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Molecular Chaperone Hsp70 and Its Constitutively Active Form Hsc70 Play an Indispensable Role During Eye Development of Drosophila melanogaster

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Abstract

In the present study, we demonstrate that molecular chaperone Hsp70 and Hsc70 is essential for normal organization and development of ommatidial cells in Drosophila melanogaster eye. An exogenously expressed dominant negative mutant of Hsp70 (K71E) and Hsc70.4 (K71S and D206S) in an eye-specific manner resulted in eye degeneration that includes loss of eye pigment, disorganized ommatidia, abnormality in bristle cell arrangement and reduction in the eye size. The developmental organization of ommatidial cells (cone, photoreceptor, pigment, and bristle cell complex) was disturbed in Hsp70 and Hsc70 mutants. Acridine orange (AO) and caspase 3 staining showed an increased cell death in Hsp70 and Hsc70 mutant eyes. Genetic interaction study of Hsp70 and Hsc70 mutants with candidate genes of JNK signaling pathway and immunocytochemistry study using phospho-JNK antibody suggested that mutation in Hsp70 and Hsc70 results in ectopic activation of JNK signaling in fly eye. Further, anti-PH3 staining in Hsp70 and Hsc70 mutant eyes revealed a reduced number of mitotic cells in second mitotic wave (SMW) of developing eye and anti-Rh1 staining showed reduced Rh1 expression, accumulation of Rh1 in the cytoplasm, and rhabdomere degeneration. Thus, on the basis of results, it was concluded that molecular chaperone Hsp70 and Hsc70 play an indispensable role during Drosophila eye development.

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Acknowledgments

We thank Prof. J. K. Roy (Banaras Hindu University, Varanasi, India), Prof. Nancy M. Bonini (Howard Hughes Medical Institute) for UAS-Hsp70 K71E, Prof. Drick Bohman (University of Rochester Medical Center, Rochester, NY USA) for UAS-Bsk DN, Prof. Martinez-Arias (University of Cambridge, UK) for puc E69, Prof. M. Miura (Graduate Institute of Pharmaceuticals Sciences, University of Tokyo) for UAS-eiger, and Bloomington Drosophila stock Centre (Indiana University, 1001 E. Third St. Bloomington, USA) for fly stocks. We also thank Prof. Patric Dolph (Dart Morth College Medical School, USA) for anti-Rh1 antibody. The financial assistance from Department of Science & Technology (DST), New Delhi, India, to AKT (SR/FT/LS-1/2010), Laser Scanning Confocal Microscope facility supported by the Department of Biotechnology (DBT), India, and support from The Puri Foundation for Education in India is duly acknowledged.

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  1. Genetics & Developmental Biology Laboratory, School of Biological Sciences & Biotechnology, Indian Institute of Advanced Research/IAR, Koba Institutional Area, Gandhinagar, Gujarat, 382007, India

    Ajay Kumar & Anand K. Tiwari

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  1. Ajay Kumar
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Conceived and designed the experiments: AK and AKT. Performed the experiments: AK. Analyzed the data: AK and AKT. Wrote the paper: AK and AKT. All authors reviewed the manuscript.

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Correspondence to Anand K. Tiwari.

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This work was supported by the Department of Science and Technology (DST), New Delhi, India, to AKT (SR/FT/LS-1/2010).

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The authors declare that they have no conflicts of interest.

Electronic Supplementary Material

Supplementary Figure 1

Mutation in Hsp70 and Hsc70 results in excessive cell death in larval and pupal eyes. (a-c) AO staining in third instar larval eye imaginal disc of Oregon R+ (a), UAS-Hsp70 K71E -GMR-GAL4/+ (b) and UAS-Hsc70.4 K71S -GMR-GAL4/+ (c). (d-f) AO staining in 50 hr pupal eyes from Oregon R+ (d), UAS-Hsp70 K71E -GMR-GAL4/+ (e) and UAS-Hsc70.4 K71S -GMR-GAL4/+ (f). Eyes from Hsp70 and Hsc70 mutants showed an increase cell death. AO staining seen at the periphery of pupal eyes in contol represents the developmental apoptosis (d, arrow). Scale Bar represents 100 μm (a-f). (Total 10 eyes (larval & pupal) were used for cell death observations). (TIFF 4717 kb)

Supplementary Figure 2

F-actin (Green) and anti-Hsp70 (5A5) (Red) staining in 50 h pupal eyes of control, Hsp70 and Hsc70 mutants. (a-d) F-actin stained 50 h pupal eyes of GMR-GAL4/+ showing proper organization of cone, pigment and bristle cell (a), a severely disorganized cone, pigment and bristle cells with actin accumulation in UAS-Hsp70 K71E -GMR-GAL4/+ eyes (b, arrow), a less severe disorganized cone, pigment and bristle cells with less actin accumulation in UAS-Hsc70.4 K71S -GMR-GAL4/+ eye was observed (c, arrow) while in GMR-GAL4/+;UAS-Hsc70.4 RNAi /+ less affected actin cytoskeleton and normally arranged cone, pigment and bristle cells were observed (d). (e-h) Anti-Hsp70 staining in GMR-GAL4/+ showing basal level of Hsp70 protein expression in GMR-GAL4/+ (e), depleted Hsp70 protein in UAS-Hsp70 K71E -GMR-GAL4/+ (f, arrow), slightly upregulated Hsp70 protein in UAS-Hsc70.4 K71S -GMR-GAL4/+ (g) and strongly upregulated Hsp70 protein in GMR-GAL4/+;UAS-Hsc70.4 RNAi /+eye (h). (i-l) are the merged images of (a & e), (b & f), (c & g) and (d & h) respectively. Scale bars represent 20 μm (a-l). (Total 10 pupal eyes were used from each group). (TIFF 4539 kb)

Supplementary Table 1

Various phenotype shown by Hsc70 (Hsc70.4) and Hsp70 mutants driven by GMR-GAL4. (DOC 37 kb)

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Kumar, A., Tiwari, A.K. Molecular Chaperone Hsp70 and Its Constitutively Active Form Hsc70 Play an Indispensable Role During Eye Development of Drosophila melanogaster . Mol Neurobiol 55, 4345–4361 (2018). https://doi.org/10.1007/s12035-017-0650-z

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