic. Citation 28 does not provide an explanation for the method used to generate Ldh-mCherryGenomic and citation 32 describes how Ldh-GFPGenomic was generated using a PCR based method and plasmid injection by Rainbow Transgenics. I recommend the authors clarify here whether the methods used for generating Ldh-mCherryGenomic were identical and cite Rainbow Transgenics if their services were used.

8. In the methods section the authors do not provide an explanation for how Ldh-mCherryGenomic was imaged. Was an antibody against mCherry required? I recommend the authors provide this information.

9. On page 8 the authors describe their confocal microscopy method. I recommend the authors provide the excitation and emission wavelengths used for detection of each fluorophore imaged, the method used for combining Z-stacks (e.g. maximum intensity projection vs average intensity projection), and the objectives used.

10. The authors describe the results from Figure one on page 10 without reference to Figure 1A or 1B. I recommend that the authors refer to those figures where they have described them.

11. On page 10 the authors acknowledge a slight decrease in lactate and 2HG in their Ldh-GFPGenomic rescue compared to control without reference to any statistical test in the figure or results. I recommend the authors provide the results of a statistical test comparing the rescue with control. Moreover, I recommend the authors add discussion of potential reasons for differences in fusion protein activity compared to endogenous Ldh. For example, protein metabolite interaction (Hicks, K.G., Cluntun, A.A., Schubert, H.L., Hackett, S.R., Berg, J.A., Leonard, P.G., Ajalla Aleixo, M.A., Zhou, Y., Bott, A.J., Salvatore, S.R., et al. (2023). Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase. Science (80-. ). 379, 996–1003. DOI:10.1126/science.abm3452 PMID:36893255.), post-translational modification (Storey, K.B. (2016). Comparative enzymology—new insights from studies of an “old” enzyme, lactate dehydrogenase. Comp. Biochem. Physiol. Part - B Biochem. Mol. Biol. 199, 13–20. DOI:10.1016/j.cbpb.2015.12.004 PMID:26688543.), or post-transcriptional regulation (Jungmann, R.A., Huang, D., and Tian, D. (1998). Regulation of LDH-A gene expression by transcriptional and posttranscriptional signal transduction mechanisms. J. Exp. Zool. 282, 188–195. DOI:10.1002/(SICI)1097-010X(199809/10)282:1/2<188::AID-JEZ21>3.0.CO;2-P PMID:9723176.).

12. In Figure 2 the image for anti-Ldh staining without the entire ventral nerve cord visible. For fair comparison, I recommend addition of an image with anti-Ldh antibody with the ventral nerve chord entirely in frame like those larval nervous systems depicted for the GFP genomic and enhancer lines.

13. On page 13 the authors summarize their findings. For clarity and to highlight differences between the methods, I recommend the authors provide a summary table comparing the methodology and Ldh characteristics identified for the different methods they recommend. Moreover, I recommend the authors comment on the utility (or lack thereof) of the Ldh-mCherry line despite having characterized it less than the other methods. If the Ldh-mCherry line does not require any antibody staining to detect the signal above autofluorescence, then this may be an advantage for studies utilizing this line outside of the context whereby the authors have identified discrepant patterns of expression (pupal development).

14. In the legend for Figure 2, 3, 4, and 5 the authors state “For Ldh-GFPGenomic and Ldh-GFPenhancer expression analysis, the CNS was fixed and stained with an anti-GFP antibody as described in the methods.” Reference to the Ldh-GFPenhancer line in Figure 3 seems to be irrelevant because the authors also state in that figure legend that only Ldh-GFPgenomic was used. In addition, having fixation and staining procedures in the methods section makes mention of this method in the figure legends redundant unless they differ from the description in the methods section. I recommend that the authors remove all statements “For Ldh-GFPGenomic and Ldh-GFPenhancer expression analysis, the CNS was fixed and stained with an anti-GFP antibody as described in the methods.“ from the figure legend unless there is a reason to distinguish a method in any figure from the method described in the methods section.

15. For all Figures, the scale bars and labels should be formatted consistently. I recommend that the authors label staining for GFP in Ldh-GFPGenomic and Ldh-GFPEnhancer in Figure 2, 3, 4, 5, and 6 consistently as either only αGFP or only LDH. I recommend the authors make all labels denoting the stain used in images below consistently displayed in a font color matching the pseudocolor in the image below, as they have done in Figure 3. I recommend that the authors remove all labels describing the length of scale bars from within the image and keep this description of scale bar length restricted to the Figure legend, as they have done in Figure 5.

16. In the legend for Figure 3, the authors state refer to (I-K) whereby they seem to be referring to (I-L). I recommend the authors change this to (I-L).

17. The label above Figure 5F and L is (αGFP). This seems to be a mistake. If this is incorrect, I recommend the authors change this to αLDH as they have done in Figure 4.

18. In the legend for Figure S2 the authors state that the brightness was increased in panel L. I recommend the authors provide the percentage the brightness was increased compared to the other images in this figure.

19. On page 13 the statement “Previous studies of the Ldh enzyme activity suggest that Ldh expression levels are relatively low in the larval salivary glands and fat body” is not cited. I recommend the authors add a citation supporting this claim.

20. The authors state “(see methods)” on page 5 where reiterating the catalog number and supplier of the antibody here would provide more clarity to the reader: (Bosterbio; DZ41222)

Reviewer #2: Dear Editor,

In the manuscript "Characterization of genetic and molecular tools for studying the endogenous expression of Lactate dehydrogenase in Drosophila melanogaster", Ray et al. provide a detailed and accurate characterization of D. melanogaster LDH reporter lines and a new anti-LDH antibody.

Although the information provided in this manuscript may be limited to a specialized audience, the experiments reported here provide an important set of data to validate l reagents previously used in several publications and now available to the Drosophila community.

I have a few minor comments below that I think should be addressed prior to publication, but overall I consider the manuscript to be of high quality and have no major comments that would preclude its publication after this minor corrections.

Sincerely

Minor comments:

- Figure 1C: The figure legend indicates that the Ldh-GFP fusion protein is able to restore steady-state levels of (C) lactate and (D) 2-hydroxyglutarate (2HG).

Although lactate and 2HG levels are significantly increased in p{Ldh-GFP} compared to Ldh null mutants, the statistical difference in metabolite levels between Ldh and p{Ldh-GFP} was not tested. The phrase "restore steady-state levels" is ambiguous in this context. The authors should either perform an additional statistical test and retain the sentence only in case of lack of statistical differences or rephrase this sentence to more accurately describe the observed results, as is already the case in the Results section.

- Figure 3C: References justifying the choice of different markers (REPO, PROS, MIRA) could be added to the text.

- The term "AMP cluster" should be defined.

**********

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PLoS One. 2024 Jan 3;19(1):e0287865. doi: 10.1371/journal.pone.0287865.r002

Author response to Decision Letter 0

29 Nov 2023

We thank the reviewer for his helpful and insightful comments.

Here are my recommended revisions in order of importance:

Thank you for noticing the discrepancy in descriptions of statistical test – Kruskal-Wallis and Dunn’s was used in Figure 1 – the text now accurately describes the test used. Data for figure panels 1B-D are now included in Table S1, as is the statistical analysis.

We appreciate the reviewer’s suggestions, however, we believe this level analysis is unnecessary. Ldh expression levels have been repeatedly quantified during larval development and our images demonstrate that Ldh-GFP qualitatively exhibits a similar expression pattern. Since the purpose of our study is to validate reagents for studying Ldh expression, we believe that such an analysis is best conducted in a future study focused on the mechanisms that temporally regulate Ldh expression.

3. Throughout the manuscript, the authors provide images from a single representative individual animal for every result. Although this may be sufficient for a binary measure whereby the outcome is only either positive or negative, instances whereby patterns are evaluated should be examined in more than a single animal. I recommend the authors provide supplementary images from additional animals for at least Figure 1, S1, and 2. At a minimum, even if additional images are not provided, I recommend that the authors discuss in the body text whether they observed any individual variation in their results from each tool they used, including Ldh-mCherry.

We completely agree. As noted in the text, we only described observations that were similar or identical across all three reagents (see Lines 267-270). And while we only presented representative images, the fact that we included images from three independent reagents for nearly all tissues far exceeds the current standards of the developmental biology community. We would also note that, in many cases, the observations were largely binary. For example, neuroblasts vs GMCs, stellate cells vs principal cells, and AMPs vs enterocytes. We’ve added additional text to emphasize this point.

Regarding the Ldh-mCherry fusion, we decided to not characterize the expression pattern on this reagent because the fusion protein appears to be excessively stabilized. We’ve added a few sentences noting that this reagent should be used with caution when quantifying Ldh expression levels.

We’ve added text to Line 63 to address this issue. We have also added the suggested reference.

Thank you for noticing the missing reference. The appropriate reference for this statement is Abu-Shumays RL, Fristrom JW. IMP-L3, A 20-hydroxyecdysone-responsive gene encodes Drosophila lactate dehydrogenase: structural characterization and developmental studies. Dev Genet. 1997;20(1):11-22. doi: 10.1002/(SICI)1520-6408(1997)20:1<11::AID-DVG2>3.0.CO;2-C. PMID: 9094207, not Gravely et. al. We have added this reference to the text (Line 80).

Thank you for suggesting these citations – Frame et al was in the near final version of the manuscript and was somehow removed. We have added the additional suggested references to our manuscript.

We feel that the methods section is clear that the Ldh-mCherry[Genomic] construct was generated using identical methods to the Ldh-GFP[Genomic], and a description of how Ldh-GFP[Genomic] was generated is available in the cited reference.

8. In the methods section the authors do not provide an explanation for how Ldh-mCherryGenomic was imaged. Was an antibody against mCherry required? I recommend the authors provide this information.

These images are from living larvae using a fluorescent microscope without fixation. We have added a new section “Whole larvae imaging” under the methods section. Lines 153-159.

The relevant information is now included in the Methods section (Line 198-200).

10. The authors describe the results from Figure one on page 10 without reference to Figure 1A or 1B. I recommend that the authors refer to those figures where they have described them.

We have added the Figure 1A and 1B in the text. Thank you for catching the mistake.

The data and statistical results can now be found in Table S1. Appropriate references regarding the manner by which Ldh functions as a tetramer have also been added (Lines 231-233).

We appreciate the suggestion, but have decided to not replace this image.

Thank you for the suggestion, but we decided not at add a summary table. We have added addition text about the use of the mCherry reagent (see Lines 329-333).

We have removed the text from the figure legends as asked by the reviewer.

We have made the recommended changes in the figures.

16. In the legend for Figure 3, the authors state refer to (I-K) whereby they seem to be referring to (I-L). I recommend the authors change this to (I-L).

We have corrected this in the manuscript.

17. The label above Figure 5F and L is (αGFP). This seems to be a mistake. If this is incorrect, I recommend the authors change this to αLDH as they have done in Figure 4.

We have corrected Figure 5F.

We’ve added this information to the figure legend.

Thank you for noticing this omission. The references seem to have been lost in the final version of the manuscript. We have added back the appropriate references.

20. The authors state “(see methods)” on page 5 where reiterating the catalog number and supplier of the antibody here would provide more clarity to the reader: (Bosterbio; DZ41222)

We have made this change to our manuscript.

Reviewer #2: Dear Editor,

Sincerely

We thank the reviewer for the kind and helpful overview of our paper!

Minor comments:

- Figure 1C: The figure legend indicates that the Ldh-GFP fusion protein is able to restore steady-state levels of (C) lactate and (D) 2-hydroxyglutarate (2HG).

We agree with the reviewer and have performed additional statistical tests. We also rephrased the figure legend for Figure 1C and D and have included the raw data for these figure panels, as well as the statistical test information, in Table S1.

- Figure 3C: References justifying the choice of different markers (REPO, PROS, MIRA) could be added to the text.

We agree and have added references to the text justifying the choice of the different markers.

- The term "AMP cluster" should be defined.

We have defined the term “AMP cluster” in the manuscript and added the appropriate references (Lines 288-292). Thank you for the suggestion.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0287865.r003

Decision Letter 1

Jyotshna Kanungo

12 Dec 2023

PONE-D-23-18419R1Characterization of genetic and molecular tools for studying the endogenous expression of Lactate dehydrogenase in Drosophila melanogasterPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: All comments were adequately addressed. There are a few very minor changes I recommend before final publication:

1) I agree that Abu-Shumays et al. is sufficient evidence for the claim that Ldh expression is relatively high during larval developement. But, it seems this citation was added to the sentence prior to this claim (Line 79). I recommend moving it to the sentence discussion Ldh expression (Line 80).

2) The method for combining Z-stacks is still not provided (e.g. maximum intensity projection / average intensity projection). I recommend this is added to the sentence on Line 196.

3) Figure 4 and 5 have the green channel legend labeled αLDH whereas figure 2 and everywhere else in the body text and figure legends denotes this without all uppercase as αLdh. I recommend changing figure 4 and 5 to match everywhere else (i.e. αLdh).

Reviewer #2: I consider that the authors have responded adequately to all my comments. I recommend the publication of this study.

**********

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Reviewer #1: Yes: Ariel K. Frame

Reviewer #2: No

**********

PLoS One. 2024 Jan 3;19(1):e0287865. doi: 10.1371/journal.pone.0287865.r004

Author response to Decision Letter 1

13 Dec 2023

Response to Reviewers Comments:

Reviewer #1: All comments were adequately addressed. There are a few very minor changes I recommend before final publication:

Done.

2) The method for combining Z-stacks is still not provided (e.g. maximum intensity projection / average intensity projection). I recommend this is added to the sentence on Line 196.

We appreciate the suggestion, but would kindly correct the reviewer and note that the methods sections is accurate and does not need edits. As already stated in the text, we did not combine Z-stacks in the figure, but rather used a representative section.

Done.

Reviewer #2: I consider that the authors have responded adequately to all my comments. I recommend the publication of this study.

Thank you!

Attachment

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PLoS One. doi: 10.1371/journal.pone.0287865.r005

Decision Letter 2

Jyotshna Kanungo

18 Dec 2023

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

PONE-D-23-18419R2

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

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Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

**********

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Reviewer #1: Yes: Ariel K. Frame

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PLoS One. doi: 10.1371/journal.pone.0287865.r006

Acceptance letter

Jyotshna Kanungo

21 Dec 2023

PONE-D-23-18419R2

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Expression of Ldh-mCherry^Genomic during larval development.

(PDF)

Click here for additional data file.^{(7.5MB, pdf)}

S2 Fig. aLdh immunostaining in control and Ldh mutant larval tissues.

(PDF)

Click here for additional data file.^{(9.4MB, pdf)}

S1 Table. Data for Fig 1B–1D.

Data and statistical analysis used to generated figure panels 1B-1D.

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Click here for additional data file.^{(19.9KB, xlsx)}

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

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

Madhulika Rai

Sarah M Carter

Shefali A Shefali

Geetanjali Chawla

Jason M Tennessen

Roles

Abstract

Introduction

Methods

Drosophila melanogaster husbandry and genetic analysis

Viability assay

Whole-larvae imaging

Immunofluorescence

Gas Chromatography-Mass Spectrometry (GC-MS) analysis

Statistical analysis of metabolite data

Results

Genetic characterization of the Ldh-GFPGenomic transgene

Fig 1. Genetic characterization of the p{Ldh-GFP} transgene.

Ldh is expressed in a complex pattern during larval development

Central nervous system

Fig 2. Characterization of Ldh expression in the central nervous system.

Fig 3. Ldh expression in ganglion mother cells, neurons, and glia.

Digestive and renal systems

Fig 4. Characterization of Ldh expression in the intestine and Malpighian tubules.

Fat body and salivary glands

Fig 5. Ldh expression pattern in the fat body and salivary gland.

Imaginal discs

Fig 6. Ldh expression pattern in imaginal discs.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Jyotshna Kanungo

Roles

Author response to Decision Letter 0

Decision Letter 1

Jyotshna Kanungo

Roles

Author response to Decision Letter 1

Decision Letter 2

Jyotshna Kanungo

Roles

Acceptance letter

Jyotshna Kanungo

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Genetic characterization of the Ldh-GFP^Genomic transgene