Regulation of tissue and development specific gene expression patterns underlies the functional specialization of organs in multi-cellular organisms. In the viviparous tsetse fly (Glossina), the female accessory gland is specialized to generate nutrients in the form of a milk-like secretion to support growth of intrauterine larva. Multiple milk protein genes are expressed specifically in the female accessory gland and are tightly linked with larval development. Disruption of milk protein synthesis deprives developing larvae of nutrients and results in extended larval development and/or in abortion. The ability to cause such a disruption could be utilized as a tsetse control strategy. Here we identify and delineate the regulatory sequence of a major milk protein gene (milk gland protein 1:mgp1) by utilizing a combination of molecular techniques in tsetse, Drosophila transgenics, transcriptomics and in silico sequence analyses. The function of this promoter is conserved between tsetse and Drosophila. In transgenic Drosophila the mgp1 promoter directs reporter gene expression in a tissue and stage specific manner orthologous to that of Glossina. Analysis of the minimal required regulatory region of mgp1, and the regulatory regions of other Glossina milk proteins identified putative homeodomain protein binding sites as the sole common feature. Annotation and expression analysis of Glossina homeodomain proteins identified ladybird late (lbl) as being accessory gland/fat body specific and differentially expressed between lactating/non-lactating flies. Knockdown of lbl in tsetse resulted in a significant reduction in transcript abundance of multiple milk protein genes and in a significant loss of fecundity. The role of Lbl in adult reproductive physiology is previously unknown. These results suggest that Lbl is part of a conserved reproductive regulatory system that could have implications beyond tsetse to other vector insects such as mosquitoes. This system is critical for tsetse fecundity and provides a potential target for development of a reproductive inhibitor.
Female tsetse flies (Diptera: Glossina) harbor and give birth to live young. To do this, they nourish their intrauterine larvae with milk secretions. This work focuses upon understanding the regulation of tsetse milk proteins, which are essential for fecundity and are expressed in a temporally and spatially specific manner by pregnant females. We identified the minimal upstream regulatory DNA sequence of the major milk protein gene mgp1, which confers tissue specific expression in the female accessory glands of reproductively active flies. This regulatory sequence functions similarly in transgenic fruit flies (Drosophila melanogaster) and drives expression of reporter gene products in the adult female accessory gland. Comparison of this regulatory sequence with sequences from other characterized milk proteins indicates that conserved homeodomain transcription factors may be responsible for regulating these genes. Analysis of Glossina homeodomain proteins identified an accessory gland/fat body specific factor, Ladybird late (lbl), which appears to regulate the expression of multiple milk proteins. Reduction of lbl levels interferes with milk protein gene expression, which in turn reduces Glossina fecundity. These results suggest that milk proteins in Glossina are regulated by a conserved regulatory system mediated in part by the homeodomain transcription factor lbl. Components of this system could provide a target for development of a tsetse reproductive inhibitor.
Citation: Attardo GM, Benoit JB, Michalkova V, Patrick KR, Krause TB, Aksoy S (2014) The Homeodomain Protein Ladybird Late Regulates Synthesis of Milk Proteins during Pregnancy in the Tsetse Fly (Glossina morsitans). PLoS Negl Trop Dis 8(4): e2645. doi:10.1371/journal.pntd.0002645
Editor: Jesus G. Valenzuela, National Institute of Allergy and Infectious Diseases, United States of America
Received: September 6, 2013; Accepted: November 27, 2013; Published: April 24, 2014
Copyright: © 2014 Attardo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study received support from NIH (http://www.nih.gov/) grants AI081774 and F32AI093023 to SA and JBB respectively. This work was also supported by an award from the Ambrose Monell Foundation (http://www.monellvetlesen.org/monell/default.htm) to SA and the European Fund for Regional Development (EFRD)(http://www.minedu.sk/european-fund-for-regional-development-efrd/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Tsetse flies (Glossina sp.) are the exclusive vectors of Human African Trypanosomiasis (HAT) and nagana in Sub-Saharan Africa. HAT transmission can be prevented through tsetse population control by baited targets and trapping . These control methods are effective due to the unusual nature of tsetse's reproductive physiology. Tsetse females reproduce via adenotrophic viviparity (the intrauterine retention and nourishment of larval offspring). Each female is capable of producing only 8–10 progeny in their 2–3 month life span . As a result, vector control strategies targeting population reduction can have a significant and quick impact upon population density and disease transmission dynamics.
Viviparity in tsetse entails the protection and nourishment of offspring for the duration of immature (larval) development by the mother . Larval nourishment is supplied as a “milk” secretion that is generated by the accessory gland (milk gland) that empties into the uterus. The tsetse milk gland is orthologous to accessory glands found in association with the female reproductive tract of other insects. Female accessory glands (also referred to as paraovaria or colleterial glands) in insects are adapted in a variety of novel ways to accommodate different reproductive strategies and life histories. In tsetse, milk gland morphology differs from the accessory glands of other insects. The milk gland expands from its connection to the uterus throughout the abdomen as a series of bifurcating tubules that intertwine with the abdominal fat body tissue . The secretory cells of the tubules synthesize and secrete large volumes of milk . At the time of parturition, larvae are almost equivalent in weight to the mother, illustrating the tremendous maternal nutritional investment in each gonotrophic cycle. Development of a control strategy to disrupt lactation in tsetse would be an effective way to reduce the vector population.
Molecular analysis of milk gland secretions has identified Milk Gland Protein 1 (MGP1 - VectorBase accession:GMOY009745) as one of the primary protein components of tsetse milk . MGP1 is a member of the lipocalin protein family, which are known for their ability to bind small hydrophobic molecules . Lipocalins are common constituents of lactation secretions in marsupials , mammals  and are a primary component of the secretion generated by the viviparous cockroach species Diplotera punctata . Synthesis of MGP1 in tsetse is exclusive to the secretory cells of the milk gland. Transcript/protein levels correlate with the demand for nutrients by the intrauterine larvae. Prior to larval development there is little to no expression of this gene. Beginning at late embryogenesis/early larvigenesis, mgp1 transcript and protein levels begin to increase and reach their maximum when the larva reaches its 3rd instar (the time of highest nutritional demand) . Knockdown of mgp1 by dsRNA treatment reduces female fecundity suggesting that this protein is required for larval development .
Transcriptomic analysis of pregnant females and proteomic analysis of milk gland secretions have provided knowledge on the protein constituents of tsetse milk . These proteins include Transferrin (trf) , Acid Sphingomyelinase (asmase1)  and nine unique proteins (mgp2-10) the function of which is to be determined ,  (see Table S11 in the Glossina genome paper) . Milk associated proteins are conserved in tissue and stage specific expression in a manner similar to MGP1 . Identification of the signals and mechanisms coordinately regulating milk protein expression is an important goal in the search for novel vector control targets.
The recent sequencing of the tsetse genome  and the advent of next generation high throughput technologies have allowed us to study the pregnancy and tissue specific nature of tsetse's most abundant milk protein gene (mgp1), focusing on the role of specific transcription factors that may be associated with the upstream promoter region. We performed our analysis of milk protein gene regulation in tsetse using a combination of classic molecular techniques and genetic analysis in transgenic Drosophila. We used in silico analysis of genomic data and multiple high throughput datasets coupled with functional studies in Glossina and in Drosophila to identify candidate homeodomain factors that are apparently involved in the regulation of multiple tsetse milk protein genes. Our results suggest conservation of a reproduction associated gene regulatory mechanism across different taxa (Glossina and Drosophila) in the context of differing reproductive physiologies. We discuss our findings in light of potential methods to disrupt the reproductive capacity of tsetse as a vector control tool.
Materials and Methods
The Glossina morsitans morsitans colony maintained in the insectary at Yale University was originally established with puparia from fly populations in Zimbabwe. Newly emerged flies are separated by sex and mated at three to four days post-eclosion. Flies are maintained at 24±1°C with 50–55% relative humidity, and receive defibrinated bovine blood every 48 h using an artificial membrane system .
Quantitative expression analysis of mgp during parturition
Samples for gene expression analysis during parturition were collected as follows. Female flies pregnant with 3rd instar larvae were collected. Flies were checked every 24 hours and individuals that had undergone parturition during the 24 hour period were collected to stage them in sample groups. Three flies were collected at the following time points, pregnant (3rd instar larva), 0–24, 24–48, 48–72, 72–96, 96–120, 120–144, 144–168, and 168–192 hrs post parturition (pp). RNA and protein were isolated from flash frozen female flies and tissue samples utilizing the standard Trizol (Invitrogen, Carlsbad CA) protocol with a modification to the final step in which the isolated protein pellets are dissolved in cracking buffer (8M urea, 3M thiourea, 1% dithiothreitol (DTT) and 4% CHAPS) . cDNA was prepared from total RNA using the Invitrogen Superscript III kit (Invitrogen). Levels of mgp1 were determined by CFX Connect Real Time PCR Detection System with SYBR Green Supermix (Bio-Rad, Hercules, CA). The data were analyzed with software version 3.1 (Bio-Rad). All samples were normalized according to Glossina tubulin (tub) expression levels.
Glossina genomic data
Genomic sequences and automated annotations were derived from the recently completed G. m. morsitans genome  and are available at Vectorbase (http://gmorsitans.vectorbase.org/Glossina_morsitans/Info/Index).
Creation of β-gal enhancer/reporter constructs and Drosophila transformation
Two constructs were created using 2 kB and 0.5 kB of the 5′ upstream from the mgp1 predicted transcription start site. These fragments were PCR amplified from G. m. morsitans genomic DNA using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and sub-cloned into the T-vector cloning vector (Promega, Madison WI). Inserts were excised from the T-vector using SphI and NotI and ligated into the p-element vector pPelican  between the SphI and NotI sites. This vector includes a lacZ reporter gene downstream of the regulatory sequence. The enhancer/reporter constructs were used to transform w1118 flies by p-element mediated transformation via a commercial transformation service (Best Gene: Drosophila Injection Services (http://www.thebestgene.com). The transformations generated 4 lines carrying the 2.0 kB construct mgp1-Bgal-2.0-1-4 and 3 lines carrying the 0.5 kB construct mgp1-Bgal-0.5-1-3. Drosophila lines were maintained according to standard protocols .
Drosophila dissection, ß-galactosidase staining, imaging and activity
Tissues from male and female Drosophila were dissected in 1× PBS. Dissected tissues were fixed and stained for β-galactosidase activity with the ß-galactosidase Staining Kit (Mirus, Madison WI) following the manufacturer's protocols.
Creation of eGFP enhancer/reporter constructs and PhiC homologous recombination mediated Drosophila line creation
Flies expressing an eGFP reporter with 509 (mgp1-egfp-509), 236(mgp1-egfp-236), 112 (mgp1-egfp-112) or 13 bp (mgp1-egfp-13) nucleotide versions of the mgp1 upstream sequence were generated utilizing the PhiC homologous recombination transformation system , . The 509 bp mgp1 upstream fragment was PCR amplified from Glossina morsitans genomic DNA using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and sub-cloned into the T-vector cloning vector (Promega). The fragment was excised with SphI and SpeI then ligated into the nuclear EGFP enhancer analysis vector pStinger  between the SphI and NheI sites by standard cloning methods. The mgp1-pStinger construct was used as a template to amplify the 509, 236, 112 and 13 base pair variants of the mgp1 promoter-egfp fusion constructs. The forward and reverse primers for the constructs were tailed with the AttB40 PhiC recombination site sequence. Amplified constructs were cloned into T-vector and sent for transformation via a commercial transformation service (Best Gene: Drosophila Injection Services (http://www.thebestgene.com). Constructs were injected into the Drosophila line genotype y1 w*; P[attP.w+.attP]JB53F (Bloomington Stock Center #27386). The AttB integration site is 53F8, 2R:12985015 . Surviving injected flies were crossed with CyO/Sco 2nd chromosome balancer flies and screened for loss of eye color as a negative marker of transgene insertion. Drosophila lines were maintained according to standard protocols .
Visual and quantitative analysis of EGFP transgene expression in transgenic Drosophila
RNA was isolated from flies using Trizol (Invitrogen). cDNA was prepared from total RNA using the Invitrogen Superscript III kit (Invitrogen). Transgene transcript levels were quantified by qPCR with iQ SYBR Green Supermix using egfp specific primers and the CFX Connect Real Time PCR Detection System (Bio-Rad). Data was analyzed using software version 3.1 (Bio-Rad). All treatments were normalized according to Drosophila beta-tubulin expression levels using gene specific primers and carried out in triplicate. Visual inspection of transgene expression was performed by tissue dissection in 1× PBS followed by fluorescent microscopy using a Axio Cam dissecting scope equipped with a X-cite series Q fluorescence system (Zeiss Microscopy & Image Analysis, Thornwood, New York).
Nutritional deprivation and stage specificity experiments on transgenic Drosophila
Stage specific quantification of egfp transgene expression was performed as described above with total RNA from 3 independent groups of larval, pupal, and 3–5 day old male and female flies from the mgp1-egfp-509 line. 3–5 day old adult females from the mgp1-egfp-236, mgp1-egfp-112 and mgp1-egfp-13 lines were also collected and analyzed in the same manner to compare transgene expression between the lines.
Nutritional deprivation experiments were performed by maintaining the mgp1-egfp-509 line on either complete (yeast extract 100 g/L, sucrose 100 g/L, Agar 27 g/L, Methlyparaben: 30 ml of 100 g/L, Proprionic acid 3 mL/L) or minimal media (yeast extract 12.5 g/L, sucrose 12.5 g/L, Agar 27 g/L, Methlyparaben: 30 ml of 100 g/L, Proprionic acid 3 mL/L) in 25×95 mm polystyrene vials. Egg deposition was monitored daily for each group. Parallel treatments were run to quantify egfp levels in stressed flies using the qPCR methods described above.
In silico transcription factor binding site prediction
Putative transcription factor binding sites were predicted within the mgp upstream regulatory region using the “MatInspector” program from Genomatix (http://www.genomatix.de/) . Upstream sequences from other characterized and predicted milk proteins were derived from the draft Glossina genome (www.vectorbase.org) . The sequences used were from the 124 bp mgp1 (GMOY009745) critical region, trf (GMOY004228), asmase1 (GMOY002246), mgp2-10 (GMOY012368, GMOY012125, GMOY001342, GMOY012016, GMOY001343, GMOY012016, GMOY012369) genes. 500 bp from the predicted transcription start site of each gene was used in a comparative analysis. This analysis was also performed using MatInspector.
Glossina homeodomain factor identification and annotation
Predicted Glossina homeodomain sequences were identified from the Glossina genome . Protein sequences from the 104 known Drosophila homeodomain factors were used to search the Glossina genomic assembly and the de novo assembly from  by tBLASTn search. All resulting hits with scores lower than 1×10−10 were collected and searched against the NCBI database by BLASTx search. Sequences were then annotated with nomenclature from the highest scoring hit result.
RNA-seq analysis of differential gene expression during pregnancy
We have recently completed a transcriptome study focusing on differences between pregnant/lactating or dry flies (non-lactating) . Raw sequence data are available from the sequence read archive at NCBI. We utilized the RNA-seq reads from that study to compare the transcript abundance of Glossina homeodomain proteins in lactating and dry flies. RNA-seq data were mapped directly to the Glossina homeodomain genes utilizing CLC Genomics Workbench (CLC bio, Cambridge, Massachusetts). Normalized gene expression was calculated as RPKM . Statistical differences of RPKM values between samples were determined by Kal's test following Bonferroni correction . Statistical significance was determined at P<0.05 and a two-fold higher or lower transcript abundance in lactating flies was utilized to determine genes of interest .
Homeodomain expression analysis
Five candidate Glossina homeodomain genes were analyzed by qPCR based tissue and stage specific expression analysis. Tissue-specific samples were acquired from females harboring 2nd instar larva. qPCR was conducted as before and transcript levels were normalized to tubulin.
lbl siRNA knockdown and phenotype analysis
Functional analysis of the ladybird late homeodomain factor was performed using synthesized siRNAs homologous to the Glossina ladybird late ortholog. siRNAs were designed by web-based tools (IDT, Coralville, IA) and ordered commercially (IDT). The siRNA consists of two Duplex sequences for lbl. Control siRNA molecules were designed as complementary to green fluorescent protein (GFP) mRNA (IDT, Cordville, IA). Concentration of each siRNA was adjusted to 700–750 ng/µl in PBS with a Nanodrop spectrophotometer. Mated female flies were injected with 1.5 µl siRNA 6–8 d after adult emergence. siRNA was shown to have no discernible effect on larval transcripts . Expression levels were determined by qPCR (as described; normalized to tubulin) 5 d after injection for lbl and 8 d after injection for mgp1.
Transcriptional regulation of mgp1 during pregnancy
Transcript abundance of the mgp1 gene is related to intrauterine larval development. Previous expression data for mgp1 was based upon samples staged by female reproductive physiology and larval development . Based upon these data, mgp1 transcripts appear to decrease after larval deposition (parturition) and increase again at the onset of intrauterine larval development in the next pregnancy cycle. To obtain a high resolution temporal analysis of milk protein gene expression during this period, pregnant flies were collected and synchronized by time of parturition in 24 hour intervals rather than by pregnancy status. Transcript levels from these samples were quantified by qPCR analysis, and protein expression was determined by Western analysis. The results show that mgp1 transcript abundance and protein levels undergo a precipitous drop after parturition and reach their lowest levels at 24–48 hours post-parturition (Figure 1A and 1B). Transcript and protein levels begin to increase again between 48–72 hours post parturition and continue to increase to the last time point in the series at 168–192 hours post parturition when there is a 2nd instar intrauterine larva in the uterus.
A. Quantitative PCR analysis of mgp1 transcript levels just prior to parturition and in 24 hour periods after parturition. Pregnant Glossina flies carrying 3rd instar larvae were collected and staged by time of parturition within 24 hour windows. The values represent the mean mgp1 transcript level from 3 individual flies at each point. Error bars represent standard error. All qPCR data was normalized to tubulin. Statistical notation: a = not significantly different than pregnant; b = significantly different from pregnant (P-value <0.05 by students t-test); c = significantly different from pregnant (P-value <0.01 by students t-test). B. Western blot analysis of MGP1 levels in flies from the same samples as described in A. Values represent quantification of the mean signal intensity from 3 individuals for each point and are normalized to tubulin levels. Statistical notation is as described above.
Analysis of mgp1 promoter functions in transgenic Drosophila
Analysis of the regulation of mgp1 expression required the development of genetic tools that facilitate the observation of Glossina promoter function in vivo. To accomplish this, we leveraged the genetic tools available in D. melanogaster to perform an in vivo analysis of the mgp1 promoter. Drosophila is related to Glossina as both are members of the dipteran “Higher Flies” (Brachycera) suborder.
The transformation constructs were developed using the pPelican enhancer/reporter plasmid . The plasmid contains a β-galactosidase (β-gal) reporter gene and the promoter/reporter fusion is flanked by gypsy insulator sequences to prevent the influence of local regulatory elements. Transformation was accomplished by p-element transposition. Reporter expression was visualized by staining for β-gal activity in dissected transgenic Drosophila tissues. Two constructs were created including either 2 kB or 0.5 kB sequence of the 5′ upstream from the predicted mgp1 transcription start site (Figure S1A). Transformation of these constructs resulted in the production of four transgenic lines for the 2.0 kB construct (mgp1-β-gal-2.0-1 to -4) and three transgenic lines for the 0.5 kB construct (mgp1-β-gal-0.5-1 to -3).
In tsetse, mgp1 expression is specific to the milk gland of adult female flies . We analyzed different tissues from both mgp1-β-gal-2.0 and mgp1- β-gal-0.5 lines with β-gal staining to understand the sex and tissue specific nature of the reporter gene expression. Transgene expression was exclusive to the accessory glands (paraovaria) of the female reproductive tract in both transgenic lines (Figure 2A) while this staining pattern was not observed in control Drosophila. Background staining was observed in the uterus, midgut and in specific cells along the midline of the dorsal abdominal surface in both sexes. This background staining pattern was also observed in untransformed control flies suggesting that this endogenous β-gal activity may result from populations of bacteria resident within the fly. The specific staining observed in the accessory glands of females suggests that regulatory mechanisms and factors driving sex and tissue specific expression of mgp1 in tsetse are conserved in Drosophila. It also demonstrates that the transcriptional regulatory elements required for sex and tissue specific expression are contained within the 0.5 kB upstream region of the mgp1 gene.
Tissue and stage specific expression of mgp1 driven ß-gal and egfp reporter genes. All qPCR analyses normalized to Drosophila tub. Error bars represent standard error. Letter groups (a,b,c) represent significant statistical differences of P-value <0.05 by students t-test. A. Accessory gland specific staining of transgenic Drosophila reproductive tissue (mgp1- β-gal-2.0). B. Florescent microscopy of accessory gland specific egfp expression in transgenic Drosophila (mgp1-egfp-509). C. qPCR analysis of sex/stage specific egfp levels in transgenic line mgp1-egfp-509. Samples represent the average of three groups of 20 flies. D. qPCR analysis of egfp levels in 3–5 day old mated adult females from the mgp1-egfp-509, mgp1-egfp-236, mgp1-egfp-112 or mgp1-egfp-13 lines. Samples represent three groups of 20 flies. E. 3–5 day old transgenic Drosophila (509 bp-mgp1/egfp) were reared on control or nutrient deficient media. Data represent the mean eggs/day from five groups of 60 flies. Statistical notation: a = statistical difference between minimal and control media (P-value <0.05 by students t-test). F. qPCR analysis of egfp levels in mgp1-egfp-509 flies reared on control or minimal media. Data represents the mean egfp transcript level from three groups of 20 flies. Statistical notation: as in E.
Transgenic analysis of the 0.5 kB mgp1 regulatory region
To identify the minimal region required for tissue and stage specific gene expression by the mgp1 promoter, additional transgenic Drosophila lines were generated. To eliminate the confounding issues of background staining with endogenous β-gal activity and variation of transgene expression due to position effect, an alternative transformation strategy was utilized. The new transgenic lines were generated using the PhiC recombination system , . This system allows for the stable incorporation of transgenes into a specific genomic locus via homologous recombination, thereby eliminating position effect derived variation in expression between lines. To enhance transgene detection and reduce background, the β-gal reporter gene was replaced with the nuclear specific enhanced green fluorescent protein (EGFP) reporter gene from the pStinger p-element transformation vector. We generated four transgenic lines, which include either 509 (mgp1-egfp-509), 236 (mgp1-egfp-236), 112 (mgp1-egfp-112) or 13 bp (mgp1-egfp-13) segments corresponding to the mgp1 regulatory region upstream of the start site (Figure S1B).
All life stages and tissues from these lines were examined by florescent microscopy for nuclear specific EGFP expression. Microscopic analysis of EGFP florescence revealed reporter gene expression is only detectable within the accessory glands of reproductively active (undergoing oogenesis and ovulation) females from the mgp1-egfp-509 line. This is almost identical to the observations in the β-gal expressing lines (Figure 2B). Of the four lines, the mgp1-egfp-509 and mgp1-egfp-236 lines were positive for female accessory specific EGFP expression by visual screening, while EGFP expression in mgp1-egfp-112 and mgp1-egfp-13 bp constructs was undetectable by visual observation. These results suggest that the minimal region for tissue specific expression and basic promoter function lies within the 236 bp upstream of the mgp1 transcription start site. The 124 bp differential region between the mgp1-egfp-236 and mgp1-egfp-112 lines appears to carry a transcriptional response element critical to the function of this promoter as a whole.
Quantitative analysis of egfp expression during different developmental and sexual stages in mgp1-egfp-509 reveals that the transgene is only significantly expressed within sexually mature adult females with only background levels present in larvae and males (Figure 2C). Comparison of egfp expression between the four lines confirms the results we obtained by microscopy analysis. Expression of the transgene is highest in mgp1-egfp-509 (Figure 2D). Transgene expression in mgp1-egfp-236 shows that the overall transcript level is significantly lower than that observed in mgp1-egfp-509. However, mgp1-egfp-236 maintains the sex and tissue specific characteristics of the mgp1-egfp-509. Transgene expression in mgp1-egfp-13 and mgp1-egfp-112 was equivalent to levels observed in male flies suggesting that regulatory function is compromised in these lines.
Nutritional and/or reproductive regulation of the mgp1 regulatory region
The reproductive cycle in Drosophila differs from that of tsetse in that oogenesis and embryo deposition occur at a steady rate once the female is sexually mature and mated as opposed to the defined cycles of oogenesis, embryogenesis and larvigenesis observed in tsetse. Nutritional stress in Drosophila results in reduction or termination of oogenesis. In the absence of appropriate nutritional stimulation yolk protein gene expression and oocyte development cease. In some cases developing oocytes may undergo apoptosis and reabsorption , . To determine if a nutritionally induced reduction in oogenesis and ovulation would influence transgene expression, 3 day old mated female mgp1-egfp-509 flies were put on a minimal media diet followed by daily observation of egg deposition and egfp transcript levels. Maintenance on minimal media relative to complete media resulted in a significant reduction in egg production in the mgp1-egfp-509 flies within 5 days (Figure 2E). Quantitative analysis of reporter egfp expression in flies maintained on minimal media during the 5 day time period shows a direct correlation between transgene expression and egg deposition over time (Figure 2F). These results correlate the down regulation of gene expression activity within the accessory gland with reduced nutritional status/oogenesis. Whether the gland is responding to reduced nutritional stimuli or reduced reproductive stimuli is not yet understood.
In silico response element predictions for the mgp1 0.5 kB regulatory region and comparative analysis with other tsetse milk proteins
We next set out to predict transcription factor binding sites that may lie within the 0.5 kb promoter region of mgp1 responsible for the sex and tissue specific expression profile we observed in both taxa. Analysis of the 0.5 kB regulatory sequence using transcription factor binding site prediction software resulted in a total of 41 predicted binding sites from a variety of factors. The matrices used in the identification included those for known insect transcription factors and eukaryotic basal promoter elements. To reduce the number of candidate factors, we narrowed our analysis to the 124 bp region between lines, mgp1-egfp-236 and mgp1-egfp-112, which is required for sex and tissue specific function. This analysis reduced the number of predictions to 11 candidate binding sites. The sites predicted include generic Drosophila homeodomain binding sites, including (DHOM: 2 sites), OVO transcription factor (DOVO: 1 site), Iroquois factor group (IRXF: 1 site), Tailless (DTLL: 1 site), Abdominal B (ABDB: 1 site), Paired homeodomain factors (PRDH: 2 sites), Dead Ringer (DRIF: 1 site), Giant (DGTF: 1 site) and Heat shock factors (DHSF: 1 site).
Previous characterizations, annotation of the Glossina genome  and RNA-Seq based analysis of lactating and non-lactating flies has identified 12 milk protein genes that function in the lactation process in tsetse, including mgp1, mgp2-10, trf and asmase1 . Comparative analysis of the 124 bp mgp1 region critical for transgene expression in Drosophila with the 500 bp sequence upstream of the transcription start site of these genes predicts that only homeodomain protein binding sites (DHOM) are common between all 12 sequences (p-value: 0.0241) (Figure 3). The only other site in common between these promoters is that of the TATA binding protein (TATA box).
This schematic represents a scale model of in silico predicted homeodomain (DHOM) binding sites. The associated table lists the number of DHOM binding sites predicted within the 500 bp upstream sequence from the 11 other milk protein genes and the associated p-value of these findings.
Annotation of Glossina homeodomain proteins
To identify homeodomain type regulatory factors that may recognize the conserved DHOM sites, we annotated and collated all sequences in Glossina bearing homology to the 106 annotated Drosophila homeodomain proteins. This was accomplished by tBLASTn search of the Glossina genome assembly  and a de novo assembly of RNA-seq libraries with Drosophila homeodomain protein sequences. All significant hits were submitted to a BLASTx comparison against the NCBI protein database. This analysis predicted a total of 96 putative tsetse homeodomain protein orthologs within the current genome assembly (see Table S36 in the Glossina genome paper).
Functional analysis of all candidate homeodomain factors was prohibitive. To reduce the number of targets, the expression profile of the 96 homeodomain genes were screened by RNA-Seq analysis using the available Illumina sequencing data from lactating and non-lactating flies . To identify differential transcript abundance between the two physiological states, we selected genes showing a relative transcript difference of greater than 2 fold higher or lower between samples, and sequence representation by at least 500 reads between libraries. Based upon these criteria, our analysis identified 5 homeodomain genes; 3 with high transcript abundance nubbin (nub – VectorBase accession: GMOY012175), teashirt (tsh – VectorBase accession: GMOY011052) and vismay (vis – VectorBase accession: GMOY000857) and 2 with lower transcript abundance ladybird late (lbl – VectorBase accession: GMOY004068), and pox meso (poxm – VectorBase accession: GMOY002525) (Table 1).
Tissue specificity of homeodomain factors
Given the tissue specific nature of expression for mgp1 (and the other milk proteins), homeodomain factors specific to that tissue were of primary interest. We performed a qPCR based expression analysis of the five candidate factors using RNA from different tissue samples obtained from lactating female flies. The expression patterns were unique for each gene. Of the genes analyzed, only the Ladybird Late gene (lbl) displayed a milk gland/fat body specific expression pattern. The fat body and milk gland are grouped together due to their interconnected nature which makes separating them by dissection next to impossible. Transcripts for lbl were ~30 fold higher in the milk gland/fat body tissue than in any other tissue (Figure 4A). The other four factors were found in multiple tissues at different levels of transcript abundance (Figure S2).
All qPCR analyses normalized to Tsetse tub. Error bars represent standard error. A. qPCR analysis of lbl tissue specificity. Samples represent 3 replicates of tissues isolated from 5 individuals. Statistical notation: a = significant difference between groups (P-value <0.05 by students t-test). B. qPCR analysis of tsetse lbl abundance 4 days post sigfp (control) or silbl injection. Statistical notation: a = significant difference between groups (P-value <0.05 by students t-test). C. Relative levels of asmase, mgp1 and mgp2 following injection of sigfp (control) or silbl. Statistical notation: a = statistical difference from control (P-value <0.01 by students t-test). All flies were injected 5–6 days after adult emergence and tested at 11–13 days for lbl suppression and 16–17 days for asmase, mgp1 and mgp2 interference.
Functional analysis of LbL by siRNA knockdown
To determine lbl involvement in milk protein gene regulation, siRNA was utilized to perform gene knockdown analyses. Flies were treated with siRNAs against lbl, or gfp (control). The siRNA injections resulted in significant reductions in target gene transcript abundance. The levels of lbl were reduced to ~30% of controls (Figure 4B). Following siRNA treatment, transcript abundance of the mgp1, mgp2 and asmase1 genes was measured by qPCR. The knockdown of lbl, resulted in a significant decrease of mgp1, mgp2 and asmase transcript levels relative to the GFP controls (Figure 4C). These data indicate that the lbl homeodomain protein functions as a positive regulator of the mgp1 gene either directly or indirectly.
Phenotypic effects of lbl knockdown on tsetse fecundity
We monitored the larviposition of siLbL treated mated females over the first gonotrophic cycle. The lbl knockdown flies showed a significant reduction in the average rate of larviposition per fly per day relative to controls (Figure 5A). Cumulative larval deposition rates between the control and lbl knockdown group revealed that the lbl group birthed about half as many larvae as the controls (Figure 5B). The reduction in fecundity in these flies is likely due to the reduced level of milk protein gene expression resulting from the knockdown of the lbl. This results in nutritional deprivation of developing larvae, larval death and abortion. This knockdown does not result in a complete disruption of tsetse fecundity in terms of the number of larvae developed per female; however we believe that this is due to incomplete penetrance of our injectable siRNA system in Glossina.
2 groups of 50 flies were injected with either sigfp or silbl at 7 days post eclosion and monitored daily for mortality and pupal deposition. A. Rate of larval deposition per fly per day after injection of either sigfp or silbl. Error bars represent standard error. Statistical notation: a = statistical difference between groups (P-value <0.05 by students t-test). B. Cumulative count of larval deposition post injection over the course of the first gonotrophic cycle.
The lactation process and the milk proteins are indispensable for intrauterine progeny development in tsetse. Recent transcriptomic/proteomic/genomic analyses in tsetse have provided a comprehensive understanding of the protein constituents of tsetse milk , . These proteins act as complete amino acid sources, lipid emulsification and transport agents, providers of enzymatic function aiding larval digestion, transporters of micronutrients/small molecules and regulators of immune function , –, –. The MGP1 protein is one of the most abundant constituents of tsetse's milk and critical to larval health , . Transcript abundance of the mgp1 gene is tightly associated with the pregnancy status of the female and is restricted to the secretory cells of the milk gland.
This pattern of regulation optimizes the utilization of resources for milk protein production by coordinating gene expression with pregnancy status and nutritional demand by intrauterine offspring. The other milk protein genes display almost identical expression profiles suggesting that these proteins are regulated by the same underlying system that controls mgp1. Our studies here indicate that Lbl is responsible for driving the tissue and stage specific expression of the mgp1 gene and most likely the other milk proteins in tsetse. Furthermore our results show that this tissue specific regulatory mechanism is conserved between Glossina and Drosophila and that Lbl likely regulates expression of the accessory gland products in the adult female Drosophila. The mechanism by which this system monitors pregnancy and larval nutritional demand of tsetse remains unknown and is a target of ongoing study. The powerful genetic capabilities of the Drosophila transgenic system will promote these studies and furthermore open up investigations into the little-known biology of insect reproductive physiology.
In tsetse, the milk gland has been a recent focus of study; however less is known regarding the function(s) of female accessory glands in Drosophila. Tsetse's physiological adaptations to accommodate viviparous reproduction are unique; but the tissues such as the milk gland and uterus in tsetse are derived from common elements of the female insect reproductive tract. Female accessory gland tissues perform essential functions in insect reproduction. Accessory gland secretions are associated with fertilization, antimicrobial activity, lubrication, embryo adhesion, uterine muscle contraction, defensive secretions and physical protection of embryos. Cockroaches and Mantids (both members of the Dictyoptera clade) have modified glands which produce 2 substances (one from the left and one from the right gland) which combine in the oviduct to generate an ootheca that encases their eggs and hardens into a protective coating , . The kissing bug (Rhodnius prolixus) uses these glands to produce an adhesive secretion to glue eggs to substrate .The accessory glands of many social non-reproductive castes of female hymenopterans have been adapted from reproductive organs to form defensive organs which secrete venom. The conservation of the female accessory regulatory system may extend beyond the higher Diptera, and could be conserved throughout the arthropod lineage as a regulator of glandular gene expression in response to reproductive events. This finding would have implications for other disease vectors such as mosquitoes where little information about accessory gland function is available.
The recent release of the Glossina genome sequence  and the availability of high throughput sequencing analysis were essential for the analysis of the milk protein promoters, and the identification and screening of the transcription factors we describe here. These in silico analyses led us to identify the homeodomain family of transcription factors and allowed us to narrow down our search from a field of 96 potential factors to a handful of 5 factors. Homeodomain proteins are a conserved family of transcription factors characterized by the presence of a helix-turn-helix DNA binding domain called the homeodomain , . These factors are associated with coordination of body plan organization during development in metazoan organisms . While the function of some of these proteins is well understood within the context of embryonic development, little is known of their function on transcriptional regulation within mature organisms. In addition, a number of homeobox genes have been identified based upon DNA binding domain conservation, yet little is known as to their functions and if the functions are orthologous between organisms .
Here, tissue specific expression profiling and RNAi analysis led us to the Ladybird Late homeodomain factor (Lbl). This factor appears critical to the expression of multiple milk protein genes in tsetse based upon our knockdown experiments. The 236 bp tsetse promoter region was sufficient to drive the synthesis of a reporter gene in an accessory gland specific manner in female Drosophila. The conservation of regulatory sequence function in tsetse and Drosophila suggests that LbL performs orthologous reproductive functions in both species.
The lbl gene was first discovered with its paralog ladybird early (lbe) during a search for novel homeodomain proteins . This factor and most of the characterized homeodomain proteins are primarily associated with developmental functions in immature organisms. Research on the ladybird genes in Drosophila have linked these genes with embryonic heart cell precursor diversification , embryonic muscle precursor cell diversification (specifically myogenesis in appendages) ,  and embryonic neuroblast diversification . Analysis of ladybird gene regulation suggests that the two genes are regulated by the wingless (wg) signaling pathway and may in turn regulate wingless expression in a feedback loop . This is the first association of Lbl function with adult regulatory processes. The data presented here indicate that Lbl is part of a system that either directly or indirectly regulates gene expression in female accessory tissues. The details of the other components of this system and the reproductive cues it responds to are unknown. The association of lbl with wingless signaling during development hints at the possibility of the wingless pathway as a regulator of reproduction associated gene regulation in adults .
The similarity in function of the mgp1 regulatory sequence between tsetse and Drosophila coupled with the ease of transgenesis in the fruit fly system allows us to use Drosophila as a surrogate in which to study female accessory gland gene expression. This promoter in combination with GAL4 mediated systems can enable in vivo tissue and stage specific knockdowns and/or ectopic expression experiments in Drosophila in order to identify components of the signaling system regulating Lbl activity. Flies carrying mgp1-GAL4/mgp1-EGFP fusions can be crossed with the available UAS-RNAi lines  of genes of interest (such as components of the wg pathway) to perform knockdowns quickly and efficiently. The presence of the mgp1-EGFP reporter will allow for rapid visual assessment of knockdown phenotypes. The findings from Drosophila can then be translated back into tsetse by RNAi experiments.
These findings are an important step in identifying the pathways, factors and signals regulating milk gland/accessory gland function in insect reproduction. Milk production in Glossina is essential for reproductive success and provides an important molecular target for the development of an insect specific reproductive inhibitor. Further analysis of mechanisms governing this system will provide important data towards controlling sleeping sickness as well as broadening the understanding of insect reproductive physiology. The knowledge gained from this work can be put towards the development of an insect specific inhibitor which disrupts milk protein production in tsetse. A substance such as this could be utilized on targets, traps and livestock as a non-toxic alternative to pesticides to reduce tsetse populations and decrease the trypanosome transmission threat.
Schematic of mgp1-reporter fusion constructs for Drosophila transformation. A. 2.0 and 0.5 kB mgp1 pPelican based transformation constructs. Constructs include 2.0 and 0.5 kB promoter fragments driving a β-gal reporter flanked by gypsy insulator sequences. The construct also includes the white transformation marker and is flanked by p-element sequences. B. 509,236,112 and 13 bp mgp1 pStinger based recombination constructs. The 0.5 kb mgp1 fragment was cloned into the pStinger transformation plasmid. pStinger is similar to pPelican with the exception that the β-gal reporter has been replaced with nuclear EGFP. Recombination constructs were amplified with primers containing AttB sites which amplify different lengths of the mgp1 promoter. Amplified fragments were cloned into the T-vector PCR cloning vector and used for recombinatorial transformation.
Tissue specific expression analysis of five putative homeodomain genes. All qPCR analyses normalized to Tsetse tub. Error bars represent standard error. Samples represent 3 replicates of tissues isolated from 5 individuals. A. qPCR analysis of nubbin (nub) tissue specificity. B. qPCR analysis of teashirt (tsh) tissue specificity. C. qPCR analysis of vismay (vis) tissue specificity. D. qPCR analysis of ladybird late (lbl) tissue specificity. E. qPCR analysis of pox meso (poxm) tissue specificity.
qPCR primer sequences. Sequences for Syber green primers utilized in the qPCR analyses performed in this work.
Construct creation primer sequences. Primer sequences utilized in the amplification of sequences for the creation of transgenic constructs.
siRNA sequences. Sequence information for siRNAs utilized within the gene knockdown experiment.
Conceived and designed the experiments: GMA JBB SA. Performed the experiments: GMA JBB VM KRP TBK. Analyzed the data: GMA JBB VM. Contributed reagents/materials/analysis tools: SA. Wrote the paper: GMA SA.
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