Home Biol 4241 Luria-Delbruck 1943 Hershey-Chase 1952 Meselson-Stahl 1958 Garapin et al. 1978 McClintock 1953 King-Wilson 1975 Sanger et al. 1977 Rothberg et al. 2011 Jeffreys et al. 1985 Bacterial Genetics Mutational Dissection Gene Regulation Cell Number: Cancer Sex Determination Complex Pattern Formation NextGen Sequencing Bioinformatics Hamer et al. 1993 "Genetic Architecture of Development" Three processes in development of early embryo: 1) Differentiation: variable gene activity, leading to synthesis of different gene products 2) Pattern Formation (regional specification): cells acquire positional information morphogen: molecule that conveys positional information and promotes developmental changes, distributed alon gradients in embryo a) cell organization along axes. We will mainly consider: hgf 1) Anterior/Posterior Axis 2) Dorsal-Ventral Axis tre b) segments are formed gfd overview of segmentation in Drosophila 3) Morphogenesis: each segment develops unique morphological features Click here for guiding questions in study of specific genes with roles in development Drosophila Somatic Anterior-Posterior Axis (A-P Axis) Formation Established via localized determinant anchored to microtubules In the syncital embryo (coenocyte) Positional information initially established via concentration gradients of two proteins Example of an intracellular cell signalling pathway BCD protein Encoded by (maternal) bicoid (bcd) gene Steep gradient of bicoid gene product in early embryo High point at anterior pole Transcription factor for GAP genes HB-M protein Encoded by hunchback (hb) gene Shallower, longer gradient More evenly distributed in coenocyte still more at anterior end Concentration gradients depend on diffusion from the localized determinant mrna is tethered to anterior and posterior ends of microtubules of coenocyte o -ve ends located at anterior pole o +ve ends located at posterior pole Results in localized concentrations of proteins at the poles BCD gradient - bcd mrna is tethered to -ve ends of microtubules (anterior end of coenocyte)
o bcd is packaged in maternal oocyte - translation of BCD proteins begins midway through early nuclear divisions of coenocyte - BCD is a transcription factor o contains signals to become localized to nuclei - therefore more BCD in nuclei nearer anterior end --> concentration gradient HB-M gradient - produced by post-transcriptional regulation by hb-m mrna o maternal in origin - hb-m mrna uniformly distributed throughout coenocyte - translation blocked by NOS protein o translational repressor o encoded by nanos (nos) gene hb-m mrna and NOS interaction - nos mrna also maternal in origin - nos mrna anchored to + ends of microtubules (posterior end) - translational of nos begins midway through coenocyte stage of embryogenesis - since nos inhibits hb-m translation, less HB-M protein at posterior end (gradient) **gradient is shallower than bcd Message > localiza on of mrna s within a cell dependent on anchoring mrna s to microtubules within cells => Microtubule Tethering How do they know bcd is essential for determination of the A-P axis? => bcd mutant experiments Drosophila Dorsal-Ventral (D-V) Axis Formation Example of an extracellular protein signalling pathway Positional information depends on proteins secreted from a localized set of cells Secreted proteinds diffuse in extracellular space and form a concentration gradient of ligands Ligands bind receptors on target cells Activates a concentration dependent receptor signal transduction system - signals passed down the sequence of events required to determine the D-V axis are concentration dependent DL protein Transcription factor Encoded by dorsal (dl) gene Two forms: 1. Active Transportation Factor located in nucleus 2. Inactive protein located in cytoplasm o inactivated by binding CACT protein - encoded by cactus (cact) gene DL Concentration Gradient
(DL) determines cell fate with regards to the D-V axis DL mrna, DL protein initially uniformly distributed throughout oocyte/embryo Late in the coenocyte stage, large gradient of active DL - Forms at ventral midpoint Message - Two Classes of Positional Information Zygotic Genes in Pattern Formation of the Early Embryo Maternal mrnas are the beginning of pattern formation, but after fertilization these maternal mrna encode transcription factors for zyotic genes (Segmentation Genes) zygotic genes include GAP genes, Pair-Rule genes, Segment Polarity Genes, and Homeotic Genes Cardinal genes (gap genes in Drosophila) first zygotic genes to respond to maternal mrnas Mutation/inactivity of these genes results in the deletion of groups of parasegments (i.e. gap FIG16-24 examples: Hunchback, Kruppel and knirps, which are acted on by bcd in Drosophila. => first responders to bcd gradient And each oth Drosophila has three classes of maternal proteins - includes about 50 proteins that set up the A-P axis alone - including bcd How many classes of zygotic? (fig 16-23) - very complex interactions with other zygotic and maternal - follows Hierarchy Why the complexity? - - you need 3 mouth, 3 thorax, 8 abdomen segments, etc. (messing up the order isn`t usually recommended... So they did) Each parasegment is unique and has defined boundaries EN/HH and many others aid in compartmentalization (see below). Gap genes act on: - pair-rule genes - segment polarity genes - homeotic genes Could you look at it step-wise? MAT -> PR -> SG -> SEL In terms of activation and hierarchy? - NO Whoa... how bad could it be? Viewer discretion is advised "The complexity of regulatory elements of the primary pair-rule genes turns an asymmetric (gap gene) expression into a repeating one." (Ch.16, p.537) Pair rule genes regulate transcription of segment polarity genes, which define individaul AP rows of cells within each segment examples: Eve (even-skipped) and Ftz (Fushi-tarazu)
Act in the alternating parasegments a mutation in a pair-rule gene will delete the corresponding parts of pattern in every other segment EN, HH and WG responsible for defining clear boundaries in parasegments (developmental units) "in a maintainable loop" (See below) Selector genes (Homeotic genes) 1894 geneticist William Bateson complied list of genetic abnormalities Bateson called these abnormalities "homeosis" ("homoio" means "same" in Greek) Homeotic = cause the change from one body part to another Homeotic genes are expressed in segments, and control segment identity Homeotic genes have a 180 bp sequence [homeobox] which codes for the protein homeodomain homeodomain binds to major groove in DNA homeotic genes encode transcription factors for the genes (realizator genes) that homeodomain proteins realizator genes and produce the morphological characteristics of each segment Homeotic genes include Hox genes In Drosophila, Hox Genes are arranged on chromosome 3 in two clusters: BX-C and ANT-C Homeotic Transformation Additional aspects of Pattern Formation Once the fate of the cell lineage has been established, it must be remembered. Maintenance is accomplished by intracellular and intercellular positive feedback loops. Two types of positive-feedback loops are regularly established: a) Transcription factors binding to an enhancer of it s own gene: Ensures that more of a particular protein will continue to be produced E.g. SxL splicing in the female development pathway b) Positive-feedback mechanisms requiring cell-cell communication: Each adjacent cell sends out a different signal, activating receptors, signal transduction pathways and transcription factors (TF) expression in another cell called mutual positive-feedback e.g. adjacent cells expressing WG and EN proteins This diagram shows both positive-feedback mechanisms - very early in embryonic development, all cells have the same fate and do not differentiate until their fate is determined. Cell fate determination can be broken down into two states: 1) Specified (or committed) determination - cell type is not yet determined - any fate that is determined can be reversed or changed to a different fate 2) Determined determination: - cell has a pre-determined fate - fate cannot be reversed or changed once it is decided For a group of cells to develop into an organ or tissue, cells must be committed in appropriate numbers and locations to the required fate Cell-cell interactions guarantee that proper commitments are made We will discuss two types of interactions: 1) Inductive interaction 2) Lateral inhibition Both interactions will be applied to the model organism Caenorhabditis elegans (C. elegans)
in the hypoderm (body wall) of the worm, several cells have the ability to become part of the worm's vulva outside the reproductive tract) all of these cells can adopt any required role; called an equivalence group one cell must become the primary vulva cell, two others become the secondary cells, and others become contributing to the overall structure of the vulva But, how do the cells know which becomes primary, secondary or tertiary cells? Two different methods: 1) Inductive Interaction One cell induces the developmental commitment of a neighbouring cell The use of an anchor cell, which lies beneath the cells of the equivalence group Anchor cell secretes a ligand that binds to the receptor tyrosine kinases present on each of the group cells The cell that receives the highest concentration of the signal activates the signal transduction pathway at a s activate transcription factors that establish this cell as the primary vulva cell Thus, the anchor cell induces a cell to adopt the primary vulva fate 2) Lateral Inhibition One cell inhibits the developmental commitment of a neighbouring cell Once the fate of the primary vulva cell is determined, this cell sends out a different signal to its This signal inhibits the immediate neighbours from interpreting the anchor cell s initial signal Prevents the neighbouring cells from becoming primary vulva cells, establishing these cells as th vulva cells Thus, the paracrine signals inhibit the lateral cells, causing them to adopt a different fate The remaining cells of the equivalence group become the tertiary vulva cells These processes call all be summarized in this diagram. Developmental Pathways are Composed of Plug-and-Play Modules Few gene products take part in pattern formation that contributes to only one developmental de Bits and pieces of different pathways are combined to determine different outcomes Different components of one developmental pathway may be found to contribute to another, being mixed and matched Can be a matter of different inputs or different outputs We will use the dpp (decapentaplegic) gene as an example of different inputs: The decapentaplegic gene is responsible for the patterning of the 15 imaginal discs (tissues that become the different parts of the body) in Drosophila melanogaster. During embryonic development of the dorsal-ventral (D-V) axis, the dpp gene is regulated by the DL (dorsal) t factor. During visceral mesoderm development, it is regulated by the Ubx (ultrabithorax) transcription factor. We will use the maintenance of EN-HH segement-polarity genes as an example of different outputs: The EN-HH (engrailed-hedgehog) segment polarity genes activate the genes that encodes the WG (wingless cells During larval wing development, the EN-HH component activates a different signalling protein (DPP) in cells that adjacently express EN-HH The DPP signal transduction pathway leads to activation of different transcription factors in dorsal-ventral patterning, wing development and visceral mesoderm development. Just to sum up this concept.
Summary of Hox Genes in Insects 1. In Drosophila, HOM-C includes two clusters arranged on the third chromosome (ANT-C and Bithorax-C) 2. In more primitive insects (e.g. flour beetle, Tribolium castanum) only one cluster of Hox genes arranged on the same chromosome Many different body plans in animal kingdom: higher taxa (phyla, classes) often characterized by unique body plans... At the genetic level, what processes might be responsible for such diversity in body plans? e.g. exon shuffling? gene duplications? gene conversion? gene or larger chromosomal delations? point mutations within genes? Compare Hox genes in insects and mammals... Hox Genes in Mammals Mammals have four paralogous clusters of homeotic genes: Hox A, Hox B, Hox C and Hox D In both mammals and insects, hox genes are arranged on the chromosome in the order that they are spatially exp à Genes at left-hand of complex are transcribed near the interior. As you move right on the chromosome, AND genes are transcribed progressively towards the posterior. à Genes in any location in mammals are more similar to genes in other clusters within mammals and insects in the same location on the chromosome relative to other genes in the same cluster in other chromosomal loc Evidence for sequence similarity from DNA-DNA hybridization studies under moderately stringent conditions. Also, evidence that Hox genes have some function in insects and mammals: Gene Knockout Experiments Conclusions: Developmental strategies in animals are evolved from an ancient common ancestor, and have been relatively conserved throughout the course of evolution. Raises the possibility that gene duplications followed by sporadic gene deletions have been important in the origin of a variety of structural forms in the animal kingdom See Phylogeny based on Hox Genes and Hox Gene Differences among animal taxa Steve Carr (instructor: scarr@mun.ca) *** Linda Lait (TA: ll1583@mun.ca) Dept of Biology, Memorial University of Newfoundland, St John's NL A1B3X9