Dynamics expression of DmFKBP12/Calstabin during embryonic early development of Drosophila melanogaster
Calcium signaling are conserved from invertebrates to vertebrates and plays critical roles in many molecular mechanisms of embryogenesis and postnatal development. As a critical component of the signaling pathway, the RyR medicated calcium-induced calcium release signaling system, has been well studied along with their regulator FK506-binding protein 12 (FKBP12/Calstabin). Lack of FKBP12 is known to result in lethal cardiac dysfunction in mouse. However, precisely how FKBP12 is regulated and effects calcium signaling in Drosophila melanogaster remains largely unknown.
In this study, we identified both temporal and localization changes in expression of DmFKBP12, a translational and transcriptional regulator of Drosophila RyR (DmRyR) and FKBP12, through embryonic development. DmFKBP12 is first expressed at the syncytial blastoderm stage and undergoes increased expression during the cellular blastoderm and early gastrulation stages. At late gastrulation, DmFKBP12 expression begins to decline until it reaches homeostasis, which it then maintains throughout the rest of development. Throughout these described changes in expression, DmFKBP12 mRNA remain stable, which indicates that protein dynamics are attributed to regulation at the mRNA to protein translation level. In addition to temporal changes in expression, dynamic expression profiles during Drosophila development also revealed DmFKBP12 localization. Although DmFKBP12 is distributed evenly between the anterior to posterior poles of the blastoderm egg, the protein is expressed more strongly in the cortex of the early Drosophila gastrula with the highest concentration found in the basement membrane of the cellular blastoderm. Fertilized egg, through the profile as under-membrane cortex distribution concentering onto basement at cellular blastoderm, to the profile as three-gem layer localization in primitive neuronal and digestion architecture of early Drosophila gastrula. By late gastrulation, DmFKBP12 is no longer identified in the yolk or lumen of duct structures and has relocated to the future brain (suboesophageal and supraesophageal ganglions), ventral nervous system, and muscular system. Throughout these changes in distribution, in situ DmFKBP12 mRNA monitoring detected equal distribution of DmFKBP12 mRNA, once again indicating that regulation of DmFKBP12 occurs at the translational level in Drosophila development.
As a critical regulator of the DmRyR-FKBP complex, DmFKBP12 expression in Drosophila fluctuates temporally and geographically with the formation of organ systems. These finding indicate that DmFKBP12 and RyR associated calcium signaling plays an essential role in the successful development of Drosophila melanogaster. Further study on the differences between mammalian RyR-FKBP12 and Drosophila DmRyR-FKBP12 can be exploited to develop safe pesticides.
KeywordsDrosophila RyR-FKBP12 DmFKBP12 dynamic profile Embryonic development
anterior midgut rudipighian
brain supraesophageal ganglion
calcium-induced calcium release
Drosophila melanogaster RyR
inositol 1,4,5-trisphosphate receptor
nerve fibers and tracts
posterior midgut rudiment
reverse transcription polymerase chain reaction
salivary gland duct
ventral nervous system
In mammals, calcium signaling plays critical roles in many biological functions with its molecular mechanism in cell [1, 2, 3]. Abnormality of the signaling leads life-threatening diseases including cancers. Extracellular environmental homeostatic calcium is regulated through cell membrane integrated protein CaSR (calcium-sensing receptor) and ITG (integrins) [4, 5], and cytoplasmic calcium is controlled by inositol 1,4,5-trisphosphate receptors (IP3R) plus ryanodine receptors (RyR) in endoplasmic reticulum (ER) via one of well-known calcium induced calcium release (CICR) signaling pathway [6, 7, 8, 9]. The CICR pathway as one of important calcium signaling pathways in cell functions through binding with their regulators such as FKBP12 (also known as Calstabin) and FKBP12.6 [10, 11, 12, 13, 14, 15]. In insect, Drosophila melanogaster ryanodine receptor (DmRyR) cDNA was cloned from lava and the physical features of DmRyR single channel were characterized with in vitro overexpression system . As a unique isoform of RyR in insect, the binding protein DmFKBP12 is essential as well in insect physiological and cellular processes. FKBP proteins are FK506 binding protein family implicating with many cellular function including calcium receptor signaling, protein folding and trafficking, transaction control, apoptotic death, and up to physiological role including embryonic development, stress response, tumorigenesis, neuronal response, angiogenesis and vascular remodeling [6, 7, 17].
Previous study unveiled that, in Drosophila melanogaster, the eight known DmFKBPs share homology with the Homo sapiens FKBP12 among its paralogues and orthologues through molecular phylogenetic analysis of FKBP family proteins . Apart from DmFKBP59 possessing two FKBP domains, all other DmFKBPs including DmFKBP12, DmFKBP13, DmFKBP14, DmFKBP39, Shutdown (CG4736) and other two unnamed DmFKBPs (CG1847 and CG5482) compose of a single FKBP domain along with Cwf/Cwc superfamily domain, EF-band domain and TPR domain individually according to references from NCBI to FlyBase . Because lack of HmFKBP12 generating the newborn defects with the cerebral edema and cardiac arrhythmia , and FKBP12 overexpression led to lethal defect of arrhythmic pathology , a critical function of DmFKBP12 is expected on Drosophila physiological role. Recently, the data from DmFKBP12 mutant (also known as Calstabin and FK506-BP2) demonstrated that DmFKBP12 is necessary for S107 to play its critical role on extension of both health and life span against oxidative stress, and DmRyR is essential for larval development in Drosophila flies [20, 21].
FKBP12 and FKBP12.6 are widely recognized as the regulators of RyRs associated many calcium signaling physiological function [6, 7, 8, 17, 21]. RyR conducted calcium sparks regulated by FKBP12 which were first found in activation of Medaka fish eggs [22, 23] in zygote early development expressing critical function of FKBP12 . To identify function of DmFKBP12 in early Drosophila development, in this study, we dynamically localized the distribution pattern of DmFKBP12 protein and mRNA at different embryonic stage. The information obtained in this research provides more comprehension on how DmFKBP12 performing its role within its dynamic distribution. Our data may benefit on developing more insect-specific pesticide targeting on early stage of insect embryo with more effective strategy related to DmRyR-FKBP12 complex.
Materials and methods
All Drosophila melanogaster flies and embryos were grown at 25 °C on standard cornmeal-molasses-agar medium until collection and fixation. The wild-type embryos were from Canton S strain. Embryos were staged as previously described . All animals maintained in natural day-night cycle. All animal maintenance and experiments were performed under guidelines approved by the Animal Care and Use Committee of Xi’an for Animal Use of Universities in Shaanxi Province, P.R. China.
Embryo histological and morphological analysis
Drosophila embryos were collected at two hrs (syncytial blastoderm, 90.8%), three hr (cellular blastoderm, 91.5%), 12 h (early gastrula, 91.1%) and 24 h (late gastrula, 93.3%) after egg lay on grape juice agar plates (2.5% sucrose, 2.5% agar, 25% grape juice, 0.5% propionic acid), then dechorionated in 50% bleach and rinsed thoroughly in phosphate-buffered saline (PBS). Embryos were fixed in 50% heptane and 50% PEMFA (100 mM PIPES, 2.0 mM EGTA, 1.0 mM MgSO4, pH to 6.9 using KOH and 4% formaldehyde) for one hr and washed 3 times with PBS. Then samples were fixed in Bouin solution, paraffin embedded and sectioned at thickness of 8-10 μm . Sections were and stained with hematoxylin-eosin (H&E) and periodic acid-silver methenamine (PASM) for regular histological analysis [26, 27].
Deparaffinised and rehydrated sections were rinsed in PBS, and the antigens of samples were retrieved with preheated (approx. 100 °C) target retrieval solution (Dako, USA) for 20 min. Sections were treated with 0.3% H2O2 in methanol for 10 min and followed by a blocking step with 1% bovine serum albumin (BSA) diluted in PBS for 60 min at room temperature (RT). Then sections were incubated with primary antibody (anti-FKBP12 at 1:1000, sc-28814, Santa Cruz). After washed with PBS, the Envision-plus detection system was applied with HRP labelled polymer conjugated with secondary antibodies (anti-Rabbit, EnVision + System-HRP, Dako). Reaction products were visualized after incubation with 3, 3′-diaminobenzidine [28, 29].
Total RNA preparation and RT-PCR
Embryos were collected and snap frozen in liquid nitrogen. Total RNA was extracted with Trizol reagent (Invitrogen) and first-strand cDNA of DmFKBP12 was amplified from 3 µg of purified total RNA through reverse transcription polymerase chain reaction (RT-PCR) (Life Technologies) [9, 30]. Forward primer (Primer-F, 5′-CTAGCTAGCCGATGGGCGTACAAGTA GTTCCA-3′) and reverse primer (Primer-B, 5′-TACGAGCTCCTATTCGACCTTGAGCAGCTC-3′) were designed according to the published cDNA sequence of DmFKBP12 as FK506-binding protein 2 of Drosophila melanogaster (NM_079068.5). Specific sequences for identifying restriction enzyme Nhe I and Sac I were included in the primer pairs at the 5′ and 3′ ends as restriction sites, respectively. The expression of DmFKBP12 mRNA was assessed and the products were visualized with gel imaging system.
RT-PCR analysis was also used to identify DmRyR in developing embryos. Two fragments (I + II, and II + IV) of DmRyR domain (I + II, and II + IV) were amplified as their first-strand cDNA and then PCR fragments were cloned into pGME-T plasmid with forward primer-1 (5′-agatgtgggctctaaaca-3′) and reverse primer-1 (5′-tgaagatctcgttgggca-3′), forward primer-2 (5′-gagacatccgatccgata-3′) and reverse primer-2 (5′-cctcgttctggaattcgt-3′). The cDNA of DmRyR domain was sequenced for confirmation on their correctness (Additional file 1: Figure S1).
RNA in situ hybridization
Drosophila embryo cDNA was amplified by PCR with two primers (5′-CTAGCTAGCCGCCACCATGGGCGTACAAGTAGTTCCA-3′, and 5′-TACGAGCTCCGCCACCTTC GACCTTGAGCAGCTC-3′), and then cloned into linearized pGEM-T Easy plasmid vector to make circular plasmid vector with the probe cDNA under down stream of SP6 and T7 promoters. The probes were synthesized for RNA labeling with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase (#11175025910, Roche). Antisense double digoxigenin (DIG) labeled RNA was used as a probe of DmFKBP12 hybridization, and DIG labeled sense RNA was used as a negative control (data not shown). The probes were quantified and applied 1 μg of probe per hybridization reaction. Probes were detected using a primary sheep anti-DIG-AP 1:10,000 (#11093274910, Roche) and NBT/BCIP for the sensitive detection .
All Drosophila embryo sections collected at syncytial blastoderm, cellular blastoderm, early and late gastrulation stages, were prepared according to the previous description above with 0.01% DEPC pre-treated water . DEPC overnight treatment was applied on all accessories used for the in situ hybridization.
Western blotting analysis
Expression analysis on the DmFKBP12 protein of Drosophila embryos was inspected by Western blotting according to the standard protocol [9, 32]. Tissue proteins from embryos was extracted using RIPA reagent. Standard BSA approach was used for protein quantification. The proteins sample were denatured after heated at 100 °C and separated on a gradient SDS-PAGE, and then transferred onto polyvinylidene difluoride (PVDF) membranes with a BioRad transfer unit at 100 V for 120 min. The membranes were blocked with 5% non-fat milk in TBST at 4 °C overnight and detected with primary antibody (anti-β-tubulin 1:5000; anti-FKBP12 1:1000, sc-28814, Santa Cruz) at room temperature (RT). After three times washing with TBST, the membranes were incubated with secondary antibody at RT. Immuno-detection was carried out with ECL followed by exposure to motored molecular imaging system (Tanon 4200, China). Images were quantitated using the Image J software downloaded from website of National Institutes of Health .
Microscopy and statistical analysis
The histochemistry images were documented and analyzed with the inverted microscopy (Carl Zeiss Microscopy GmbH) and manufacturer software ZEN. Data were presented as mean ± SEM, and were analyzed with SPSS 22.0 and Graphpad Prism 7. A one-way ANOVA followed by a post hoc comparison Tukey was employed to analyze the data. A P value less than 0.05 and 0.01 was considered statistically significant [16, 27].
DmFKBP12 during Drosophila embryogenesis
The Drosophila embryonic samples were collected at four different stages i.e. syncytial blastoderm, cellular blastoderm, early and late gastrulation stages according to the approach described in the previous publication . These four-stage Drosophila embryos harvested and sorted at following four timing-points for inspection of DmFKBP12 expression during Drosophila development. The embryos from syncytial blastoderm stage were sorted from collection of 2 h after fertilization (syncytial blastoderm, 90.8%; n > 400). The embryos of cellular blastoderm stage (cellular blastoderm, 91.5%; n > 300), early gastrulation stage (early gastrula, 91.1%; n > 200) and late gastrulation stage (late gastrula, 93.3%; n > 200) were done individually from 3 h, 13 h and 24 h collection after fertilization.
RT-PCR analysis of DmFKBP12 gene expression supported the result of protein analysis (Fig. 1b). In the total RNA samples of syncytial blastoderm, cellular blastoderm, early and late gastrulation stages, the DmFKBP12 cDNA fragment amplified from transcripted first strain cDNA were clearly detected in four-stage Drosophila embryos. The above inspection demonstrated that the Drosophila DmFKBP12 expression was examied at both translation and transcription levels. Our data extend the detected expression of DmFKBP12 from adult Drosophila  to very early embryonic syncytial blastoderm stage of its development with multiple nuclei of first 13 mitosis after fertilization .
As we know that DmFKBP12 is the regulator of DmRyR, the major component of Calcium signaling on CICR pathway, we further examined its existence within the Drosophila embryos by cloning its most conserved RyR domain of all RyR orthologues. The cDNA sequencing confirmation provided the evidence on the appearance of DmRyR in Drosophila embryos (Additional file 1: Figure S1).
Dynamic profile of DmFKBP12 protein from syncytial blastoderm to cellular blastoderm of the Drosophila embryo
Dynamic profile of DmFKBP12 protein from early to late gastrulation stages of the Drosophila embryo
As the fundamental events of early differentiation, association of cell proliferation and migration are tightly relevant with cellular calcium signaling as discussed previously. It is noticeable that DmFKBP12 is essential within stage transition from early gastrula to late gastrula.
In Drosophila late gastrulation stage, a major recognizable difference from previous stages is that the non-distribution areas of the DmFKBP12 protein are much more noticeable beside yolk-zone surrounding central portion of nerve fibers and tracts (NF), luminal portion of the most tubular structures in entire body cavity (Fig. 5e–h). At the end of late gastrulation stage, the non-expression zone of DmFKBP12 protein is getting more and more discernible. Furthermore, inner germ layer differentiated organs/tissues expressing less DmFKBP12 are extremely eminent (Fig. 5i–l). Compared to our inspection on DmFKBP12 expression pattern in previous three stages of Drosophila development listed above, the protein distribution in the late gastrula is significant less than that in early gastrulation and other two stages according to our immunological detection. The DmFKBP12 protein expression distributed more in the cell-composed primary viscera including mouth hook, brain (supraesophageal ganglion), midgut caecum, proventriculus, anterior midgut rudipighian, middle midgut and posterior midgut rudiment, less located in their lumens (Fig. 5m, all p < 0.001). Weak signal of DmFKBP12 expressed in cavity of posterior and anterior poles can be detected.
Dynamic mRNA profile of DmFKBP12 gene expression of Drosophila embryogenesis from syncytial blastoderm via cellular blastoderm to gastrulation
As our dynamic data on expression of DmFKBP12 in Drosophila embryogenesis, the distribution pattern of DmFKBP12 protein developed from syncytial blastoderm, via cellular blastoderm, to early and late gastrulation stages along with growing construction of primitive tissues/organs from three-germ layers. All differentiation from a single-cell embryo, via syncytial thousand-nuclei cell embryo, to classic single-nuclear cell embryo after cellularization, the Drosophila development accompanying with cellular DmFKBP12 is critical on morphological architecture. In data of this session, we focus on investigation of DmFKBP12 gene expression on mRNA level to confirm the function of evolutional conceived DmFKBP12 gene in Drosophila embryogenesis.
RyRs plus IP3R are mainly responsible for calcium sparks in skeletal muscle, cardiac myocytes, smooth muscle, neurons and other excitation cells [6, 7]. This calcium sparks can transform in different appearances or forms to perform their diverse functional molecular mechanism in cells of tissues where sparks locate. Ca2+ releasing via single tetramer RyR generating Ca2+ quark, via multiple adjacent complex producing Ca2+ wave and ER/SR origin Ca2+ local discretting Ca2+ puff play critical role in myocytes [6, 7]. In neuronal presynaptic terminal, RyR mediated Calcium releasing as Ca2+ syntilla, through Ca2+ synapse of a crossing-membrane structural and functional complex, are essential for neuronal transition [36, 37]. In vertebrate, three isoforms of RyR comprehend their biological compound commission with molecular mechanism in cells, in which regulations from FKBP12 and/or FKBP12.6 is required. In this study, our data obtained with dynamic localization of DmFKBP12 protein and DmFKBP12 mRNA (Fig. 7), demonstrate functional essentiality of DmRyR-FKBP12 complex in embryonic development of Drosophila melanogaster.
It has been well recognized that FKBP12 and FKBP12.6 are critical regulators for Calcium-induced Calcium releasing through RyRs [6, 38, 39]. RyR1, RyR2 and RyR3 represent skeletal muscle specific isoform, cardiac isoform and brain specific isoform. Traditionally, FKBP12 is in charge of regulating skeletal muscle RyR1 Calcium channel, and FKBP12.6 interacts with cardiac muscle RyR2 by controlling EC coupling [38, 39]. Because of recent data demonstrated that FKBP12 functions in transgenic heart with its cardiac overexpression by generating lethal arrhythmia [19, 39, 40, 41], it is more reasonable that multifunctional regulation of these two proteins on skeletal or/and cardiac RyR could be much miscellaneous or complexed at tissue specific cellular microenvironment. Recently, FKBP12 was linked to IP3R by regulating calcium release function in cancer cells . The data indicated that FKBP12 could play as multi-functional regulator working for both RyR and IP3R in charge of associating these two critical calcium channel proteins while cell carrying on the pathophysiological function. It is certain that DmRyR-FKBP complex contributes its important role within Drosophila development according to our result although no report make sure yet on embryonic function of IP3R in Drosophila.
The Drosophila homolog of mammalian RyRs has been investigated previously, and comprehensions on DmRyR are relatively well known such as that an only RyR gene was identified in insect instead of three isoforms in mammalian [16, 42, 43, 44]. However, very limited information documented from published data about insect RyR regulator DmFKBP12. The DmFKBP12 was identified on function of extension both health and life spans using loss of DmFKBP12 gene function model in Drosophila muscle . This work claimed that the absence of DmFKBP12 in the cytoplasmic of DmFKBP12 mutant muscle dissociated RyR from response to oxidation and resulted in earlier aging. Our data of DmFKBP12 cloning from earlier embryo (Fig. 1) extended that the function of DmFKBP12 perform its early contribution to fly embryo. The DmRyR-FKBP12 complex may already initiate their role not only in muscle but also in brain because DmRyR is only one RyR Drosophila homolog representative of mammalian three isoforms as we discussed above.
Molecular cloning of DmRyR cDNA provided the solid evidence of DmRyR expression in Drosophila lava . As the crucial regulator of this CICR pathway along with binding protein of IP3R as well , in which four DmFKBP12 control opening of ER/SR DmRyR tetramer, the expression and function of DmFKBP12 should be carried out in the lava stage. Because of, as discussed above, RyR-mediated CICR exhibiting with calcium spark and calcium wave robusting from fertilization of many animals [48, 49, 50, 51], it is judicious that the DmRyR-FKBP12 complex functions as early as the same stage. Our data in this investigation demonstrated that the DmFKBP12 distributed in subsurface cortex apposition to the plasma membrane in syncytial blastoderm (Figs. 1, 2, and 6a–c). We believe that the Drosophila DmRyR-FKBP12 as an ancient conserved complex performs its essential role throughout entire life of Drosophila melanogaster. Our data showed that the protein expression of Drosophila FKBP12 is significant different at four embryonic stages with the same mRNA level. This result strongly indicated that the existence of regulation pathway in the Drosophila embryonic development could link to the involvement of more proteins such as BAP1 and PTEN, which regulate by targeting on IP3R3-mediated Ca2+ flux to mitochondria in mammal cell [52, 53, 54].
Furthermore, our results suggested a proficient strategy that binding ligands such as Tacrolimus (FK506), sirolimus (rapamycin) and chemicals derived from them, have potential to utilize on developing pesticide against early development stage Drosophila melanogaster by targeting on the embryonic DmRyR-FKBP12 complex. When tacrolimus and sirolimus as immunosuppressant molecule bind on FKBP proteins including DmFKBP12, this binding would decline or even fail the formation of DmRyR-FKBP12 or DmIP3R-FKBP12. Therefore, the cellular calcium signaling pathway would be disrupted accompanying with death of muscular and neuronal tissues while the dysfunction of DmRyR FKBP12 occurs on Drosophila embryo in four stages in development.
Calcium signaling plays critical roles in many biological functions with Ryanodine and IP3 Receptor mediated CICR mechanism at cell molecular level. Calcium spark along with its other appearances evidently performs function with RyR/IP3R-FKBP complex associated calcium signaling from fertilization, via skeletal, cardiac and smooth muscle contraction, to presynaptic terminal neuronal cells. Our data suggested that DmRyR-FKBP12 complex plays an essential role as the regulator of DmRyR through CICR pathway. As absence of FKBP12 directly causes early newborn lethality with cerebral edema and severe arrhythmic defect representative with conduction error of cardiomyocyte in vertebrate, our data approved the present of DmFKBP12 in Drosophila early embryonic development and investigated the dynamic distribution of DmFKBP12 protein. The DmFKBP12 subsurface distribution apposition to the plasma membrane and cortex distribution within the multinuclear plasma directly supports the critical role performed through DmRyR-FKBP12 complex in Drosophila melanogaster embryo as early as at the stages of syncytial blastoderm, via cellular blastoderm and to late gastrula. Our data propose a unique potential to develop insect specific and long-term (from early embryo and adult) effective pesticide by targeting on insect embryonic RyR-FKBP12 complex against insect CICR calcium signaling pathway.
XHX conceived and designed the study. RF, XZ, WZ, TP, YS, RY, DW, XZ, ZC, YG, YL, QY, YW, XL, ZL, LW, and YS developed protocols and analyzed all the data. RF, OJ, YS, LW, WBI, JM and XHX prepared the manuscript and all authors edited it. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
This work was supported by the National Natural Science Foundation of China (#31571273/31771277/31371256), the Foreign Distinguished Scientist Program from the National Department of Education (#MS2014SXSF038), the National Department of Education Central Universities Research Fund (#GK20130100/201701005/GERP-17-45), US Maryland Stem Cell Research Fund (2009MSCRFE008300), and the Outstanding Doctoral Thesis fund (#X2014YB02/X2015YB05).
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