Issues of authorship prac ce and policy in experimental science research labs
Cassandra Extavour Associate Professor Organismic and Evolu onary Biology Harvard University Members of the research team Principal Inves gator
Graduate Postdocs Students
Research Undergraduate Assistants Students Members of the research team have different goals
Principal • Publish • Obtain funding Inves gator • Get tenure/promo on/other honors • Help grad students graduate • Help postdocs get jobs Graduate Postdocs Students • Achieve career stability • Graduate • Publish • Obtain post-graduate • Obtain post-postdoc job job • Publish
Research Undergraduate Assistants Students • Keep good job • Graduate • Obtain next career opportunity • Obtain post-graduate posi on • Contribute to research/publish • Contribute to research/publish Authorship has different values for different members of the team
Principal • Demonstrates ability to lead produc ve research team Inves gator • Indicates intellectual contribu on • CRITICAL
Graduate Postdocs Students • Demonstrates ability to • Demonstrates ability to carry out and complete carry out research independent research • MAYBE IMPORTANT • MAYBE IMPORTANT (depending on career (depending on career coals) goals) Research Undergraduate Assistants Students • Stepping stone for next career • Stepping stone for next career opportunity opportunity • BONUS • BONUS Authorship order has specific meaning, which is
different in different fields of scholarship RESEARCH ARTICLE 5015
• DevelopmentAuthorship is merited if you have contributed 138, 5015-5026 (2011) doi:10.1242/dev.073395 © 2011.substan ally to Published by The Company of Biologists Ltd Notch/Delta– Experimental design signalling is not required for segment generation– Carrying out experiments in the basally branching insect Gryllus bimaculatus– Analyzing data – Wri ng the manuscript Franz Kainz1,2, Ben Ewen-Campen1, Michael Akam2 and Cassandra G. Extavour1,* – Obtaining funding for the research • SUMMARYWho decides who has contributed substan ally? First authorArthropods: carried out and vertebrates display a segmental body organisation along all Last authoror part of the anterior-posterior: senior author axis. Whether this reflects– aDifferent team members may differ in their defini on of shared, ancestral developmental genetic mechanism for segmentation is uncertain. In vertebrates, segments are formed bulk of experimental and/sequentially by a segmentation ‘clock’ of oscillating gene expression involving Notch= PI. Usually major pathway components. Recent studies in or analy cal work. Usually spiders and“substan al” basal insects have suggested that segmentation in these arthropodscontributor of intellectual also involves Notch-based signalling. These grad student or postdoc. observations have been interpreted as evidence for a shared, ancestral gene networkcontent and funding. for insect, arthropod and bilaterian segmentation.– Ul mately the PI makes this decision However, because this pathway can play multiple roles in development, elucidating the specific requirements for Notch– signallingPI should is important for understanding the ancestry of segmentation. Here we show that Delta, a ligand of the Notch Second authorpathway, is not: likely carried out some required for segment formationOther middle author in the cricket Gryllus bimaculatus: could have contributed in any , which retains ancestral characteristics of of experimental and/or analy cal work. arthropod •embryogenesis. Clarify expecta ons/lab publica on policy Segment patterningway but how is unclear; less experimental/analy cal genes are expressed before Delta in abdominal segments, and Delta expression doesUsually student or technician. not oscillate• guide other team members in effec ve communica on/nego a on of in the pre-segmental regioncontribu on than preceding authors AND less senior or in formed segments. Instead, Delta is required for neuroectoderm and mesectodermauthorship goals formation; embryos missing these tissues are developmentally delayed and show defects in segment morphology but normal segment number. Thus, what initially appear to berole played than last author. ‘segmentation phenotypes’ can in fact be due to developmental delays and cell specification errors. Our data do not support an essential or ancestral role of Notch signalling in segment generation across the arthropods, and show that the pleiotropy of the Notch pathway can confound speculation on possible segmentation mechanisms in the last common bilaterian ancestor.
KEY WORDS: Arthropod, Segmentation clock, Evolution, Neurogenic phenotype, Gryllus bimaculatus
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epartment of Organismic and Evolutionary Biology, Harvard University, 16 Divinity /"#$"%&( ,.&&"0%1%#( 1%( /3*0&J#"0$( .0&30*,*'/6( 1%2-5'1%# Avenue, Cambridge, MA 02138, USA. 2Laboratory for Development and Evolution, University Museum of Zoology, Department of Zoology, University of Cambridge, %"50*#"%"/1/6(.>1/("-*%#.&1*%(.%'(.,*,&*/1/8(Z>,0"//1*%(*4(G*&23 Downing Street, Cambridge CB2 3EJ, UK. ,.&3:.+( #"%"/( 1%( &3"( /3*0&J#"0$( 1%/"2&/( 6"'5#(',1* 2)$0).-,1 ?)""&-"F(.%'(72&'$0#2-"2)*8"-8)"') ?-*25/&F('*(%*&(/5##"/&(0*-"/(1% *Author for correspondence ([email protected]) ".0-+(/"#$"%&(#"%"0.&1*%(?@".0'"%(.%'(;P.$6(CDDDI(;0.%'.("&(.-86 Accepted 13 September 2011 CDDEF8(R*0"*7"06(1%(39*5'1)2,()0,$6(G*&23(/1#%.--1%#(1/($.&"0%.--+ Germ cell selection in genetic mosaics in Drosophila melanogaster Cassandra Extavour* and Antonio Garcı´a-Bellido†‡
*Museum Molecular Laboratory, University Museum of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, United Kingdom; and †Laboratorio Gene´ tica del Desarrollo, Centro de Biologı´a Molecular, Universidad Auto´ noma de Madrid, 28049 Madrid, Spain
Contributed by Antonio Garcı´a-Bellido, August 3, 2001 Heritable mutations in the germ line lead to genetically heteroge- ref. 8). Given the large number of genes likely to be required by neous, or mosaic, gonads. Many of the genes used in germ-line the germ line, and the complexity of germ-line development, Author's personal copy development also play roles in somatic development [Saffman, particularly the loss of 50% of germ-line cell precursors early in E. E. & Lasko, P. (1999) Cell. Mol. Life Sci. 55, 1141–1163]. Mutations development (1) (see below), we undertook clonal and mosaic in these genes may have cellular phenotypes throughout germ-line analyses to ask whether or not germ-line cells undergo cellular development leading to their differential elimination or survival, as competition in genetic mosaics. Cell competition and selection has been observed in somatic cells [Morata, G. & Ripoll, P. (1975) in the germ line have important population and evolutionary 288 D.P. Sarikaya et al. / Developmental Biology 363 (2012) 279–289 Dev. Biol. 42, 211–221]. We investigate whether mutations in implications (9, 10). heterozygosis are subject to pregametic selection in the germ line. To date, only two mosaic and clonal analyses in the germ line, We initiated clones of wild-type homozygous cells at different concerned with proliferative parameters of germ cellsdevelopmental over process subject to evolutionary change in Drosophi- Acknowledgments stages of development in gonads heterozygous for eight different developmental time, have been published (11, 12). Wieschauslid ovariole number. Because developmental studies on this group recessive chromosome deficiencies. Here we show that cell selec- and Szabad (11) performed a quantitative germ-line clonal tion takes place in mosaic germ-line populations. This phenomenon analysis, initiating clones from blastoderm through pupalhave stages. thus far been limited, future work could take advantage of Thanks to Dorothea Godt, the Hartl Lab, Jessica Cande, and Tassos represents a level of selection that precedes and conditions sub- By using the chorion phenotype of the mutation fs(1)K10thisas clade a as an opportunity to study the developmental basis for Pavlopoulos for kindly sharing reagents, Adam Bahrami for advice on sequent zygotic selection by affecting the genes available in the marker, they measured clone size in the number of eggsovariole laid with number variation across shorter evolutionary time scales. statistics, Michelle Ang for performing preliminary experiments that gametic population. the K10 phenotype. Perrimon (12) carried out mosaic analyses of BIOLOGY
DEVELOPMENTAL contributed to Figure S4, Tripti Gupta and members of the Extavour the dominant female sterile mutations ovoD1, ovoD2,andovoD3, erm-line development in animal species is a complex se- initiating clones throughout larval development. Clones of re- lab for discussion of the data and manuscript. This work was partially Gquence of events involving many cellular processes and combinant wild-type homozygous cells initiated at earlyCell larval types and evolutionary change supported by funds from Harvard University. DPS is supported by a requiring large numbers of genes and gene functions (1). Often, stages in ovoD1͞ϩ females were larger (in number of ovarioles) Postgraduate Scholarship from the Natural Sciences and Engineering in both invertebrates and vertebrates, only a fraction of the than ϩ ϩ clones initiated at the same age in K10 ϩ females ͞ ͞ Evolutionary change in Drosophila wing size occurs through Research Council of Canada (NSERC). DAG is supported by an NSF progeny of initially established germ-line precursor cells, or (11). This finding led Perrimon to suggest that recombinant ϩ͞ϩ primordial germ cells (PGCs), actually contribute to the gametic gonia undergo more larval divisions than ovoD1͞ϩ germchanges cells. At in both cell number and cell size, where selective pressures predoctoral fellowship. AD was supported by a research fellowship population. The genetic requirements that determine which later larval stages, however, clones were measured in numberare proposed of to act on the size of the entire wing, rather on specific from the SROH/MCO Summer Research Program at Harvard University PGCs will contribute to the functional gonad could be affected adult progeny produced by females containing germ-linemechanisms clones. of cell proliferation or growth (Zwaan et al., 2000). Dip- as part of the Leadership Alliance Consortium. by mutation in the germ line and subsequent gonial selection. We have performed germ-line mosaic and clonal analyses mea- Clearly, which germ cells finally give rise to gametes is of utmost suring clone size in the number of ovarioles containingteran recom- wing development comprises a continuous, interlocked set of importance with respect to the genetic makeup of the progeny. binant gonia, thus directly quantifying the germ cells contribut-processes, in which proliferation, growth and patterning of all wing Germ-line development (1), which involves processes such as ing to the gametic population. Our results demonstratedisk cell cells show a high degree of coupling throughout development References specific proliferation programs, passive and active gonial migra- competition leading to selection in mosaic gonads( betweenBaena-Lopez and Garcia-Bellido, 2006; Garcia-Bellido and Garcia- tion, cell–cell contact, signaling among germ cells and with recombinant ϩ͞ϩ germ cells and neighboring cells heterozygous Aboïm, A.N., 1945. Développement embryonnainre et post-embryonnaire des gonades various somatic cell populations, cell rearrangement and inter- for several different chromosome deficiencies. Bellido, 1998; Rafel and Milan, 2008; Resino and Garcia-Bellido, normales et agamétiques de Drosophila melanogaster. Rev. Suisse Zool. 3, 53–154. calation, stem cell division, and gamete differentiation, along 2004). By contrast, in ovariole development discrete steps of prolifer- Ashburner, M., Golic, K.G., Hawley, R.S., 2005. Drosophila: A Laboratory Handbook, 2nd with basic cellular metabolism, is sure to require the expression Materials and Methods ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ation, patterning, movement and sorting by one of many distinct of a significant fraction of the genome, also active in somatic Generation of Recombinant Chromosomes. All deficiencies and Azevedo, R.B.R., French, V., Partridge, L., 2002. Temperature modulates epidermal cell development. Genetically heterogeneous, or mosaic, cell popu- hs:FLP chromosomes used (FLP12 and FLP22 on the firstovarian chro- cell types are required to produce TFs. Each step of TF forma- size in Drosophila melanogaster. J. Insect Physiol. 48, 231–237. lations may arise as a result of mutation in the germ line. In mosome) were obtained from the Bloomington stocktion center is relatively autonomous with respect to the behaviors of other Baena-Lopez, L.A., Baonza, A., Garcia-Bellido, A., 2005. The orientation of cell divisions determines the shape of Drosophila organs. Curr. Biol. 15, 1640–1644. mosaic populations in certain somatic tissues, proliferating cells (http:͞͞fly.ebi.ac.uk:7081͞). 2L recombinants were createdovarian by cell types during morphogenesis, and to global body-wide have been shown to display differential developmental success standard meiotic recombination. 3L recombinants were created Baena-Lopez, L.A., Garcia-Bellido, A., 2006. Control of growth and positional information processes of growth and patterning (Green & Extavour, unpublished by the graded vestigial expression pattern in the wing of Drosophila melanogaster. according to genotype, a phenomenon known as cellular com- by inducing mitotic recombination in the male germ line with X Proc. Natl. Acad. Sci. U. S. A. 103, 13734–13739. petition. A well known example of this is the effect of the rays. Male larvae at 70–80 h after egg-laying (h AEL)observations; were Boyle and DiNardo, 1995; Gilboa and Lehmann, 2006; Minute Barcelo, H., Stewart, M.J., 2002. Altering Drosophila S6 kinase activity is consistent with haploinsufficient mutations in cells of the wing of irradiated with 1,500–1,700 rads (300 rads͞min, 10 kV,Kerkis, 15 mA, 1931; King, 1970; Li et al., 2003; Riechmann et al., 1998). a role for S6 kinase in growth. Genesis 34, 83–85. Drosophila melanogaster (2). Mutations, which may be recessive with a 2-mm filter). inAuthor list does not always clarify contribu on to research adult organisms, may display dominant phenotypes caused by TFC behavior may thus be able to change in response to a particular Barnes, A.I., Boone, J.M., Jacobson, J., Partridge, L., Chapman, T., 2006. No extension of evolutionary pressure, without large effects on the other aspects of lifespan by ablation of germ line in Drosophila. Proc. R. Soc. Lond. B Biol. Sci. 273, haploinsufficiency at the cellular level. In some somatic mosaic Immunohistochemistry. For embryonic germ cell counts, embryos 939–947. populations, this can lead to cell behavior phenotypes that differ were collected on fruit juice plates at intervals of2handallowedovarian or general somatic development. In this context, Drosophila between wild-type homozygous and heterozygous mutant cells, Bergland, A.O., Genissel, A., Nuzhdin, S.V., Tatar, M., 2008. Quantitative trait loci affect- to develop at 25°C. Embryos were collected, fixed, and stained resulting in cell competition and selection (2, 3). Gametic ovaries provide an interesting model for addressing the role of different ing phenotypic plasticity and the allometric relationship of ovariole number and according to standard protocols. The anti-Vasa Ab was a gift (sperm) selection in Drosophila (4) and other organisms (5, 6) cell types in organ size evolution. thorax length in Drosophila melanogaster. Genetics 180, 567–582. from P. Lasko (McGill University, Montreal, Canada). Staining Pre y clear: last author is senior author fi Germhas been described, cell as well selection as germ-line and somatic in genetic negative mosaics in In summary, we have taken a developmental approach to a long- Boyle, M., DiNardo, S., 1995. Speci cation, migration and assembly of the somatic cells selection of deleterious mutations in homozygosis (7). It is not fi of the Drosophila gonad. Development 121, 1815–1825. standing question= PI; first author likely graduate student regarding the evolution of a quantitative tness Brand, A.H., Perrimon, N., 1993. Targeted gene expression as a means of altering cell known whether wild-type homozygous germ cells compete with Abbreviation: h AEL, h after egg laying. Drosophila melanogaster trait, and shed new light on the specific cell population likely to be fates and generating dominant phenotypes. Development 118, 401–415. heterozygous mutant cells in genetic mosaics. Mosaic analyses ‡ To whom reprint requests should be addressed. E-mail: [email protected]. or postdoc but is also corresponding Bremer, M., Doerge, R.W., 2010. Statistics at the Bench: A Step-by-Step Handbook for have shown that a larger fraction of zygotic lethal point†‡ muta- the target of evolutionary change in ovariole number. We hypothe- Cassandra Extavour* and Antonio Garcı´a-Bellido The publication costs of this article were defrayed in part by page charge payment. This Biologists. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. tions (67%) or deficiencies (88%) are homozygous lethal in germ article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. size that the mostauthor (*), sugges ng much higher promising candidate pathways for future investiga- Büning, J., 1994. The Insect Ovary: Ultrastructure, Previtellogenic Growth and Evolu- *Museumcells than Molecular in somatic Laboratory, (tergite) University cells (20% Museum and of 58%, Zoology, respectively; University of Cambridge,§1734 solely Downing to indicate Street, this fact. Cambridge CB2 3EJ, United Kingdom; and †Laboratorio Gene´ tica del Desarrollo, Centro de Biologı´a Molecular, Universidad Auto´ noma de Madrid, 28049 Madrid, Spain tion of species-specific genetic changes affecting ovariole number are tion. Chapman and Hall, London. pathways thatdegree of intellectual/technical control growth and cell proliferation in TFCs. These Büning, J., 1998. The ovariole: structure, type, and phylogeny. In: Harrison, F.W. (Ed.), Contributedwww.pnas.org by͞cgi Antonio͞doi͞10.1073 Garcı´a-Bellido,͞pnas.201409198 August 3, 2001 PNAS ͉ September 25, 2001 ͉ vol. 98 ͉ no. 20 ͉ 11341–11346 Microscopic Anatomy of Invertebrates. Wiley-Liss, Inc., pp. 897–932. may include cell cycle genes, long-range signaling molecules, and Heritable mutations in the germ line lead to genetically heteroge- ref. 8). Given the large number of genes likely to be required by contribu on rela ve to PI Cabrera, G.R., Godt, D., Fang, P.Y., Couderc, J.L., Laski, F.A., 2002. Expression pattern of neous, or mosaic, gonads. Many of the genes used in germ-line the germ line, and the complexity of germ-line development,organ-level proliferation and growth control pathways. Consistent Gal4 enhancer trap insertions into the bric a brac locus generated by P element re- development also play roles in somatic development [Saffman, particularly the loss of 50% of germ-line cell precursorswith early this in hypothesis, several such genes, including the insulin receptor, placement. Genesis 34, 62–65. E. E. & Lasko, P. (1999) Cell. Mol. Life Sci. 55, 1141–1163]. Mutations development (1) (see below), we undertook clonal andare mosaic contained in the Drosophila QTL that have been identified as linked Capella, I.C.S., Hartfelder, K., 1998. Juvenile hormone effect on DNA synthesis and apo- Pre y unclear: first and corresponding ptosis in caste-specific differentiation of the larval honey bee (Apis mellifera L.) in these genes may have cellular phenotypes throughout germ-line analyses to ask whether or not germ-line cells undergoto cellular inter- and intraspecies variation in this trait (Orgogozo et al., 2006; development leading to their differential elimination or survival, as ovary. J. Insect Physiol. 44, 385–391. ARTICLE competition in genetic mosaics. Celldoi:10.1038/nature10811 competition and selection author(s) are PIs, who were unlikely to have Chakir, M., Moreteau, B., Capy, P., David, J.R., 2007. Phenotypic variability of wild living and has been observed in somatic cells [Morata, G. & Ripoll, P. (1975) in the germ line have important population and evolutionaryWayne and McIntyre, 2002; Wayne et al., 2001), and insulin pathway laboratory grown Drosophila: consequences of nutritional and thermal heterogeneity Dev. Biol. 42, 211–221]. We investigate whether mutations in implications (9, 10). genes are present in some honeybee QTL linked to ovariole number dif- carried out experiments. What was the role/in growth conditions. J. Thermal Biol. 32, 1–11. heterozygosis are subject to pregametic selection in the germ line. To date, only two mosaic and clonal analyses in the germferences line, (Hunt et al., 2007). Intriguingly, differential activity of the insu- We initiated clones of wild-type homozygous cells at different Cohet, Y., David, J.R., 1978. Control of the adult reproductive potential by preimaginal concerned with proliferative parameters of germ cells over thermal conditions. Oecologia 36. stagesThe of developmentDrosophila in gonads heterozygous for melanogaster eight different lin pathwayposi on of Gibbs (last author)? What were can alter ovariole number in both D. melanogaster (Green & developmental time, have been published (11, 12). Wieschaus Cooke, J., 1975. Control of somite number during morphogenesis of a vertebrate, Xenopus recessive chromosome deficiencies. Here we show that cell selec- Extavour, unpublished observations; Richard et al., 2005; Tu and Tatar, and Szabad (11) performed a quantitative germ-line clonal laevis. Nature 254, 196–199. tion takes place in mosaic germ-line populations. This phenomenon the role(s) of middle authors? Genetic Reference Panelanalysis, initiating clones from blastoderm through pupal2003 stages.) and in honeybees (Mutti et al., 2011; Patel et al., 2007; Coyne, J.A., Rux, J., David, J.R., 1991. Genetics of morphological differences and hybrid represents a level of selection that precedes and conditions sub- By using the chorion phenotype of the mutation fs(1)K10 as a Trudy F. C. Mackay1*, Stephen Richards2*, Eric A. Stone1*, Antonio Barbadilla3*, Julien F. Ayroles1{, Dianhui Zhu2, Wolschin et al., 2011). Our work provides novel developmental and sterility between Drosophila sechellia and its relatives. Genet. Res. 57, 113–122. sequent zygotic3 selection2 by affecting the genes1 available in the4 marker, they measured5 clone size in the6 number of eggs3 laid with
So`nia Casillas {,YiHan, Michael M. Magwire , Julie M. Cridland , Mark F. Richardson , Robert R. H. Anholt , Maite Barro´n , Clarified by “author contribu ons” sec on: BIOLOGY David, J.R., 1970. Le nombre d'ovarioles chez Drosophila melanogaster: relation avec la gametic population.2 2 1 3 2 1 cell1 biological tools to test the hypotheses that these and other genes Crystal Bess , Kerstin Petra Blankenburg , Mary Anna Carbone , Davidthe CastellanoK10 phenotype., Lesley Chaboub Perrimon, Laura(12) carried Duncan out, Zeke mosaic Harris analyses, of fécondité et valeur adaptive. Arch. Zool. Exp. Gen. 111, 357–370. Mehwish Javaid2, Joy Christina Jayaseelan2, Shalini N. Jhangiani2, Katherinethe dominant W. Jordan female1, Fremiet sterile Lara mutations2, Faye LawrenceovoD1, ovo1,D2,andhaveovoD3 been, theDEVELOPMENTAL direct targets of evolutionary change leading to ovariole 2 7 5 1 8 2 2 last author is senior author = PI; first author Delpuech, J.-M., Noreteau, B., Chiche, J., Pla, E., Vouidibio, J., David, J.R., 1995. Phenotyp- Sandraerm-line L. Lee , development Pablo Librado in, Raquel animal S. species Linheiro is, a Richard complex F. Lyman se- initiating, Aaron J. clonesMackey throughout, Mala Munidasa larval, development. Donna Marie Muzny Clones, of re- Lynne Nazareth2, Irene Newsham2, Lora Perales2, Ling-Ling Pu2, Carson Qu2, Miquel Ra`mia3, Jeffrey G. Reid2, number variation. ic plasticity and reaction norms in temperate and tropical populations of Drosoph- quence of events1 involving many7 cellular processes2 and combinant1 wild-type2 homozygous cells2 initiated at early1 larval GStephanie M. Rollmann {, Julio Rozas , Nehad Saada , Lavanya Turlapati , Kim C. Worley , Yuan-Qing Wu , Akihiko Yamamoto , Supplementarylikely graduate student or postdoc; roles of materials related to this article can be found on- ila melanogaster: ovarian size and developmental temperature. Evolution 4, requiringYiming Zhu large2, Casey numbers M. Bergman of genes5, and Kevin geneAuthor's R. Thorntonfunctions4 (1).,personal David Often, Mittelmanstages copy9 & in RichardovoD1͞ A.ϩ Gibbsfemales2 were larger (in number of ovarioles) 670–675. line at doi:10.1016/j.ydbio.2011.12.017. in both invertebrates and vertebrates, only a fraction of the than ϩ͞ϩ clones initiated at the same age in K10͞ϩ femalesmiddle authors detailed. Dong, J., Feldmann, G., Huang, J., Wu, S., Zhang, N., Comerford, S.A., Gayyed, M.F., Anders, R.A., progeny of initially established germ-line precursor cells, or (11). This finding led Perrimon to suggest that recombinant ϩ͞ϩ Maitra, A., Pan, D., 2007. Elucidation of a universal size-control mechanism in Drosophila primordial germ cells (PGCs), actually contributeDevelopmental to the Biology gametic 363 (2012) 279gonia–289 undergo more larval divisions than ovoD1͞ϩ germ cells. At and mammals. Cell 130, 1120–1133. population.A major challenge The genetic of biology requirements is understanding that determine the relationship which between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determineslater our larval ability stages, to predict however, phenotypes clones werefrom measuredgenotypes in and numberAuthor of contributions Engstrom, L., Caulton, J.H., Underwood, E.M., Mahowald, A.P., 1982. Developmental le- PGCsto understand will contribute how to evolutionary the functional forcesContents gonad shape could lists available variation be affected at SciVerse withinadult ScienceDirect and progenybetween produced species. Previous by females efforts containing to dissect germ-line the clones. sions in the Agametic mutant of Drosophila melanogaster. Dev. Biol. 91, 163–170. bygenotype–phenotype mutation in the germ map line and were subsequent based on gonial incomplete selection. genotypicWe have information. performed Here, germ-line we describe mosaic and the clonalDrosophila analyses mea- Extavour, C.G., García-Bellido, A., 2001. Germ cell selection in genetic mosaics in Drosophila Clearly,melanogaster which germGenetic cells finally Reference give rise Panel toDevelopmental gametes (DGRP), is ofa communityutmost Biologysuring resource clone for size analysis in the number of population of ovarioles genomics containing and recom-CE conceived the idea for the research; AAB (initial S6K analyses), melanogaster. Proc. Natl. Acad. Sci. U. S. A. 98, 11341–11346. importancequantitative with traits. respect The to DGRP the genetic consists makeup of fully of sequenced the progeny. inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomalbinant gonia, regions thus and directly the X quantifyingchromosome, the germevidence cells for contribut-AA (hpo:RNAi analyses of TF number and TFC/TF number), DAG Fenton, T.R., Gout, I.T., 2011a. Functions and regulation of the 70kDa ribosomal S6 ki- Germ-linepositive and development negative selection, (1),journal which and homepage: involves rapid evolution processes www.elsevier.com/developmentalbiology of such the asX chromosome.ing to the Manygametic variants population. in novel Our genes, results most demonstrate at low cell nases. Int. J. Biochem. Cell Biol. 43, 47–59. specificfrequency, proliferation are associated programs, with passive quantitative and active traits gonial and migra- explain acompetition large fraction leading of theto phenotypic selection variance. in mosaic The gonads DGRP(analysis between of the bab:GAL4 expression pattern), AD (D. yakuba analy- Fenton, T.R., Gout, I.T., 2011b. Functions and regulation of the 70kDa ribosomal S6 ki- facilitates genotype–phenotype mapping using the power of Drosophila genetics. tion,Evolution cell–cell of Developmental contact, signaling Control among Mechanisms germ cells and with recombinant ϩ͞ϩ germ cells and neighboring cells heterozygoussis) and DS (all other experiments) performed experiments and col- nases. Int. J. Biochem. Cell Biol. 47–59. various somatic cell populations, cell rearrangement and inter- for several different chromosome deficiencies. lected data; CE, AAB and DS analyzed data; CE and DS wrote the Fisher, R.A., 1918. The correlation between relatives on the supposition of Mendelian calation,The roles stem of cell cell division, size and gamete cell number differentiation, in determining along ovariole number in Drosophila inheritance. Trans. R. Soc. Edinb. 52, 399–3433. Understanding how molecular variation maps to phenotypic variation of 192 inbred strains derived from a single outbred population. Thepaper with input from AA and DAG; CE obtained funding for the withforDidem quantitative basic Pelin cellular Sarikaya traits metabolism, is centrala, Abel foris A. understanding sure Belay toa require, Abha evolution,the Ahuja expression animala, AishaDGRP DortaMaterials containsb, and a representativeMethods sample of naturally segregating Forbes, A.J., Spradling, A.C., Ingham, P.W., Lin, H., 1996. The role of segment polarity c 1,2 a, genetic variation, has an ultra-fine-grained recombination map ofandDelbert a plant significant André breeding, fractionGreen and personalized II of, the Cassandra genome, medicine G. also Extavour. The active principles in⁎ somatic of Generation of Recombinant Chromosomes. research. genes during early oogenesis in Drosophila. Development 122, 3283–3294. mapping quantitative trait loci (QTLs) by linkage to, or association suitable for precise localization of causal variants,All and deficiencies has almost and development.a Genetically heterogeneous, or mosaic, cell popu- 12 22 with,Department marker of Organismic loci are and conceptually Evolutionary Biology, simple Harvard1,2. However, University, we16 Divinity have notAvenue, yet Cambridge,completehs:FLP MA 02138, euchromatic USAchromosomes sequence used information. (FLP and FLP on the first chro- lationsb Brooklyn College,may ariseCity University as a New result York, 2900 of Bedfordmutation Avenue, in Brooklyn, the NY germ 11210, line. USA In achievedc Department our of Molecular goal of and explaining Cellular Biology, genetic Harvard variation University, for 16 Divinityquantitative Avenue, Cambridge,traits MAHere, 02138,mosome) USA we describe were molecular obtained and from phenotypic the Bloomington variation in 168stock re- center mosaicin terms populations of the underlying in certain genes; somatic additive, tissues, epistatic proliferating and pleiotropic cells sequenced(http:͞͞ linesfly.ebi.ac.uk:7081 comprising Freeze͞). 2L 1.0 recombinants of the DGRP, were population created by haveeffects been as well shown as phenotypic to display plasticity differential of segregating developmental alleles; and success the genomicstandard inferences meiotic of recombination. patterns of polymorphism 3L recombinants and divergence were created accordingmoleculararticle nature, to genotype, info population a phenomenonfrequency andabstract evolutionary known as cellulardynamics com- of and their correlation with genomic features, local recombination rate causal variants. Efforts to dissect the genotype–phenotype map in andby selection inducing acting mitotic on this recombination population, genome-wide in the male germ association line with X petition. A well3,4 known example5–7 of this is the effect of the modelArticle history: organisms and humans haveAll revealed insect ovaries unexpected are composed com- of functionalmappingrays. units analyses Male called larvae ovarioles,for three at whichquantitative 70–80 contain h sequentiallyafter traits, egg-laying and developing tools facilitating (h AEL) were haploinsufficientplexities,Received for publication implicating 17Minute August many, 2011mutations novel loci,egg pervasivein chambers. cells pleiotropy,of The the number wing of and ovarioles of the varies use of between this resource. and within species. Ovariole number is an important Revised 9 December 2011 determinant of fecundity and thus affectsirradiated individual fitness. with Although 1,500–1,700Drosophila radsoogenesis (300 has rads been͞min, inten- 10 kV, 15 mA, context-dependentAccepted 10 December 2011 effects. Drosophila melanogaster (2). Mutations,sively which studied, may the genetic be recessive and cellular basis for determination of ovariole number remains unknown. Ovar- Available online 19 December 2011 Molecularwith a 2-mm variation filter). in the DGRP in adultModel organisms, organism reference may display populations dominant ofiole inbred formation phenotypes strains begins that during caused can larvalbe by development with the morphogenesis of terminal filament cells (TFCs) shared among laboratories studying diverseinto stacks phenotypes, called terminal andfilaments for (TFs).Weconstructed We induced changes the DGRP in ovariole by number collecting in Drosophila mated melanogaster females from the haploinsufficiencyKeywords: at the cellular level. In some somatic mosaic whichDrosophila environmental melanogaster conditions can be controlledby genetically and altering manipulated, cell size andRaleigh, cell numberImmunohistochemistry. North in the Carolina, TFC population, USA,For andpopulation, embryonic analyzed TFfollowed morphogenesis germ by cell 20 generations counts, in embryos populations, this can lead to cell behaviorthese ovariesphenotypes to understand that the differ cellular basis for the changes in ovariole number. Increasing TFC size contributed greatlyOvariole number facilitate efforts to dissect the genetic architecture of quan- of full-siblingwere collected inbreeding on fruit of their juice progeny. plates atWe intervals sequenced of2handallowed 168 DGRP betweentitativeReproductive traits wild-typefitness3,4. Measuring homozygous many individuals and heterozygousto higher of the ovariole same homozygous number mutant by increasing cells, lines TF number. using a Similarly, combination increasing of Illumina total TFC number and 454 led tosequencing higher ovariole technology: Fecundity number via an increase in TF number. Byto analyzing develop ovarian at morphogenesis 25°C. Embryos in another wereDrosophila collected,species fixed, we and stained resultinggenotypeCell number increases in cell competition the accuracy and of the selectionshowed estimates that TFC (2,of number 3). genotypic Gametic regulation is29 a target of the of linesevolutionary were changesequenced that affects using ovariole both number.platforms, In contrast, 129 lines have 1 according to standard protocols. The anti-Vasa Ab was a gift (sperm)valueCell size and selection the power in toDrosophila detect variants,(4) andtemperature-dependent genotypes other organisms of molecular plasticity (5, 6) in ovarioleonly Illumina number was sequence, due to changes and in 10 cell lines–cell sorting have during only 454TF morpho- sequence. We markers need only be obtained once. Wegenesis, constructed rather thanthe Drosophila changes in cell sizemapped or cellfrom number. sequence P. Lasko We have reads (McGill thus identito thefi University,edD. two melanogaster distinct Montreal,developmentalreference Canada). pro- genome, Staining hasmelanogaster been described,Genetic Reference as well as Panel germ-line (DGRP)cesses andas that such regulatesomatic a community ovariole negative number:re-calibrated establishment base of total quality TFC scores, number, and and TFC locally sorting re-aligned during TF Illumina selectionresource. Unlike of deleterious previous mutationspopulations in ofmorphogenesis. homozygosis recombinant Our inbred (7). data It suggest lines is not thatreads. the genetic Mean changes sequence underlying coverage species-speci was 21.4fic ovariole3 per number line formay Illumina fi fi derived from limited samples of genetic variation,alter the total the number DGRP of consists TFCs availablesequences to contribute and to TF formation.12.13 per This line work for provides 454 for sequences the rst time (Supplementary speci c known whether wild-type homozygousand germ quantitative cells developmental compete with tools to investigateAbbreviation: the evolution h AEL, h after of a highly egg laying. conserved reproductive structure.
1 2 © 2011 Elsevier Inc. All rights reserved. heterozygousDepartment of Genetics, mutant North Carolina cells State in University, genetic Raleigh, mosaics. North Carolina Mosaic 27695, analysesUSA. Human Genome‡To Sequencing whom reprint Center, requests Baylor College should of be Medicine, addressed. One Baylor E-mail: Plaza, [email protected]. Houston, Texas 77030 haveUSA. 3Genomics, shown Bioinformatics that a larger and Evolution fraction Group, Institut of zygotic de Biotecnologia lethal i de point Biomedicina muta- - IBB/Department of Genetics and Microbiology, Campus Universitat Auto`noma de Barcelona, 08193 Bellaterra, Spain. 4Department of Ecology and Evolutionary Biology, University of California - Irvine, Irvine, CaliforniaThe publication 92697, USA. costs5Faculty of ofthis Life article Sciences, were University defrayed of Manchester, in part by Manchester page charge M13 payment. 9PT, This tionsUK. 6Department (67%) of or Biology, deficiencies North Carolina (88%) State University, are homozygous Raleigh, North Carolina lethal 27695, in USA. germ7Moleculararticle Evolutionary must thereforeGenetics Group, be hereby Department marked of Genetics, “advertisement Faculty of” Biology, in accordance Universitat with de 18 U.S.C. Introduction 8 1979). As follicles leave the germarium, they move9 posteriorly and cellsBarcelona, than Diagonal in somatic 645, 08028 Barcelona,(tergite) Spain. cellsCenter (20% for Public and Health 58%, Genomics, respectively; University of Virginia,§1734 PO Box solely 800717, to indicate Charlottesville, this fact. Virginia 22908, USA. Virginia Bioinformatics Institute and Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061, USA. {Present addresses:continue FAS Society to develop of Fellows, to Harvard form University, mature 78 oocytes. Mt AuburnD. Street, melanogaster Cambridge, Massachusettsovaries 02138, USAAll (J.F.A.); insect Functional ovaries Comparative are composed Genomics Group, of highly Institut conserved de Biotecnologia functional i de Biomedicinaconsist - IBB, Campus of approximately Universitat Auto`noma 16 de to Barcelona, 23 ovarioles 08193 Bellaterra, (depending Spain (S.C.); on Department the of Biologicalunits called Sciences, ovarioles University (Büning, of Cincinnati, 1994 Cincinnati,). Ovariole Ohio 45221, number USA varies (S.M.R.). within strain). Ovariole number is determined during larval development www.pnas.org*andThese between authors contributed͞cgi species͞doi͞ equally10.1073 (Büning, to this͞pnas.201409198 1998; work. Markow and O'Grady, 2007; through the morphogenesis PNAS ͉ ofSeptember somatic structures 25, 2001 called͉ vol. terminal98 ͉ no.fil- 20 ͉ 11341–11346 Telonis-Scott et al., 2005). Because each ovariole produces eggs au- aments (TFs), each of which is composed of a stack of seven to ten 9FEBRUARY2012|VOL482|NATURE|173 tonomously (Extavour and García-Bellido, 2001; R' kha et al., 1997), terminal filament cells (TFCs) (Godt and Laski, 1995; King et al., ©2012 Macmillan Publishers Limited. All rights reserved the number of ovarioles is an important determinant of fecundity 1968). TFC specification begins at the second larval instar (L2; (Cohet and David, 1978; David, 1970; R' kha et al., 1997), thereby Fig. 1A), and proceeds until the onset of the pupal stage (LP; influencing evolutionary fitness (Orr, 2009). It is therefore important Fig. 1D) (Godt and Laski, 1995; Sahut-Barnola et al., 1995). TFs to understand the developmental mechanisms that regulate ovariole form during the late third larval instar (L3; Fig. 1B, C) by intercalation number. This will inform our understanding of how evolutionary of TFCs in a medial to lateral progression across the ovary (Godt and changes in these mechanisms might lead to ovariole number differ- Laski, 1995). As TF formation is completed, apical somatic cells mi- ences, and thus fitness differences, within and between species. grate posteriorly between the TFs, secreting a basement membrane Ovariole development and function are best understood in Drosophila that separates TFs from each other. The progressive posterior migra- melanogaster. Each ovariole consists of an anterior germarium and ma- tion of these apical cells encapsulates two to three germ line stem turing egg chambers, or follicles. The germarium houses germ line cells, and several early oogonia, into each forming ovariole. Finally, stem cells that divide to produce oocytes (Wieschaus and Szabad, a stack of basal stalk cells is incorporated into the posterior end of each ovariole. These stalk cells ultimately connect ovarioles to the oviduct, providing an outlet for the oocytes formed in each ovariole ⁎ Corresponding author. Fax: +1 617 496 9507. (King, 1970; King et al., 1968). Because TFs serve as beginning points E-mail address: [email protected] (C.G. Extavour). for ovariole formation, elucidating how TF number is established is
0012-1606/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2011.12.017