Synopsis. Introduction

Transcription

1 Introduction

2 Synopsis In the fruitfly Drosophila melanogaster, wings and halteres are the dorsal appendages of the second and third thoracic segments, respectively. In the third thoracic segment, wing development is suppressed by the homeotic selector gene Ultrabithorax (Ubx) to mediate haltere development. Loss of Ubx function from developing haltere discs induces haltere-to-wing transformations, whereas ectopic expression of Ubx in developing wing discs leads to wing-to-haltere transformations. The differential development of wing and haltere constitutes a good genetic system to study cell fate determination at different levels such as growth, cell shape, size and its biochemical and physiological properties. They also represent the evolutionary trend that has established the differences between fore and hind limbs in mammals including human, wing and legs in birds and fore and hind wings in insects. Homeotic genes encode proteins containing DNA-binding domains and function by regulating downstream target genes. The structure and function of these genes are highly conserved across a wide range of animals including human. Although considerable information is available about the molecular and biochemical nature of homeotic genes, comparatively little is known regarding the mechanism/s that are used to generate segmental diversity. We have employed two complementary approaches to experimentally address the questions on the mechanisms by which Ubx specifies haltere fate. First, examination of genetic control of morphological events during haltere development. The second approach is to identify modulators and targets ofhomeotic gene function by enhancer-trap method. Introduction 1.1 Appendage Development: a common theme Development from fertilized eggs to adults and execution of many life activities requires the proper functioning of adult appendages. Tremendous morphological diversity exists among animal appendages, both wit~in a single organism and between, species. Such variations facilitate the many functions of appendages, such as sensing their environments, swimming, feeding, crawling, walking and flying. Drosophila antenna, for example, has both auditory and olfactory functions, while the leg is used primarily for locomotion. Nonetheless, loss-of-function mutations in a The 1

3 variety of Drosophila genes lead to antenna to leg or leg to antenna transformations (Balkaschina, 1929; Strohl, 1981; Strohl, 1982; Sunkel and Whittle, 1987; Casares and Mann, 1998; Pai eta/., 1998), supporting the idea that the antenna and leg are homologous structures. The current understanding of the molecular basis of limb development in arthropods is based almost exclusively on studies in Drosophila melanogaster with a few studies in Crustaceans, a group of hemimetabolous insects such as grasshoppers, whose limbs arise directly from the body wall. However, Drosophila and other holometabolous insects possess a rather derived mode of development in which the limbs, and much of the adult body, arise from small, specialized populations of cells in the larvae known as imaginal discs. In Drosophila, limb discs are specified along the lateral body wall of the embryo, then invaginate from the ectoderm and eventually evert as legs/wings during pupation. The discs are subdivided into compartments by discrete expression domains of multiple gene products, which interact to pattern the limb. The vertebrate limbs are similar to fruit fly antennae/legs in many ways, but at the same time the two are markedly different from each other. Insect wings are analogous to both bird and bat wings, although the two are not drawn from historically homologous structures. Vertebrate limbs develop as an outgrowth of the embryonic body wall, consisting of mesenchyme derived from the somites and the somatic portion of the lateral plate mesoderm, surrounded by an ectodermal jacket. In the fully developed limb, the muscle, cartilage, connective tissue, skin and skin derivatives (in addition to nerves, circulatory elements etc) are integrated into a complex functional structure. Although human (vertebrate) limbs bear no resemblance to their fruit fly counterparts various studies have suggested that homologous genes are responsible for the development of these seemingly divergent structures. The Dll/Dlx gene family was originally identified in Drosophila, where it is required for proximal-distal axis formation in the legs and antennae. Members of the Dll/Dlx gene family encode transcription factors: DNA-binding proteins that regulate the expression of other genes. Dll/Dlx family members are involved in the limb formation of a variety of vertebrate species. The Dlx5/6 knockout mice displayed defects in bone, inner ear and craniofacial development. But most interestingly, the mice were also born with the claw-like limb deformities characteristic of split hand/split foot malformations (SHFM) (Robledo eta/, 2002). 2

4 A principal feature of the organization of Drosophila appendage is the distinction between the distal part (the appendage proper) that contains the full activity of Hh!Dpp/Wg morphogens and the proximal part that contains the hthlexd (homeodomain containing protein) activity. Extensive work comparing Drosophila appendages with mouse and chicken limbs has shown a striking degree of conservation of this configuration: the vertebrate homologues of hth and exd; meis and Pbxl, respectively; are functional in the proximal but not in the distal limb. Even the sub-cellular regulation of exd by hth in Drosophila is also observed for Pbxl and meis in mouse limb development. In both Drosophila and chicken, ectopic expression of hth or meis in the distal component blocks limb development (Abu-Shaar and Mann, 1998; Rieckhofet. al., 1997; Mercaderetal.l999). The distal part of Drosophila appendages, which is the focus of the present study, requires wingless (wg) function for proper growth and patterning. This member of a secreted glycoprotein family acts as a long-range morphogen activating target genes in a threshold dependent manner. Homologs of Wg in vertebrates, known as Wnts express in the mouse/chick limb buds. Their over-expression in the flanks of chick embryos using a viral vector causes activation offgfs, which control both limb initiation and the formation of apical ectodermal ridge (AER), that acts as the "organizer" of limb development. Furthermore, there is evidence that Hh signaling is required in mouse limbs: limbs fail to develop properly in Sonic hedgehog (Shh) knockouts. The mode of action of Shh in chick limb development also resembles the situation in Drosophila: it acts as a short-range activator of bone morphogenetic protein (BMP2), a vertebrate homologue of Decapentaplegic (DPP), and much of the effect of SHH is mediated by BMP2 and other BMPs (TableH) (for revtew see Graham et. al., 1999; Jhonson et. al, 1997 or 3

5 A Dorso N entral Axis specification f '.,...,...,...,...,...,..., -~ "''"' D -~~~hn l n:.;~::i!::... j I Droso~hi~aj[ii_to Apterous J. Wg and_!''!..~?~. _ J!.. ~~-~e-~~~~.~ ~~~-~~- t~-~-~~... J B Anterior/Posterior Axis Specification -~~~i=~~-~!:~~~=--~.~~~....1 ~ Posterior L... _I Expression 1...,... --~ I Drosophila: i I IL...,... _, "',....,, II Vertebrate: i hedgehog (hh) Decapentaplegic(BMP) sonic hedgehog (shh) BMP-2 Experimental Embryology Evidence anterior misexpression of both hh I & dpp results in muror image ' duplication ' ~'' "',, ~ ~ -- _..,.,_~, "'"' -- '... -,.-... _,... '... _'.. '"' - -~.. ~.-.. '. - ~ ~-' ~-1 0 antenor shh 0 0! mtsexpresslon r results in mirror image duplicate, l not BMP-2 Key difference: Drosophila wing/limb is formed primarily from ectoderm while vertebrate limb initiates in the meso/ectoderm. This means hh is expressed in ectoderm while its homologue shh is expressed in lateral mesoderm. C Distal/Proximal Axis Specification lr=d=-= =-- =h=~=l :;;;.;;;:: I D- -~- II , form limbs in novel locations,! rosop l a.: -----~-~~ J ~~~i?~~ -~~~~~?-~ista~l~b_s. j Vertebrate: jjd~~-(distal-less!ike_~rotein)j _mutation.= no distalli~b j Additionally, FGF is expressed in Ascidians, a vertebrate sister group, along with Dll. This is similar to expression to vertebrates. Tablel.l Comparison of gene expression in the developing discs of Drosophila and the vertebrate limb buds. The molecular homology in the evolution of limbs is astonishing. In both arthropods and vertebrates the three major axes of limb development seem to employ homologous genes. A brief outline of their expression domains and mutant and/or over-expression phenotypes is shown above. 4

6 Together these observations indicate that there is a universal mechanism to form a limb, shared by all animal groups. Studies on the mechanisms of appendage development are of interest, not only because of the general conservation of the process, but also because of their importance for understanding the general mechanisms controlling growth and proliferation in higher animals. A number of congenital limb development abnormalities are linked/mapped to genes known to have a conserved role in appendage development across species. In addition, mutations of human homologues of exd and hth (Pbxl and meis) cause leukemias in humans and mutant forms of genes of the Wnt and Hh signaling pathways are involved in tumorigenesis. Fully understanding appendage development in Drosophila, therefore, has implications that go beyond developmental and evolutionary biology, to biomedicine (Table 1.2). Tahlel. 2 A. i Vertebrate/! Drosophila 1 -- g~~e~ - - J. HoxD13 : (AbdB) ' r-~~ -~--"- ~.,...-,_,...-~- 1 Dlx5-6 (D/1) t I Diseases 1 Synpolydactyly II l f_ ' i. f Spht hand-foot I syndrome Associated Defects in Vertebrates Split hand split foot malformations with nystagmus an oncogene, implicated in various tumors an oncogene, carcinomas T~~phoc~i~-~~~~~j~ of. - - I B-cell I.... Appears to be important m tumour progression especially in colon cancers Overexpression causes basal cell carcinomas in mice skin, Table 1.2 shows a few representatives of congenital defects(a) and tissue specific tumor(b) associated with malfunctioning of genes required for appendage development across species 5

7 1.2 Specifying appendage identity Two major genetic components seem to act in combination for specifying appendage identity. They are, (a) a segment specific property provided by the Hox/ Homeotic (HOM/HOX) class of genes and (b) tissue specific patterning and growth properties provided by various morphogens Figl.l The body of Drosophila indicating the trunk and the different appendages, colour coded based on their position on the dorsal or ventral surface and the... ~ omeotic genes expressed in each of those segments : in addition to wings legs and halteres, antennae mouthparts and annalia can also be considered as Adopted and modified from Morata, segment specific appendages Homeotic genes HOM/HOX genes play a causal role in the regionalization of the body plan of all bilaterally symmetric animals (de Rosa et.al, 1999). HOM/HOX genes encode highly conserved homeodomain transcription factors and are instrumental in conferring segmental identities along the primary body axis (reviewed by McGinnis and Krumlauf, 1992). Similar to the specification of cephalic and trunk segments, appendage development is also specified by HOM/HOX genes. The Drosophila legs provide the best example: the identity of the first leg is specified by the homeotic gene Sex combs reduced (Scr), the second leg is specified by Antennapedia (Antp) and the third leg by Ultrabithorax (Ubx). In the absence of homeotic gene function, all segments develop the same 'ground' pattern, a mixture of thoracic and cephalic pattern elements; no morphological diversity is generated along the AlP body axis. The HOX/HOM genes were first characterized in the fruitfly, where mutations that cause ectopic expression of Antp gene product result in antenna to leg transformations suggesting that master regulatory genes act as selectors for regional 6

8 identities. Eight linked Antennapaedia-class homeobox genes make up the homeotic complex and are arranged in two clusters. The Antennapaedia complex (ANT-C) comprising more anteriorly expressed five genes that defme the head and the anterior thorax and the bithorax complex (BX-C) constituting three genes expressed in the third thoracic segment onwards. The only homeotic gene that does not fall into one of these clusters is Caudal (cad), which is responsible for analia development. The clustered organization of the homeotic genes shows a relationship to their mode of expression. The expression domains along the primary axis of the developing embryos reflect the location of individual genes within the clusters, such that the more proximal (to the centromere) located genes have more anterior expression domains. This orderly relationship has been termed spatial colinearity, and in invertebrates there is also a temporal colinearity, such that the most proximal genes have the earliest onsets of expression, with the sequential activation of distal genes. The clustered organization is believed to be vital for the establishment of colinear expression, although the mechanisms for this process are still obscure (reviewed by Duboule, 1998). Extensive functional studies in flies and mice have established that the basic functions of HOX/HOM genes are well conserved. Mutational analyses in Drosophila have established that gain-of-function mutations tend to cause more posteriorizing homeotic transformations, where the identity of a segment anterior to the normal expression domain is altered to resemble the more posterior segment (Lewis, 1978); Antp (dominant, gain-of-function) mutations being a classical example. Conversely, loss-of-function mutations cause anteriorizing transformations. Homeotic transformation of T3 to T2 in Drosophila caused by mutations in Ubx locus is the most cited example of such phenotypes. In the tetrapod vertebrates, particularly mouse, mis-expression and null mutant analyses have revealed that similar rules apply, For example, the knockout of Hoxc-8, a gene expressed in the thoracic region, produces anteriorizing homeotic transformations in the vertebral column of the mouse (Le Moulleic et a!, 1992; reviewed by Bruke, 2000). Over-expression of Hoxd-11 in the chick limb bud using retroviral vectors leads to homeotic transformations of anterior foot digits into more posterior ones (Morgan et a!, 1994). Thus (although studies are incomplete), the general conclusion that has emerged from the various functional studies is that the A p axis is specified by similar arrays of homeobox genes in fruit flies and mammals. 7

9 Supporting this, the mammalian Hox proteins have been shown to be functional in transgenic flies and can, remarkably alleviate the effect of homeotic mutations (McGinnis et a/, 1990). Figl.2 Anterior Pooterior DI'OIC()hllfl Hti!K d.jsmr A.nocr;b;ll Htu riu"-far latj ~ =r Dfl1 Scr ftl Nr4' UDX fltlrl-11 NJ/1-IJ -o II!;...a.-a--o-- I l l t l 1 HoJcl HDX2 IC-3 Hoic4 Hox5 HcJIItj (GII.trlr8!) HaK7(~Jto rj i I r I r I I I Iii.. HOXA Bt B2 83 B4 85 EM B7 B I-IOXB -CIII Ill o-o o- E C4 ('._<; C8 C8 C'O C t1 C 12 C t3 :z; ~Q)(C FfOXO D1 ru D,O Dt D1J Anteriol' Fig1.2. Hox/Hom gene organization and domains of expression m Drosophila melanogaster and vertebrates. Ahypothetical ancestral condition is also shown. Colours represent the most closely related genes. Current models suggest that the variations in Hox gene numbers between species reflect an evolutionary history characterized by two kinds of duplication events: tandem duplications that gave rise to a single cluster organization as in flies and whole cluster duplications resulting in four clusters ofhox e:enes in vertebrates. There are few differences between flies and mammals in expression and function of HOM/HOX genes. For example, a transgenic mouse line expressing 8

10 Hoxc-6 (an anteriorly expressed HOX gene) induces to the formation of extra pairs of ribs in lumber vertebrae (Jegalian & DeRobertis, 1992). Such anteriorizing phenotypes in the context of gain-of-function are not observed in Drosophila. The situation in mammals is rendered more complex in by the existence of not one but four HOX/HOM clusters. In addition, the complexity of the morphogenesis of the anterior head regions make true homeotic transformations difficult to detect, which could be the reason for the failure to detect such transformations in earlier knockout studies. In spite of the knowledge of the molecular and biochemical nature of these proteins (homeodomain-containing transcription factors), even in tractable systems like Drosophila, the entire range of targets are not known and it is not clear how these genes function as developmental switches Morphogens Morphogens are "form generating" substances, whose configuration within a tissue "prefigures" the pattern (Meinhardt, 1983; Lawrence and Struhl, 1996). These substances set up an extracellular concentration gradient. The concentration gradient orchestrates a coherent set of cellular behaviors that will eventually result in the proportionate growth of an organ, including the finest details. For example, different scalar concentrations may specify the type of cells and their relative position within the field; the slope of the gradient may be correlated to the degree of growth of the intervening cells, and the direction of the gradient with respect to the compartment may determine polarity. All this information (cell fate, position, polarity and growth) is in principle specified by the induction of target genes by the morphogen that are activated in a concentration dependent manner. Organizers were first identified in vertebrate embryos. Their long-range signaling activities were postulated to be mediated by secreted signaling molecules acting as morphogens or via relay of secondary signals (recently reviewed in Weinstein and Hemmati-Brivanlou, 1999; Niehr, 1999). Subsequently the same concepts have been applied to other systems to explain how positional information and differentiation programs are imparted: other localized organizing regions were identified, as well as the molecules mediating their functions (reviewed for example in Lawrence and Struhl, 1996; Neumann and Cohen, 1997). Some of the processes where morphogens have been shown to be important in vertebrates include: patterning 9

11 of the early embryonic axes (mostly studied in Xenopus, reviewed in McDowell and Gurdon, 1999; Dale and Wardle, 1999), patterning of the DN axis of the neural tube (reviewed in Briscoe and Ericson, 1999) and patterning of the limbs (mostly studied in the chick and mouse embryo, reviewed in Tickle, 1999). Here I present the paradigm case of Activin in mesoderm patterning of the frog Xenopus. In the early Xenopus embryo, cells with prospective endodermal and ectodermal fate can be identified as the 'vegetal pole' and the 'animal pole' respectively. Signals emanating from the vegetal pole induce mesodermal tissue in the overlying animal pole cells. Activin, a member of the TGF-~ family of secreted signaling proteins, has been purified from early Xenopus embryos and has been shown to induce and pattern mesodermal tissues when applied to animal caps ( explants of animal pole tissue). Several lines of evidence show that Activin can signal directly to distant cells and can elicit different responses in cells as a function of its local concentration. When dissociated animal cap cells are exposed to different concentrations of Activin, cells respond by activating different target genes (shown for example in Green et a/., 1992). When intact animal cap tissue is cultured in contact with beads soaked in Activin, target genes are expressed in distinct domains: distant cells activate genes that are turned on by low levels of Activin and cells close to the beads activate genes turned on by high levels of Activin (McDowell et a/., 1997). Cell movement or cell division do not seem to account for the long-range effect of Activin, as shown by lineage tracing of responding cells. Moreover, expression of dominant negative forms of Activin-receptors interferes with the activation of target genes in distant cells (McDowell et a/., 1997), arguing in favour of direct long-range signaling by Activin. In Drosophila, morphogens have been proposed to act in early embryonic axial patterning, in embryonic segmentation and in limb patterning (reviewed in Morata, 2002). Two classical examples have been discussed below. Bicoid was the first protein proposed to act as a morphogen in flies. bicoid function is required for anterior patterning of the oocyte and consequently of the early embryo; its activity was shown to be localized to the anterior of the embryo by cytoplasmic transplantation experiments. Increasing the dosage of bicoid expands anterior structures at the expense of the posterior ones (reviewed by Driever, 1993). Bicoid mrna is tightly localized at the anterior pole of the embryo; this localization 10

12 1s critical for its function. The protein then diffuses posteriorly, forming a concentration gradient (Struhl et.al, 1989). Bicoid has been shown to directly control the activity of downstream genes in a concentration dependent manner, m cooperation with Hunchback, whose distribution is also uneven in the embryo (Hulskamp eta/., 1990). Both proteins are transcription factors, but can diffuse in the early syncytial Drosophila embryo to reach distant nuclei. Dorsal is another transcription factor. It is required for dorso-ventral patterning of the early embryo. It is distributed throughout the embryo, but its activity is graded, being higher in ventral regions. This is achieved by the graded levels of transport of the protein into the nucleus (reviewed in Chasan & Anderson 1993). 1.3 The Drosophila model system The life cycle of Drosophila melanogaster Drosophila melonogaster is a holometabolous insect: the fertilized egg develops into an adult fly through different stages and moults (Fig 1.3), including a characteristic Fig1.3 dramatic transformation between the larval and adult stages (metamorphosis). The Drosophila life cycle includes an embryonic stage (that lasts about 24 hours at 25 C), three larval stages also called instars (24, 24 and 72 hours long), the pupal stage Drosophtla Life Cycle (about 5 days long), and the adult stage. Although adult tissues and organs are completely different from the larval ones, the general body plan is the same in all stages: in carotlna Biological SUDDIV Gomoanu ~1!'.. ~ lkoll l 11...,..., """''" c... _.. ol', J t ~ (._..., 1 _ ~1'1d!ti'Ctlr, particular along the AlP axis it is easy to identify distinct head and tail regions and eleven intermediate repeating segmental units: three thoracic segments and eight abdominal Development of the Drosophila wing The following section reviews the literature regarding the embryological and larval development of the wings in Drosophila -- an appendage which shares a common 11

13 developmental origin as the haltere and leg and utilizes the same set of developmental pathways to establish positional information in the three axes (anterior-posterior, dorso-ventral and proximo-diatal) but undergoes distinct developmental processes to function in flight. Recent studies find remarkable conservation of the genes expressed during appendage formation, suggesting that the basic mechanisms for appendage development are similar be it wing, leg or haltere. Drosophila limbs (legs, wings, halteres, antennae, mouth parts) derive from structures called imaginal discs. Imaginal discs begin as small clusters of cells, which are set aside during embryogenesis. These cells proliferate during larval development to form folded, single layer, epithelial sacs. Each imaginal disc gives rise to a separate structure so there is a disc for each leg, wing, haltere, eye and antenna. The cells in the disc stop dividing just prior to differentiation, which begins at the time of pupation. As the discs begin to differentiate, they evert (or unfold) and fuse to form a continuous adult head and thoracic cuticle. The Drosophila wing has been primarily used as the model system for the research in this thesis and hence merits a brief description. Adult morphology The adult Drosophila wing is a relatively simple epithelial sheet that is demarcated by specific landmark veins, both in a latitudinal (anterior to posterior) and longitudinal (proximal to distal) direction (Milan and Cohen, 2000). 3 Fig1.4 Drawing of Drosophila wing indicating vein labels (Sturtevant eta/., 1995). Vein size and direction are relatively constant and are seen in most species (Milan and Cohen, 2000). Longitudinal veins are labeled L1-L6, beginning from the veins most anterior, as seen in figure

14 Molecular basis of appendage development The cells that give rise to the thoracic imaginal disc primordia are initially part of the embryonic ectoderm. The primordia are established straddling the parasegmental boundaries in each of the three thoracic segments. They are specified in response to two different secreted signals: W g and Dpp. W g is expressed as a stripe just anterior to the parasegmental boundary. Dpp is expressed in a lateral stripe running perpendicular to the cells expressing W g. The cells in the vicinity of the intersection between the W g and Dpp stripes are exposed to both secreted signals and become specified as imaginal disc cells. This was demonstrated by correlating the spatial pattern of genes expressed in imaginal discs (such as Distal-less) with the expression patterns of W g and Dpp and the effects of wg and dpp mutations on the formation of imaginal discs. Fig1.5 Specification of imaginal primordial in early Drosophila embryos. Imaginal disc progenitors m the embryo are drawn in blue. Experimentally they can be visualized by escargot or Distal-less expression in the three thoracic segments of stage 15 embryos. The primordial are specified at the intersection of W g (grey) and Dpp (green) stripes. A subset of the cells in this cluster is derived from en expressmg cells, which confer posterior identity to the developing disc. A er Cohen The newly formed primordium inherits the AlP and DN axis position information from the ectoderm by virtue of differential gene expression and grows as an independent entity. Early in Drosophila larval development, the wing primordium separates from the imaginal cells that will form the future leg structures. 13

15 First lns.tar 0 Second IMtar v Late Third lnstar 0 margut v I Adult 0 v A p (Adopted from Blair, 1995) Fig 1.6 Gene expression and growth of wing compartments during development. Compartments are established and maintained by differential gene expression and all cells strictly respect compartment boundaries. The earliest identified primordium already has posterior identity marked by the expression of engrailed in red. En expression is clonally inherited and maintained throughout development. A subset of the cells during second instar express Apterous, which confers dorsal identity and give rise to the dorsal surface of the adult wing Wing Morphogens: compartment boundaries as sources of positional information During wing development, first the wmg imaginal discs are divided into four compartments by the activities of certain selector genes, following which the morphogens are expressed in the compartment boundaries. The anterior-posterior decision is controlled by the expression of the homeobox gene engrailed (en). Engrailed expression results in cells becoming posterior in identity. The later subdivision between dorsal and ventral cells is controlled by the expression of the homeobox gene apterous (ap) in dorsal cells. en and ap are called selector genes because they are necessary and sufficient for posterior versus anterior and dorsal versus ventral fate, respectively. These initial differences are used to establish the localized expression of secreted molecules that further pattern the AlP and DN axes. 14

16 Figl. 7 Compartment boundaries as sources of morpho gens. The selector genes en and ap (in grey) specify the posterior and ventral identity respectively. The cells in the interface are influenced by the gene expressions in both compartments. As a result long-range morpho gens, Dpp and W g are expressed in a narrow stripe (thick black line in the figure) along the AlP and DN boundaries respectively. (Modified from Brook et a/., 1996) Along the AlP axis, a number of genes determine the positioning of vein and intervein regions. Posterior cells that express En interact with the anterior cells that do not express En leading to the expression of the secreted factor Dpp at the boundary between them. The expression of En directly patterns the location of the fourth longitudinal wing vein (la). Expression of En in the posterior compartment initiates production of Hedgehog (Hh), which diffuses to the anterior compartment cells (Milan and Cohen, 2000; See Figl.8). In addition to directly patterning the third longitudinal wing vein (L3) at the anterior-most edge of its expression, Hedgehog works through Smoothened and Patched receptors to stimulate production of Dpp. Dpp regulates production of the Spalt, which plays a role in placement of the second (L2) and fifth (L5) longitudinal wing veins (Milan and Cohen, 2000). 15

17 Fig1.8 Expression of Engrailed, Hh, Dpp, and Sal LS (from Milan and Cohen, 2000) Growth and patterning during wmg development are also mediated by signaling from its dorso-ventral (DN) organizer. Interactions between dorsal and ventral cells of the wing pouch set up the organizer by activating Notch (N) in the DN boundary (Diaz-Benjumea and Cohen, 1993, 1995; Williams eta/ 1994; Irvine and Wieschaus, 1995; Kim eta/., 1995; de Celis eta/., 1996). N, in tum, activates Wingless (Wg), Cut (Ct) and Vestigial (Vg) in the DN boundary (Couso eta/., 1995; Kim eta/., 1995; Rulifson and Blair, 1995; Kim eta/., 1996; Neumann and Cohen, 1996). Ct keeps Delta (Dl) and Serrate (Ser) up regulated in the adjacent ventral and dorsal cells, respectively (de Celis and Bray, 1997). This ensures sustained N activation in the DN boundary and its simultaneous inactivated in the adjacent non DN cells. Wg is known to diffuse to non-dn cells from the DN boundary and acts as a morphogen (Zecca eta/., 1996; Neumann and Cohen, 1997). High levels ofwg are required for activating Achaete (Ac), whereas moderate levels are enough to activate Distal-less (Dll) and low-levels to activate Vg (Neumann and Cohen, 1997). 1.4 Tools for the study of gene function in flies In Drosophila, tools are available for the in vivo analysis of gene function. In loss of function (LOF) genetic analyses, the protein or its activity is eliminated and in gain of function (GOF) experiments, a protein is expressed elsewhere or at higher levels. These approaches have been refmed and have become more sophisticated. Detailed explanations about standard techniques and more recent developments can be found in laboratory manuals (de Robertis CSHL Protocols, 1999) In this section I intend to briefly introduce some of these techniques with emphasis on the ones that have been extensively used in the course of the experimental work discussed in this thesis. 16

18 1.4.1 Loss of function of a given gene can be analyzed: i) in homozygous mutant animals ii) iii) in mosaic mutant animals by expressing the dominant negative version of a gene product In the first case analysis of the phenotype is possible if the mutation is viable, or it is otherwise limited to the stages before lethality occurs. By looking at the phenotype of the mutant one can ask whether the activity of that gene is required for the formation of the given tissue, organ or structure within an organ, etc. in the second case 'mosaic animals' are generated where only some cells are homozygous for a given mutation in an otherwise heterozygous organism. Apart from overcoming potential early lethality, generating clones of mutant cells that develop in heterozygous background can be used to address many biological questions including autonomy of gene action. Fig 1.9 A m / A :n I ~ x rrutont Am Am ~"-[8 : Am/ m: cellular mar~er A: mutation wild type + + I + + after Xu and Rubin, 1993) The generation of mosaics is based on events of somatic recombination between homologous chromosomes, an event that takes place after DNA duplication but before chromatid separation. Genetic mosatcs were originally generated by X-ray induced mitotic recombination. The rate of recombination is very low though and the cells in the clones can be distinguished from surrounding wild type cells only in very specific cases. The method that is now most commonly used is based on the FLP/FRT site-specific recombination system, schematized in the adjoining figure. FLP is a yeast site-specific recomomase mar recogmses a target sequence called FR T and mediates recombination between two of these sites. Depending on the orientation of the FRT sites and whether they are present on the same DNA strand or in trans, the recombination event will have different outcomes including excision, inversion or strand exchange. For the purpose of making clones homozygous for a previously identified mutation and distinguishirlg them from surrounding cells, homologous chromosomes carrying the FRT site in identical positions, each associated with a more distally located mutation or marker are required. Exchange 17

19 between homologous chromosomes leads to the production of one daughter cell homozygous for the mutation but lacking the marker, and one daughter cell (twin) carrying two copies of the marker but homozygous for the wild type allele. The surrounding cells are still heterozygous for both the mutation and the marker. When cells proliferate, a clone of mutant cells lacking the marker and a neighbouring clone of wild type cells containing two copies of the marker will be generated and can be easily distinguished from the surrounding heterozygous tissues (schematic). The word clone hereafter refers to the progeny of a given cell; in the context of mosaic analysis it is used to refer to the (marked) cells that can be tracked back to one of the two daughter cells generated after the recombination event; by defmition all cells in a clone are related by lineage and share the same genetic material. Clonal analysis of limb development has lead to many conclusions about how the founder cells in the limb primordia give rise to mature imaginal discs. Cells in a clone stay together, implying that there is no cell mixing in development. This observation led to the conclusion that cells are confined to territories on the disc called "compartments". (iii) One can generate loss-of-function phenotypes by expressing a edna encoding a dominant negative variant of the gene of interest. By regulating the expression of dominant negative form of the gene product, one could generate tissue- and developmental stage-specific phenotypes. It provides an alternate means for mosaic analysis Gain of function analysis is based on: i) mis-expression of a gene. In terms of space and time- known as ectopic expression and or levels (over-expression) ii) expression of a constitutively active form of a protein (e.g. a constitutively active receptor that signals independent of the presence of the ligand). Ectopic expression of a given gene can be achieved in flies by different means. One by regulatory mutations of genes that result in aberrant patterns of expression without altering the gene function. Such mutations of Ubx have been utilized in Chapter3. Although these mutations have proven to be extremely powerful, one depends completely on luck to fmd one and for most genes they are not available. 18

20 The second method is by expressing the gene of interest in a transgenic system. Here the edna of interest would be cloned downstream to an inducible or tissue-specific promoter. Figl.lO The Gal4- UAS system for tissue specific gene expression. A P-element either carrying a GAIA transactivator downstream of a X tissue specific promoter or a ~AS.1t~ged to minimal promoter but inserted in the ' gene of mterest vicinity of a tissue specific enhancer will express in the pattern of the promoter or enhancer trapped. When ~.. nr L. endogenous expression in the wing disc of a hypothetical protein is crossed to UAS GFP will mimic the endogenous regulation of the enhancer/ promoter. The shown in black. An enhancer trap GAIA mediated expression of GFP marker is represented in green. Instead of the marker if a gene of interest is present downstream ofuas, it will be expressed in the pattern identical to the hypothetical protein. The binary GAIA-UAS system, in which the target gene and the transcriptional activator are separated in two distinct transgenic lines (Figl.l 0) (Brand and Perrimon, 1993), has been extensively used in the current work. This overcomes problems related to toxicity of a given transgene. In this method, a transgene in which the edna of interest is coupled to the upstream activating sequences (UAS) of the yeast transcription factor GAIA. Since GAIA is a heterologous protein the gene is normally kept silent. The other transgenic line contains the GAIA transcription factor coupled to a tissue specific promoter. Only when a cross combines the two elements in the progeny, the desired target gene is expressed in a pattern defmed by the GAIA (schematized in Figl.lO) Regulation of gene expression In addition to analysis of gene function, in vivo analysis of gene expression is also possible. The enhancer trap technique is widely used for studying tissue-specific gene expression in Drosophila. Classically, a P element containing the /acz reporter gene under a minimal promoter is mobilized throughout the genome using a transposase 19

21 source. The P-element construct is sensitive to the regulatory elements of the region that it inserts. As a result, detection of ~-galactosidase activity reflects the tissue type and timing of the endogenous gene activity (O'Kane and Gehring 1987). A large number of enhancer-trap lines with distinct staining patterns have been generated in this manner (e.g., Bellen et al., 1989; Bier et al. 1989; Wilson et al. 1989; Klambt and Goodman 1991 ). Analyses of such enhancer trap using molecular techniques have resulted in the cloning of many new genes. A major improvement to the enhancer trap employs the GAIA transcriptional activator (Brand and Perrimon 1993). In this system, the GAIA gene is included in the enhancer trap P-element construct and expression is under control of the local regulatory region just like the expression of the lacz gene in earlier stable enhancer traps. The GAIA system is far more flexible than earlier lacz reporter systems. GAIA enhancer traps can be used to drive the expression of any other gene placed downstream of the upstream activation sequence (UAS; Brand and Perrimon 1993)(Fig1.10). 1.5 Objectives of the current study: The current work has been undertaken to understand the mechanisms by which homeotic gene Ubx modifies wing fate into haltere fate. The approach has been by identifying novel targets of Ubx function in the developing haltere. This approach had the advantage of identifying new gene/s required for wing development, whose expression is modified by Ubx to specify haltere development. Thus, the objectives of the current study were, 1. To study the effect of clonal removal of Ubx from distinct regions of haltere disc. 2. To identify target/s of Ubx function by enhancer-trap approach. 3. To study the mechanism of Ubx-mediated regulation of the identified target, which would result in differential expression of the latter in wing and haltere discs. 4. Genetic and molecular characterization of the identified target to understand its role in appendage development. 5. Identification of interacting genes to position the identified gene m the contexts of developmental and genetic pathways. 20