Paper No. : 11 Module : 6 Development Team Principal Investigator: Prof. Neeta Sehgal Head, Department of Zoology, University of Delhi Paper Coordinator: Prof. Namita Agrawal Department of Zoology, University of Delhi Content Writer: Dr. Om Prakash Sri Venkateswara College, University of Delhi Content Reviewer: Prof. Shweta Saran Jawaharlal Nehru University, Delhi 1
Description of Module Subject Name Paper Name ; Zool 011 Module Name/Title Module ID Keywords Genetics of 6; Axis-specification, Drosophila, Morphogen, Homeotic selector gene, Dorsal protein, Syncytial blastoderm, Bicoid Contents 1. Learning Outcomes 2. Introduction 3. Anterior Posterior Axis Formation 4. Anterior Posterior Body Plan 4.1 Bicoid as the Anterior Morphogen 4.2 Nanos mrna Localization in the Posterior Pole of the Oocyte 4.3 Terminal Gene torso specify the Anterior and Posterior Extremities 4.4 Gap Genes 4.5 Pair Rule Genes 4.6 Segment Polarity Genes 4.7 Homeotic Selector Genes 5. Dorsal-Ventral Patterning in the Oocyte 6. Dorsal and Ventral Pattern in the Embryo 7. Effect of Dorsal Protein in Embryo 7.1 Mutation in Dorsal and Ventral Maternal Genes 8. Summary 2
1. Learning Outcomes After studying this module, you will come to learn about: Fertilization in drosophila Anterior posterior axis formation Maternal genes and zygotic genes Effect of mutations in the zygotic genes Gap, pair-rule, segment polarity and homeotic selector genes Dorsal ventral patterning in the oocyte Effect of dorsal protein in embryo 2. Introduction The fruit fly Drosophila melanogaster is a small dipteran insect measuring approx. 3 mm as an adult. The embryonic development of this insect occurs inside an egg which hatches as a larva. Drosophila is easy to breed, hardy, abundant, can withstand diverse conditions. Cellular membranes do not form in Drosophila until after the thirteenth nuclear division. In Drosophila fertilization occurs when sperm enters an egg which is already activated. The Drosophila egg is sausage- shaped which has micropyle, at the future anterior end of the embryo. Sperm enters the egg at micropyle end, which allows entry of one sperm at a time. As the sperm enters the egg fertilization takes place and the zygote undergoes a series of rapid mitotic division (karyokinesis) but without cytokinesis. After 12 nuclear divisions the embryo is called a syncytial blastoderm, it is equivalent to the blastula or blastoderm stage of the other animals. After thirteenth division blastoderm becomes fully cellular, by the invagination of cell membranes. The cellular blastoderm will give rise to all the future tissues. Gastrulation starts at about 3 hours after fertilization. During gastrulation, endodermal and mesodermal tissues move to their future positions inside the embryo, leaving the ectoderm as the outer layer. The general body plan of Drosophila is same in the embryo, the larva, and the adult possessing a distinct head end, repeating segmental units and a distinct tail. In the repeating segmental units three segments form thorax, while another eight segments form the abdomen. The first thoracic segment has legs; the second thoracic segment has legs and wings and the third thoracic segment has legs and halters (balancing organs). In early 1990 s a powerful forward genetics approach was used for identification of most of the genes involved in shaping the larval and adult forms of Drosophila. Flies were randomly mutagenized and screening is done for the mutations that disrupted the normal formation of the body. The genes responsible for the mutant phenotype were cloned and are characterized according to their expression patterns and their functions. Molecular events critical for Drosophila embryogenesis occur during oogenesis and the single female germ cell- oogonium is the descendant for a single oocyte. The oogonium, before oogenesis begins 3
divides four times with incomplete cytokinesis, to give rise to 16 interconnected cells: 15 nurse cells and the single oocyte precursor. Numerous mrnas are made in the nurse cells which are transported on microtubules through the cellular interconnections into the enlarging oocyte. 3. Anterior-Posterior Axis Formation The follicular epithelium surrounding the developing oocyte is broken by two signals which involve the same gene, gurken organized by the oocyte nucleus. In the oocyte nucleus, gurken gene is localized between the nucleus and the cell membrane and is translated into Gurken protein. The time at which oocyte nucleus is very close to the posterior tip, Gurken signal which results in the posteriorization of the follicle cells is received by these follicle cells through a receptor protein encoded by the torpedo gene (Figure 3.1). Figure 3.1: Specification of anterior-posterior axis during oogenesis http://vignette1.wikia.nocookie.net/mmg-233-2013-geneticsgenomics/images/b/b6/gurken.jpg/revision/latest/scale-to-width/396?cb=20131104212132 4. Anterior-Posterior Body Pain Maternal effect genes produce messenger RNAs that are placed in different regions of the egg and encode transcriptional and translational regulatory proteins which after diffusion through the syncytial blastoderm activate or repress the expression of certain zygotic genes. Maternal genes are expressed by the mother but not by the embryo and during oogenesis they are expressed in the tissues of the ovary. Zygotic genes in contrast are expressed during the development of the embryo in the nuclei of the embryo itself (Figure 4.1). The zygotic genes express in a sequential manner that establishes the body plan along the anteroposterior axis (Table 4.1). 4
Figure 4.1: Sequential expression of different sets of genes for establishment of the body plan along the antero-posterior axis http://www.mun.ca/biology/desmid/brian/biol3530/devo_02/ch02f08.jpg Gap genes are the first such zygotic genes to be expressed and encode transcriptional factors. These are called so because mutations in gap genes cause gaps in the segmentation pattern. Pair rule genes divide the embryo into periodic units and their transcription is regulated by differing combinations and concentrations of the gap gene proteins. Their transcription results in a striped pattern of seven transverse bands perpendicular to the anterior-posterior axis. Segment polarity genes are activated by the pair-rule proteins. The mrna and protein products of the segment polarity genes divide the embryo into 14-segment-wide units and establish the periodicity of the embryo. Homeotic selector genes which determine the developmental fate of each segment are regulated by the interaction of the protein products of the gap, pair-rule, and segment polarity genes at the same time. 5
Table 4.1: Genes affecting segmentation pattern in Drosophila Category Gap genes Pair-rule genes Segment polarity genes Homeotic selector genes Gene name Kruppel (kr) hunchback (hb) giant (gt) knirps (kni) hunckebein (hkb) empty spiracles (ems) orthodenticle (otd) buttonhead (btd) hairy (h) runt (run) even-skipped (eve) fushitarazu (ftz) odd-paired (opa) odd- skipped (odd) sloppy- paired (slp) paired (prd) engrailed (en) wingless (wg) hedgehog (hh) cubitusinterruptusd (cid) fused (fu) armadillo (arm) patched (ptc) pangolin (pan) gooseberry (gsb) labial (lab) Antennapedia (Antp) sex combs reduced (scr) deformed (dfd) proboscipedia (pb) Ultrabithorax (Ubx) abdominal A (abd A) abdominal B (abd B) There are three classes of maternal genes which specify the antero-posterior axis (Figure 4.2). 6
Figure 4.2: Effects of mutations in the maternal gene system http://www.mun.ca/biology/desmid/brian/biol3530/devo_02/ch02f09.jpg Bicoid gene is anterior class gene and mutations in these genes lead to reduction or loss of head and thoracic structures, even in some cases their replacement with posterior structures. Mutation in nanos which is the gene of the posterior group cause the loss of abdominal regions, results in a smaller than normal larva. Mutations in the terminal class genes such as torso causes loss of both acron and telson. 4.1. Bicoid as the Anterior Morphogen Bicoid mrna is located at the anterior end in the unfertilized egg and after fertilization it is translated and the Bicoid protein diffuses from the anterior end. It forms a concentration gradient along the antero-posterior axis which provides the positional information required for further patterning along the axis. When bicoid-deficient embryos are injected with bicoid mrna, the point of injection forms the head structures and developed normal anterior-posterior polarity. Figure 4.3: Representation of bicoid gene as a morphogen responsible for head structures in drosophila http://courses.biology.utah.edu/bastiani/3230/db%20lecture/lectures/flymaternal/slide26.jpg http://courses.biology.utah.edu/bastiani/3230/db%20lecture/lectures/flymaternal/slide27.jpg 7
Moreover, if the bicoid message is injected at any location in an embryo that location become the head. When a normal anterior cytoplasm is injected into the center of the fertilized bicoid mutant egg, head structures develop at the site of injection and the adjacent segments become thoracic segments and sets up a mirror image body pattern at the site of injection (Figure 4.3). Bicoid is a transcription factor and acts as a morphogen which forms a gradient with the high point at the anterior end of the egg. As it diffuses through the embryo, it breaks down and this breakdown is necessary to establish the antero-posterior concentration gradient. 4.2. Nanos mrna Localization in the Posterior Pole of the Oocyte Oskar which is one of the maternal posterior group genes localize nanos mrna at the extreme posterior pole of the unfertilized egg and specify the posterior germplasm in the egg which gives rise to the germ cells (cells that will give rise to sperm and eggs). Nanos after translation give a concentration gradient of Nanos protein with the highest level at the posterior end of the embryo. Nanos suppress translation of maternal mrna of hunchback gene by binding to a complex of hunchback mrna and the protein encoded by the posterior group gene pumilio. Subsequently a clear gradient of zygotically expressed hunchback protein is established which acts as a morphogen for the next stage of patterning (Figure 4.4). Figure 4.4: Anterior-posterior patterning model by maternal effect genes http://10e.devbio.com/images/ch09/550a.jpeg 4.3. Terminal Gene torso specify the Anterior and Posterior Extremities Torso proteins generate the unsegmented extremities of the anterior-posterior axis: the acron (the terminal portion of the head that includes the brain) and the telson (tail) (Figure 4.5). The torso mrna which is synthesized by the ovarian cells, deposited in the oocyte and translated after fertilization. Torso must normally be activated only at the ends of the egg because the gain-offunction mutation of torso converts the entire anterior half of the embryo into an acron and the entire posterior half into a telson. 8
Figure 4.5: Specification of the terminal regions of the embryo by Torso receptor protein http://mol-biol4masters.masters.grkraj.org/html/developmental_biology1-drosophila_files/image048.jpg 4.4. Gapgenes Gap genes define regional differences and are activated or repressed by the maternal effect genes. These genes are expressed in the anterior-posterior domain (Figure 4.6). Examples of gap genes are hunchback, kruppel, and knirps, giant, tailless etc. Figure 4.6: Expression of gap genes in the early drosophila embryo http://www.stolaf.edu/people/colee/studentprojects/drosophila/developmental/pictures/gap%20genes1%20- %20not%20for%20use.jpg Certain threshold level of of Bicoid enhances the level of Hunchback in the anterior region of the embryo. Hunchback protein is a transcription factor and acts as a morphogen to which other gap genes respond. Kruppel gene: High level of Bicoid and low level of Hunchback induces its expression, but high concentration of Hunchback represses expression of kruppel gene. Due to this the expression of kruppel gene is restricted at the centre of the embryo (Figure 4.7). 9
Figure 4.7: Specification of kruppel gene activity by Hunchback protein http://scienceblogs.com/pharyngula/wp-content/blogs.dir/470/files/2012/04/i- 4c8a116afee4eda51928f554265d5732-kruppel.jpg When high concentration of Hunchback is present kruppel is repressed and it is also repressed when Hunchback is present below the threshold concentration. In mutants where bicoid is not present, zygotic hunchback gene expression is also absent, so maternal Hunchback is present at the anterior end of the embryo in low level, so expression of kruppel gene is restricted to the anterior end. 4.5. Pair-Rule Genes The primary pair-rule genes, hairy, even-skipped, and runt are expressed in seven stripes where each stripe corresponds to every second parasegment. There are pair-rule genes which define odd numbered parasegments (e.g, even-skipped), whereas others define even-numbered parasegments (e.g, fushitarazu). Bicoid and hunchback proteins activates the even-skipped gene, though the boundaries of the stripe are defined by kruppel and giant proteins by repressing even-skipped at posterior and anterior edge of the stripe respectively. In contrast, fushitarazu are not regulated by the gap genes, but they may depend on the prior expression of primary pair-rule genes such as even-skipped and hairy. 4.6. Segment Polarity Genes Segment polarity genes are activated in response to pair-rule gene expression and have two important functions to perform: They reinforce the parasegmental periodicity established by the earlier transcription factors. Establishing cell-to-cell signaling and cell fates within each parasegment. Segmentation genes acts in a cellular environment as during pair-rule gene expression the blastoderm becomes cellularized. One of the segmentation genes is engrailed which has a key role in segmentation and is activated by the pair rule genes. It is activated in cells that have high levels of the even-skipped, fushitarazu, or paired transcription factors. Transcription of engrailed gene marks the anterior compartment of each parasegment and the posterior compartment of a segment. Mutation in engrailed gene cause transformation of posterior compartment in clones of wing cells whereas in the minute clones, the anterior and posterior parts of the segment are not confined and there is no compartment boundary (Figure 4.8). 10
Figure 4.8: Demonstration of the boundary between between anterior and posterior compartments in the wing by marked cell clones http://www.mun.ca/biology/desmid/brian/biol3530/db_02/fig2_34.jpg An intercellular signaling circuit sets up between the adjacent cells and delimits the boundary between the parasegments. Three segmentation genes are involved in this circuit. These are wingless, hedgehog and engrailed, which are expressed in restricted domains within the parasegment. The secreted signal hedgehog is expressed in the cells expressing engrailed. Subsequently in the row of cells immediately anterior to the engrailed-expressing cells the expression of the signal protein is activated. And this secreted wingless protein gives a signal that feeds back over the parasegment boundary to maintain hedgehog and engrailed expression. This signal stabilizes and maintains the compartment boundary. 4.7. Homeotic Selector Genes Specification of each segment is defined by homeotic selector genes. In Drosophila two homeotic gene clusters are present named as bithorax complex and Antennapedia complex and the chromosome region containing these complexes are referred to as the homeotic complex (Hom-C) (Figure 4.9). 11
Figure 4.9: Expression of homeotic selector gene in Drosophila http://test.classconnection.s3.amazonaws.com/446/flashcards/416446/jpg/14-2.jpg Bithorax complex: these are responsible for diversification of posterior segments. It contains three genes, Ultrabithorax: mutation in this gene causes transformation of haltares into wings, i.e. fly results in four wings (Figure 4.10). Figure 4.10: Mutations in the ultrabithorax produce a four-winged fly http://qph.is.quoracdn.net/main-qimg-2caf89330953c03958aada0ec43cbf0f?convert_to_webp=true Abdominal A (abd A) and Abdominal B (abd B): segmental identities of abdominal segment is regulated by these two genes. Antennapedia complex: It is a complex of five genes, labial (lab) deformed (dfd) Antennapedia (Antp) sex combs reduced (scr) proboscipedia (pb) labial and deformed genes are involved head segment specification, whereas Antp and scr are responsible for thoracic segment specification. Gene pb is active only in the adult flies, due to its mutation transformation of labial palp of mouth into legs occurs. 12
Due to mutation in the gene Antennapedia homeosis occurs, i.e. antenna transforms into legs in head sockets. 5. Dorsal-Ventral Patterning in the Oocyte The movement of oocyte nucleus occurs towards anterior dorsal position with increase in volume of oocyte. Message of gurken gene is localized in crescent between the oocyte nucleus and the oocyte cell membrane. The product of gurken gene is Gurken protein which forms an anterior-posterior gradient along the dorsal surface of oocyte. Gurken gene is present only in oocyte, whereas torpedo is active only in the somatic follicule cells. Follicle cells contain Torpedo receptor protein which receives Gurken signals. The Torpedo signal inhibits the expression of pipe gene, because of which Pipe protein is produced only in the ventral follicle cells. Figure 5.1: Dorsal-ventral patterning in Drosophila http://www.dls.ym.edu.tw/lesson3/dros.htm During formation of ventral region of oocyte with the help Pipe protein which is only activated in the ventral cells modifies an unknown factor X. Nudal protein and factor X splits Gd (Gastrulation defective) and Snake protein, which in turn activates Spatzle protein. Spatzle protein binds to Toll receptor protein (which is present throughout embryonic cell membrane). The dorsal cell receives Toll signal and separates the Cactus protein from the Dorsal protein due to which Dorsal protein gets translocated into the nuclei which leads to ventralization of cells (Figure 5.1). Note: Deficiency of maternal gene gurken or torpedo causes ventralization of the embryo. 6. Dorsal and Ventral Pattern in the Embryo Product of the gene dorsal is involved in the Dorsal-ventral patterning of the embryo. Mother gene dorsal produces Dorsal protein which which is placed in the oocyte by nurse cells. Dorsal protein is also known as ventral morphogen. In the syncytial blastoderm of early Drosophila embryo Dorsal protein is present everywhere, but in late embryo Dorsal protein is translocated only in the ventral part. During ventral specification Dorsal enters the nucleus and represses the genes 13
responsible for the dorsal specification, so the embryo becomes specified as ventral cell. But if this does not happen all the cells of embryo becomes specified as dorsal cell. Note: If the dorsal gene or Dorsal protein is absent, all the ventral cells becomes dorsalized. During ventral patterning the product of dorsal gene is translated in Dorsal protein which remains complexed with Cactus protein in cytoplasm. When the Spatzle binds to Toll protein it gets activated and in turn it activates a protein kinase called Pelle. Pelle remains in bounded form with Tube protein. Once activated Pelle phosphorylates Cactus, which is then degraded and in turn Dorsal protein becomes free to enter the nucleus. This process establishes Dorsal protein gradient in the nucleus of embryo. Note: In the ventral cell nuclei the gradient of Dorsal protein is highest. 7. Effect of Dorsal Protein in Embryo As the Dorsal protein enters the nuclei, it defines the dorsal-ventral axis on the basis of gene expression. At this stage Dorsal protein also distinguishes the germ layers and specifies the ventral most cells as prospective mesoderm. In the nucleus Dorsal protein acts as transcriptional activator of ventralizing genes (snail, twist, and rhomboid) and a transcriptional repressor of the dorsalizing (decapentapelagic, zerknullt and tolloid) in the ventral region. twist and snail are activate where the internuclear concentration of Dorsal is highest. These genes are responsible for development of cells as mesoderm and for gastrulation. rhomboid gene is activated where low level of Dorsal protein is present, and it acts as future neuroectoderm. In more ventral region they are repressed by Snail protein. decapentapelagic, tolloid and zerknullt are repressed by Dorsal proteins (which is present mainly in the ventral region), so are expressed in the dorsal region of the embryo (where there is no Dorsal protein in the nuclei). Zerknullt gene is expressed in the dorsal most region of the embryo and forms aminoserosa. decapentapelagic gene is involved in the specification of dorsal part of embryo where no Dorsal protein is present and also specifies dorsal ectoderm. dpp is a member of TGF-β family of cytokines. Decapentapelagic is homolog of bone morphogenetic protein-4 (BMP-4) present in vertebrates (Figure 7.1). Figure 7.1: Nuclear gradient of Dorsal protein http://mol-biol4masters.masters.grkraj.org/html/developmental_biology1-drosophila.htm 14
7.1. Mutation in Dorso-Ventral Maternal Gene Dorsalized embryo: When Dorsal protein is excluded uniformly from the nuclei, decapentapelagic is no longer repressed and this leads to its expression everywhere. Whereas twist and snail are no longer expressed in dorsalized embryos, as they require high level of internuclear Dorsal protein level. Ventralized embryo: When Dorsal protein is present is present at high concentration in all nuclei, twist and snail areal so expressed everywhere, whereas decapentapelagic is not present at all. 8. Summary The embryonic development of Drosophila melanogaster occurs inside an egg which hatches as a larva. In drosophila fertilization occurs as the sperm enters the egg at micropyle end and then the zygote undergoes a series of rapid mitotic division. By the invagination of cell membrane blastoderm becomes fully cellular and gives rise to all the future tissues. During gastrulation (which starts at about 3 hours after fertilization), endodermal and mesodermal tissues move to their future positions inside the embryo, leaving the ectoderm as the outer layer. The general body plan of drosophila possesses a distinct head end, repeating segmental units and a distinct tail. Maternal genes are expressed by the mother and produce messenger RNA that encodes transcriptional and translational regulatory proteins. Zygotic genes are expressed during the development of the embryo in the nuclei of the embryo itself and establish the body plan along the antero-posterior axis. Mutations in Bicoid and nanos gene cause loss of head and thoracic structure and loss of abdominal regions respectively. Mutations in the terminal class gene such as torso causes loss of both acron and telson. Bicoid act as a morphogen and forms a concentration gradient along the antero-posterior axis which provides the positional information required for further patterning along the axis. The genes responsible for the anterior portion of the fly are activated by the Bicoid and Hunchback whereas Caudal activates genes responsible for posterior development. The gap genes are regulated by the concentrations of the maternal effect gene proteins. In turn the gap genes regulate the piar-rule genes. The pair-rule genes activate engrailed and wingless expression, where the engrailed expressing cells form the anterior boundary of each parasegment. There are two complexes for homeotic selector genes in drosophila. Together, these regions are called Hom-C, the homeotic gene complex. The entry of Dorsal protein into the nucleus regulates the dorsal-ventral polarity. It forms a gradient as it enters the various nuclie: nuclie at the most ventral surface incorporate the most Dorsal protein and become mesoderm; those more lateral become neurogenic ectoderm. 15