A Novel Cell Death Gene Acts to Repair Patterning Defects in Drosophila melanogaster | Genetics

A Novel Cell Death Gene Acts to Repair Patterning Defects in Drosophila melanogaster

  1. Kentaro M. Tanaka*,†,1,
  2. Aya Takahashi*,‡§,
  3. Naoyuki Fuse** and
  4. Toshiyuki Takano-Shimizu-Kouno*,††,1
  1. *Department of Population Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka, 411-8540, Japan
  2. †Department of Biological and Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP, United Kingdom
  3. ‡Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, 192-0397, Japan
  4. §Research Center for Genomics and Bioinformatics, Tokyo Metropolitan University, Hachioji, 192-0397, Japan
  5. **Department of Biophysics, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan
  6. ††Drosophila Genetic Resource Center, Kyoto Institute of Technology, Saga Ippongi-cho, Ukyo-ku, Kyoto, 616-8354, Japan
  1. 1Corresponding authors: Drosophila Genetic Resource Center, Kyoto Institute of Technology, Saga Ippongi-cho, Ukyo-ku, Kyoto, 616-8354, Japan. E-mail: fruitfly{at}kit.ac.jp; and Department of Biological and Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP, UK. E-mail: p0037755{at}brookes.ac.uk

Abstract

Cell death is a mechanism utilized by organisms to eliminate excess cells during development. Here, we describe a novel regulator of caspase-independent cell death, Mabiki (Mabi), that is involved in the repair of the head patterning defects caused by extra copies of bicoid in Drosophila melanogaster. Mabiki functions together with caspase-dependent cell death mechanisms to provide robustness during development.

  • Drosophila melanogaster
  • developmental robustness
  • embryogenesis
  • bicoid
  • p35-insensitive cell death

ORGANISMS are surprisingly robust to various stresses and perturbations. Elimination of undesirable cells is one mechanism that ensures robust development. For example, compensatory cell death is observed in the expanded prospective head region of embryos from mothers carrying extra (six) copies of bicoid (6xbcd) in Drosophila melanogaster. bcd mRNA is localized to the anterior pole of the oocyte, forming an anteroposterior gradient of BCD protein in the embryo (Frohnhöffer and Nüsslein-Volhard 1986; Berleth et al. 1988). 6xbcd embryos show a posterior shift in expression of the downstream genes and the position of the cephalic furrow located near the head/trunk junction. Nevertheless, many embryos still survive to adulthood (Namba et al. 1997). More importantly, the final pattern and size of the adult structures are mostly normal (but for an exception, see Busturia and Lawrence 1994). Drosophila has repair mechanisms for these patterning defects, and one is cell death in the expanded head region of embryos. However, the mechanism leading to this compensatory cell death remains largely unexplored.

To identify genes involved in this repair, we first screened a panel of 152 autosomal deficiencies for those with significantly lower viability in 6xbcd than in normal (2xbcd) condition, namely, for haploinsufficient genes in the 6xbcd condition, and obtained two candidate regions, 29A2-A3 and 34A7-B6 (Supporting Information, Table S1 and Table S2). To complement this haploinsufficiency screen, we performed a microarray expression analysis to identify genes with differential expression between the two conditions at embryonic stage 11–12, when extensive cell death occurs in the expanded head region of 6xbcd embryos (Table S3 and Table S4; Namba et al. 1997). Twelve genes showed more than twofold higher expression in 6xbcd compared to 2xbcd. Cross-referencing the genetic positions and changes in gene expression from these two complementary screens allowed us to identify a candidate gene, CG15479, involved in the repair. We named this gene Mabiki (abbreviated as Mabi). The intronless Mabi gene contains a 615-bp open reading frame and encodes a potential member of the basic region-leucine zipper (bZIP) family of transcription factors that binds specifically to DNA as dimers (Fassler et al. 2002). While it is found only in the Diptera lineage, there are three paralogs, CG14014, CG16813, and CG16815, in the D. melanogaster genome (Figure S1).

The higher expression of Mabi in 6xbcd embryos compared to 2xbcd was validated by real-time quantitative PCR (4.7-fold difference). Mabi was expressed throughout the embryo at stage 11, but a stronger signal was detected in the anterior region in both 2xbcd and 6xbcd (Figure S2). At stage 12, a few cells showed Mabi expression; however, some 6xbcd embryos with head defects (3/79) showed strong and broad Mabi expression in the head domain.

Since there is no mutant available for Mabi, we conducted RNAi knockdown experiments. A 1-hr heat shock during early embryogenesis resulted in embryonic lethality of UAS-MabiRNAi; hsp70-GAL4 and the frequency of embryos with head defects (expanded head domains or abnormality in mouth hook formation, or both) was significantly greater in Mabi (RNA interference, RNAi) embryos than in control embryos in both 2xbcd and 6xbcd conditions (Figure 1), implying that Mabi is essential even under normal conditions. While the injection of 449-bp Mabi dsRNA molecules also effectively prevented eggs from hatching in both conditions (Pilot et al. 2006), the hatchability of embryos injected with either of two shorter dsRNA molecules was significantly reduced only in 6xbcd (Figure 2). Together with the observation that the relative viability of Df(2L)ED784 heterozygotes lacking the Mabi gene was reduced in 6xbcd to about 60% of that in 2xbcd (Table S1), this result suggests that the development in the 6xbcd condition depends on higher expression of Mabi.

Figure 1
Figure 1

Defects in Mabi knockdown embryos. After a 2-hr egg collection and 1-hr incubation at 25°, embryos were heat shocked at 37° for 1 hr and then incubated at 25°. (A and C) hsp70-GAL4/+ embryos at stage 17. (B and D) Examples of head defects of unhatched UAS-MabiRNAi/+; hsp70-GAL4/+ embryos. (A and B) 2xbcd, and (C and D) 6xbcd embryos. Small black arrowheads indicate abdominal segments (A1–A8). Large open arrowheads indicate mouth hook. Anterior is to the left in all images. (E) Hatchability (%) of Mabi knockdown embryos. (F) Frequency (%) of embryos showing head defects among dead embryos. (E and F) White and black bars represent 2xbcd and 6xbcd embryos, respectively. Error bar represents the standard error of the mean of four experiments. * and *** indicate statistical significance at the 5% and 0.1% levels, respectively.

Figure 2
Figure 2

Reduced hatchability of embryos injected with Mabi dsRNA. (A) The Mabi gene structure (top) and three constructs used for dsRNA-mediated RNAi. The coding region is shown as a black box, while white boxes represent UTRs. All constructs are designed within the coding region. (B–D) Effect of dsRNA injection was assessed by measuring the relative hatchability = (hatchability of Mabi-dsRNA injected embryos) / (hatchability of dsRNA-free water injected embryos). Error bar represents the standard error of the mean of four experiments. (B) Construct #1 (449 bp); (C) construct #2 (221 bp); and (D) construct #3 (230 bp). * and ** indicate statistical significance at the 5% and 1% levels, respectively; ns, not significant.

What is more, after a 1-hr heat shock, the number of acridine orange (AO) positive cells was fewer in UAS-MabiRNAi; hsp70-GAL4 embryos than in control embryos (Figure 3, A–D), implying a requirement for Mabi in cell death. Mabi was indeed able to trigger cell death in both embryos and imaginal discs. Heat shock induction of Mabi expression in hsp70-GAL4/UAS-Mabi embryos produced many AO positive cells (Figure 3, E and F); AO positive cells were also detected in the posterior region of small wing discs of en-GAL4/+; UAS-Mabi/+ larvae and the final size of the posterior region of the wings was reduced by 40% (Figure S3).

Figure 3
Figure 3

Mabi knockdown reduces cell death in embryos, while ectopic expression of Mabi induces cell death without activation of rpr. (A–D) Acridine orange (AO) staining of stage-12 embryos. (A and B) hsp70-GAL4/+ and (C and D) UAS-MabiRNAi/+; hsp70-GAL4/+. (A and C) 2xbcd and (B and D) 6xbcd embryos. (E–H) AO staining (E and F) and rpr expression visualized by whole mount in situ hybridization (G and H) in stage-11 embryos. (E and G) hsp70-GAL4/+ and (F and H) hsp70-GAL4/UAS-Mabi. All embryos were heat shocked for 1 hr. Anterior is to the left in all images.

It has been shown that cell death in 6xbcd embryos involves the proapoptotic reaper (rpr) gene, which triggers the canonical cell death pathway through caspase activation (Namba et al. 1997; Bangs and White 2000). However, rpr expression was not altered in the hsp70-GAL4/UAS-Mabi embryos (Figure 3, G and H). Consistent with this observation, coexpression of p35 did not rescue the eye defects observed in ey-GAL4/+; UAS-Mabi/+ (Figure 4). Thus, cell death induced by Mabi is likely to be caspase independent. In addition, unlike p53, ectopic expression of Mabi by the glass-multimer-reporter (GMR)-GAL4 driver did not affect eye size (Figure 4). Mabi presumably requires an as-yet-unidentified factor or factors to induce cell death.

Figure 4
Figure 4

Effects of ectopic expression of the Mabi gene in eye imaginal discs. Mabi or p53 are ectopically expressed by ey-GAL4 (A–E) or GMR-GAL4 (F–H). (A) ey-GAL4/+, (B) ey-GAL4/+; UAS-Mabi/+, (C) ey-GAL4/+; UAS-p53/+, (D) ey-GAL4/+; UAS-Mabi/ UAS-p35, (E) ey-GAL4/+; UAS-p53/UAS-p35, (F) GMR-GAL4/+, (G) GMR-GAL4/+; UAS-Mabi/+, and (H) GMR-GAL4/+; UAS-p53/+. Bar gives the relative scale.

Here, we describe the identification and characterization of Mabi, a novel regulator of cell death that is involved in the elimination of excessive cells in the expanded head region of 6xbcd embryos. The findings suggest that elevated expression of rpr (Namba et al. 1997) and the ensuing caspase-dependent cell death are not sufficient to repair head patterning in the conditions with elevated concentrations of bcd. Both the caspase-dependent and independent cell death pathways act to confer developmental robustness in 6xbcd conditions.

Acknowledgments

We acknowledge A. P. McGregor for useful discussions and experimental assistance and S. Tamura, K. Suzuki, and Y. Ishii for technical assistance. We also thank C. Desplan, R. Ueda, T. Hayashi, Y. Yuasa, S. Arif, Szeged Drosophila Stock Center, Vienna Drosophila RNAi Center, and Drosophila Genetic Resource Center at Kyoto Institute of Technology for flies. K.M.T. was a research fellow of the Japan Society for the Promotion of Science. This work was supported in part by Grants-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T.T.-S.-K., 20570100 and 23570123).

Footnotes

  • Communicating editor: I. Hariharan

  • Received February 21, 2014.
  • Accepted March 24, 2014.

Literature Cited

    1. Bangs P.,
    2. White K.
    , 2000 Regulation and execution of apoptosis during Drosophila development. Dev. Dyn. 218: 68–79.
    1. Berleth T.,
    2. Burri M.,
    3. Thoma G.,
    4. Bopp D.,
    5. Richstein S.,
    6. et al.
    , 1988 The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo. EMBO J. 7: 1749–1756.
    1. Busturia A.,
    2. Lawrence P. A.
    , 1994 Regulation of cell number in Drosophila. Nature 370: 561–563.
    1. Fassler J.,
    2. Landsman D.,
    3. Acharya A.,
    4. Moll J. R.,
    5. Bonovich M.,
    6. et al.
    , 2002 B-ZIP proteins encoded by the Drosophila genome: evaluation of potential dimerization partners. Genome Res. 12: 1190–1200.
    1. Frohnhöfer H. G.,
    2. Nüsslein-Volhard C.
    , 1986 Organization of anterior pattern in the Drosophila embryo by the maternal gene bicoid. Nature 324: 120–125.
    1. Namba R.,
    2. Pazdera T. M.,
    3. Cerrone R. L.,
    4. Minden J. S.
    , 1997 Drosophila embryonic pattern repair: how embryos respond to bicoid dosage alteration. Development 124: 1393–1403.
    1. Pilot F.,
    2. Philippe J.-M.,
    3. Lemmers C.,
    4. Chauvin J.-P.,
    5. Lecuit T.
    , 2006 Developmental control of nuclear morphogenesis and anchoring by charleston, identified in a functional genomic screen of Drosophila cellularisation. Development 133: 711–723.