DEVELOPMENTAL DYNAMICS 244:21–30, 2015 DOI: 10.1002/DVDY.24214
RESEARCH ARTICLE
MicroRNA miR-7 Contributes to the Control of Drosophila Wing Growth a
Ricardo Aparicio, Carolina J. Simoes Da Silva, and Ana Busturia*
DEVELOPMENTAL DYNAMICS
Centro de Biologıa Molecular “Severo Ochoa” CSIC-UAM, Madrid, Spain
Background: The control of organ growth is critical for correct animal development. From flies to mammals, the mechanisms regulating growth are conserved and the role of microRNAs in this process is emerging. The conserved miR-7 has been described to control several aspects of development. Results: Here, we have analyzed the function of miR-7 during Drosophila wing development. We found that loss of miR-7 function results in a reduction of wing size and produces wing cells that are smaller than wild type cells. We also found that loss of miR-7 function interferes with the cell cycle by affecting the G1 to S phase transition. Further, we present evidence that miR-7 is expressed in the wing imaginal discs and that the inactivation of miR-7 increases the expression of Cut and Senseless proteins in wing discs. Finally, our results show that the simultaneous inactivation of miR-7 and either cut, Notch, or dacapo rescues miR-7 loss of function wing size reduction phenotype. Conclusions: The results from this work reveal, for the first time, that miR-7 functions to regulate Drosophila wing growth by controlling cell cycle phasing and cell mass through its regulation of the expression of dacapo and the Notch signaling C 2014 Wiley Periodicals, Inc. pathway. Developmental Dynamics 244:21–30, 2015. V Key words: miR-7; dacapo; cut; Drosophila; wing; growth Submitted 16 May 2014; First Decision 30 September 2014; Accepted 30 September 2014; Published online 9 October 2014
Introduction Proper control of organ growth requires the coordination of both the initiation of cell division and its termination upon reaching the correct organ size. The Drosophila wing imaginal disc is an excellent model system to investigate the molecular pathways controlling growth (for recent reviews, see Neto-Silva et al., 2009 and Herranz and Milan, 2008), which are highly conserved between flies and mammals (Gerhart, 1999; Dekanty and Milan, 2011). The wing imaginal disc is the rapidly growing wing primordium, increasing over 96 hr from approximately 50 to 50,000 cells (Garcia-Bellido and Merriam, 1971). Cell cycle regulators such as dacapo (de Nooij et al., 1996) and pathways such as the EGFR, Wingless, and Notch signaling pathways (Gerhart, 1999) function to organize and coordinate the growth and morphogenesis of the wing (Herranz and Milan, 2008). The wing imaginal disc becomes subdivided during development into different cell populations defining the anterior/posterior (A/P) and the dorsal/ ventral (D/V) compartments. Cell interactions at the boundary between D and V compartments induce the activation of Notch in “boundary cells” and consequent activation of Wingless pathway in adjacent “non boundary” cells. Both Notch and Wingless con*Correspondence to: Ana Busturia, Centro de Biologıa Molecular “Severo Ochoa” CSIC-UAM, Nicol as Cabrera 1, 28049 Madrid, Spain. E-mail: [email protected] Grant sponsor: Ministerio de Ciencia e Innovaci on; Grant numbers: BFU2008-01154 and CSD 2007-00008; Grant sponsor: Fundaci on Ramon Areces. Ricardo Aparicio and Carolina J. Simoes Da Silva contributed equally to this work.
trol and organize the growth of the entire wing until late in development when a different set of cell interactions at the boundary promotes cell cycle arrest and the cessation of wing disc growth (Baker, 2007; Herranz and Milan, 2008; Herranz et al., 2008). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional repressors (Bushati and Cohen, 2007; Inui et al., 2010) most often by binding to the 3’UTR mRNA sequences and whose function allows organisms to quickly modify gene expression in response to intra- and extra- cellular signals. The biogenesis of miRNAs as well as the mechanisms of miRNAs mediated repression of protein expression have been extensively studied (Stark et al., 2003; Brennecke et al., 2005; Yang and Lai, 2011). To date, 238 miRNAs have been predicted in Drosophila (www.mirbase.org, www.microRNA.org, Stark et al., 2003; Brennecke et al., 2005; Yang and Lai, 2011) but, surprisingly, the functions of only a few have been reported in detail (Smibert and Lai, 2010). Evidence exists suggesting several miRNAs are involved in the control of wing growth (Herranz and Milan, 2008; Becam et al., 2011; Bejarano et al., 2012; Waldron and Newbury, 2012) and it has also been shown that miRNAs target transcripts that encode proteins directly involved in cell cycle progression (Hatfield et al., 2005; Carleton et al., 2007; Banisch et al., 2012). For example, down-regulation of dacapo expression by miR-7 permits the G1S transition, thereby allowing germline stem cells (GSCs) division Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy. 24214/abstract C 2014 Wiley Periodicals, Inc. V
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Fig. 1. The microRNA miR-7 controls wing growth. A: Wild type wing. B: Class 1 Df (2R) exu1/miR-7D1 wing. B’: Class 2 Df (2R) exu1/miR-7D1 wing. B”: Class 3 Df (2R) exu1/miR-7D1 wing. C: Graph representing the Class 1, Class 2, and Class 3 wings sizes measurements (n¼40) of Df (2R) exu1/miR-7D1. **P
RESEARCH ARTICLE
MicroRNA miR-7 Contributes to the Control of Drosophila Wing Growth a
Ricardo Aparicio, Carolina J. Simoes Da Silva, and Ana Busturia*
DEVELOPMENTAL DYNAMICS
Centro de Biologıa Molecular “Severo Ochoa” CSIC-UAM, Madrid, Spain
Background: The control of organ growth is critical for correct animal development. From flies to mammals, the mechanisms regulating growth are conserved and the role of microRNAs in this process is emerging. The conserved miR-7 has been described to control several aspects of development. Results: Here, we have analyzed the function of miR-7 during Drosophila wing development. We found that loss of miR-7 function results in a reduction of wing size and produces wing cells that are smaller than wild type cells. We also found that loss of miR-7 function interferes with the cell cycle by affecting the G1 to S phase transition. Further, we present evidence that miR-7 is expressed in the wing imaginal discs and that the inactivation of miR-7 increases the expression of Cut and Senseless proteins in wing discs. Finally, our results show that the simultaneous inactivation of miR-7 and either cut, Notch, or dacapo rescues miR-7 loss of function wing size reduction phenotype. Conclusions: The results from this work reveal, for the first time, that miR-7 functions to regulate Drosophila wing growth by controlling cell cycle phasing and cell mass through its regulation of the expression of dacapo and the Notch signaling C 2014 Wiley Periodicals, Inc. pathway. Developmental Dynamics 244:21–30, 2015. V Key words: miR-7; dacapo; cut; Drosophila; wing; growth Submitted 16 May 2014; First Decision 30 September 2014; Accepted 30 September 2014; Published online 9 October 2014
Introduction Proper control of organ growth requires the coordination of both the initiation of cell division and its termination upon reaching the correct organ size. The Drosophila wing imaginal disc is an excellent model system to investigate the molecular pathways controlling growth (for recent reviews, see Neto-Silva et al., 2009 and Herranz and Milan, 2008), which are highly conserved between flies and mammals (Gerhart, 1999; Dekanty and Milan, 2011). The wing imaginal disc is the rapidly growing wing primordium, increasing over 96 hr from approximately 50 to 50,000 cells (Garcia-Bellido and Merriam, 1971). Cell cycle regulators such as dacapo (de Nooij et al., 1996) and pathways such as the EGFR, Wingless, and Notch signaling pathways (Gerhart, 1999) function to organize and coordinate the growth and morphogenesis of the wing (Herranz and Milan, 2008). The wing imaginal disc becomes subdivided during development into different cell populations defining the anterior/posterior (A/P) and the dorsal/ ventral (D/V) compartments. Cell interactions at the boundary between D and V compartments induce the activation of Notch in “boundary cells” and consequent activation of Wingless pathway in adjacent “non boundary” cells. Both Notch and Wingless con*Correspondence to: Ana Busturia, Centro de Biologıa Molecular “Severo Ochoa” CSIC-UAM, Nicol as Cabrera 1, 28049 Madrid, Spain. E-mail: [email protected] Grant sponsor: Ministerio de Ciencia e Innovaci on; Grant numbers: BFU2008-01154 and CSD 2007-00008; Grant sponsor: Fundaci on Ramon Areces. Ricardo Aparicio and Carolina J. Simoes Da Silva contributed equally to this work.
trol and organize the growth of the entire wing until late in development when a different set of cell interactions at the boundary promotes cell cycle arrest and the cessation of wing disc growth (Baker, 2007; Herranz and Milan, 2008; Herranz et al., 2008). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional repressors (Bushati and Cohen, 2007; Inui et al., 2010) most often by binding to the 3’UTR mRNA sequences and whose function allows organisms to quickly modify gene expression in response to intra- and extra- cellular signals. The biogenesis of miRNAs as well as the mechanisms of miRNAs mediated repression of protein expression have been extensively studied (Stark et al., 2003; Brennecke et al., 2005; Yang and Lai, 2011). To date, 238 miRNAs have been predicted in Drosophila (www.mirbase.org, www.microRNA.org, Stark et al., 2003; Brennecke et al., 2005; Yang and Lai, 2011) but, surprisingly, the functions of only a few have been reported in detail (Smibert and Lai, 2010). Evidence exists suggesting several miRNAs are involved in the control of wing growth (Herranz and Milan, 2008; Becam et al., 2011; Bejarano et al., 2012; Waldron and Newbury, 2012) and it has also been shown that miRNAs target transcripts that encode proteins directly involved in cell cycle progression (Hatfield et al., 2005; Carleton et al., 2007; Banisch et al., 2012). For example, down-regulation of dacapo expression by miR-7 permits the G1S transition, thereby allowing germline stem cells (GSCs) division Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy. 24214/abstract C 2014 Wiley Periodicals, Inc. V
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Fig. 1. The microRNA miR-7 controls wing growth. A: Wild type wing. B: Class 1 Df (2R) exu1/miR-7D1 wing. B’: Class 2 Df (2R) exu1/miR-7D1 wing. B”: Class 3 Df (2R) exu1/miR-7D1 wing. C: Graph representing the Class 1, Class 2, and Class 3 wings sizes measurements (n¼40) of Df (2R) exu1/miR-7D1. **P
