Embryology PDF Lecture Notes

Gene Networks and the Early Body Plan: Fly Lecture 15 Why Fly???! Genome been sequenced Genome is relatively simple and small Three autosomes Two sex chromosomes 15,700 genes (compared to 20,400 for human) Most genes present and conserved - directly comparable to other anima...

Gene Networks and the Early Body Plan: Fly Lecture 15 Why Fly???! Genome been sequenced Genome is relatively simple and small Three autosomes Two sex chromosomes 15,700 genes (compared to 20,400 for human) Most genes present and conserved - directly comparable to other animals Long tradition of mutant stocks on hand (chemical- and radiation-induced) Genes can be easily knocked-out, knocked-in, and conditionally expressed Many mutant lines are available for study genetic map Fly is cheap to rear been hasextensively laid out Has a short generation time (breeding genetics is possible), and is fecund Females can lay up to 100 eggs every 20 days In 10 days the flies fully mature Development is external and can be easily monitored Amenable to manipulation as embryos (injections etc) Not as subject to ethics approval Some drawbacks been around for genes have long time a Develop very differently from us – hard to make direct mechanistic comparisons Drosophila are Diptera: possess only two wings (second pair reduced to balancing organs – halteres) Not typical of most arthropods – compared to others, relatively recent derivation Their developmental pattern is a bit unusual too – they produce long versus - short germ band embryos. Flies develop Segments all at once Short germ band embryos develop a little more like us insofar as segments arise incrementally In Drosophila , segments arise pretty much simultaneously Short vs Long Germ Band Development segments arise Segments arise incrementally simultaneously Compared to Mice… another genetic model Genome 85% similar to that of humans Genes and gene networks highly conserved Developmental process quite similar to our own Genome has been characterized (fully mapped and sequenced, In-bred lines AND cell lines are available Multiple mutant lines are available for study Genes can be knocked in, knocked out, mutagenized (CRISPR etc) Mice can be bred to do genetic crosses 1 litter consists of 5-6 babies. A female can birth between five and 10 litters per year Gestation is 19-21 days 4-7 weeks to become sexually mature, but rarely very viable breeders  develop best in Mouse and mammalian model drawbacks Mero > - to maintain expensive Generation time longer than that of Drosophila: typically about 8 months - Although IVF is similar to humans, most of development occurs in utero – - later stage embryos are more or less unobservable and difficult to work on. Must be housed according to strict guidelines (highly regulated) - Once born, mice much be fed specific foods at specific times Levels of stress/pain must be within ethical protocols Housing is labor-intensive, and very expensive Most institutions require a centralized and well funded facility - Enter/Exit air through tunnel ↳ careful to bring in disease not mouse Spread diseases ↳ alter ↳ genes hepafiller Compared to fish? easy eggs to collect slower to produce than mice Compared to mice, cheap to rear Genome has been fully characterized, and multiple mutant lines are available Genes and gene networks highly conserved Can be genetically manipulated using mutagens, CRISPR, morpholinos, or injected transcripts. Embryos develop externally, are optically transparent, and are large enough to do some surgery on Standard of care required is a little less than for mammals, but that is changing May eggs easily available, easily fertilized Embryos easy to collect and observe Fish – some drawbacks of good lines lot not a cell - not easy petri dish at lower ↳ maintained temperatures Generation times longer than that of Drosophila, but still fast: 2-4 months - comparable to mouse Modular housing units are fairly expensive, but cheaper to maintain than for mice - Few tissue culture models - Compared to Amphibians Spread semen over eggs -> all fertilized within minutes (large quantities) Cheap to collect and rear Single fertilization can produce hundreds of embryos that develop as a cohort in synchrony. Genome has been fully characterized, and multiple mutant lines are available Can be genetically manipulated using CRISPR, morpholinos, or injected genes or transcripts. Embryos develop externally, and are larger than fish embryos, so they are accessible to more surgical interventions Developmental processes and gene regulatory networks similar to our own Standard of care required is a little less than for mammals, but that is changing. conserved gene networks Amphibian down-side I only cell line available Generation time is too long to be practical for breeding purposes 1-2 years - d to hard Few tissue culture models transfer to Culture Studies Advantages of Chicks known genetics not as well Cheap to obtain in quantity - Easy to obtain developmentally synchronized embryos - Fast embryological development – 21 days - Reproductive maturity at 4-5 months Amniotes – develop very much as we do, but flattened out - Easy to observe - Easy to surgically manipulate - Lineage tracking easy – chick/quail, vital dye - Genome characterized, beginning to be as amenable to manipulation as other - systems A hen will lay an egg every 25-27 hours or so, this cycle goes on every day - Chicks: the down side Expensive to house in large number - Slower reproductive cycle - Genetic manipulations not as advanced as in mammalian, fish, and insect systems Up-Coming Themes for Embryo Mapping tiers act on Hierarchies – genes and tissues ( cross talk eachother on turn)to between levels that between goes both directions Modularity – genes, gene networks, tissue and organogenesis Conservation – genes, networks, and processes  Fly (Drosophila) Developmental Genetic Hierarchies post-gastrulation is happens as egg M 1. Maternal Effect Genes being made G 2. Gap Genes > - big gaps mutale = P 3. Pair Rule Genes > - affect alternate S 4. Segment Polarity Genes H 5. Homeotic Selector Genes > - specific adress Genes at one level refine the boundaries and expression dynamics of each other They then activate the next suite of genes in the hierarchy (ie; maternal effect turn on gap genes etc.) That next hierarchy of genes regulate each other as well as the genes that turned them on  Fly Hierarchies Cont’d 1. Maternal Effect Genes deposited as transcripts or proteins by mom These genes are the mother’s genes, and provide products during construction of the egg. Products such an mRNA or a protein are packed into the egg prior to fertilization. - - Embryo rely exclusively on the maternal effect genes for its early development until the zygotic genome is activated. before MBT Maternal products help with metabolism and regulation of early embryonic patterning - - and morphogenesis. - Maternal effect genes help to identify the anterior and posterior ends of the embryo - - head vs tail thorax vs abdomen 2. Gap Genes Among the first patterning genes activated during zygotic mid blastula transition (MBT). - Gap genes define the location of the head, thorax, and abdominal region. If there is a - - mutation in one of the different gap genes, then there is a loss of a large segment in that - - region. – creates a ‘gap’ in the normal body plan - Fly Hierarchies Cont’d 3. Pair-Rule Genes It diffuse pattern s refinedmore boundaries Divide the embryo into seven evenly spaced regions. - First evidence of a segmental body plan. Pair-rule genes encode transcription factors that regulate the expression of segment polarity genes. - - - - 4. Segment Polarity Genes Divide the segments formed by the pair-rule genes into two - anterior/posterior (total segments/domains now 14). - 7 14 - V 5. Homeotic Selector Genes conserved role These genes are known as HOX genes in mammals and as antennapedia (ANT) or HOM (homeotic) - complex in flies - - Encode transcription factors which endow segments with a discrete address & Ex bearinghead seg grows that antennas Direct segments to form various parts of the body. Direct downstream genes to implement the agenda to make specialized parts appropriate to segment location (mandible versus leg versus wing etc) Hierarchies! II II Fig 2.9, p. 47 But First! Some Definitions/Disambiguation Sound similar, but are different 1. Homeotic Transformation/Homeosis - changes a part of the body (bodySegment into another 2. Homeotic Gene mutated transforms > - When body part into another body part 3. Homeobox - conserved 1806p sequence > motif that encodes a homeodomain HOMEOBOX GENE -one subset of transcription 4. Homeodomain Goaa > - by motif encoded a homeobox motif 3 alpha helicies I binds DNA (TAAT) genes eg. Zinc finger , leucine Zipper 5. Hox Genes , > - homeobox subclass of genes 1. Homeotic Transformation/Homeosis costume ↳ party body part dresses as another mistakenly to mutation up Mutations that profoundly change a part of the body. EX transformation of. antenna into Fruit flies Transforms a body segment into the likeness of another. - legs in Can be caused to defect in a secreted factor (ie;Wnt, BMP) or a transcription. & secreted factor homeobox induce homeotic mutation Not gene > - Example – Krüppel – (German for Cripple) encodes a zinc finger domain transcription factor – one of the Gap Genes that plays a role in Antero-posterior patterning. ↳ can be caused by any gene or chemical Some Definitions/Disambiguation Blueprint for body plans Mutate design for 3 - swap 2. Homeotic Gene CHANGE ONE car wheels for PART INTE its headlights ANOTHER Any gene which, when mutated, results in the transformation of one body - - part into the likeness of another. Can be a Hox genes, or a flower MADS gene… - & mutate and get diff flower parts  Some Definitions/Disambiguation Cont’d Genetic blueprint that Subdomain helps shape the body plan 3. Homeobox only > - one of organism an - cells know their roles Conserved 180 bp sequence motif that encodes a homeodomain. Gives Homeobox genes their name – but can be one of several sequence motifs contained within the gene 3 a. Homeobox gene Master switches of other controlling genes the production shape development to Is only one subset/class of transcription factor genes (POU, zinc finger, High Mobility Group, Helix Loop Helix, Leucine Zipper etc) Homeobox-containing genes are usually transcription factors – they activate or repress other downstream genes. > - helps form eyes Eg; Drosophila pax6 gene. Pax6 is a transcription factor that contains a homeobox motif, but also contains a second DNA binding motif called the paired domain. Pax6 is highly conserved (flies through molluscs, fish, reptiles, amphibians, mammals), and activates suites of genes necessary to the development of optical organs in these organisms Homeodomain 60 aa subdomain only one T a # N-TERM can't c - Some Definitions/Disambiguation Cont’d fits key that into a specific lock With its three (DNA) 4. Homeodomain helicies helping on/off to turn gene expression by alterin 9) tightly how wrapped DNA is The 60 aa acid motif encoded by a homeobox motif. Can be one of several subdomains encoded by a gene Structurally forms three alpha helices, the third of which binds the major groove of DNA where is modified chromatin conformation to effectuate changes to the target gene expression Typically, homeodomains interact with a specific consensus DNA sequence such as TAAT Other domains within the encoded protein might act to bind DNA at different sites, to interact with other transcription factors, or even to dimerize with other homeodomains HOX genes dimers specificity lend Example – Homeodomain of Pax, Hox, Ant, Nkx Some Definitions/Disambiguation Cont’d blueprint for body plans - determine where limbs and features develop 5. Hox Genes A subclass of homeobox genes Also called: Homeotic selector genes Antennapedia/bithorax complex genes HOM genes Example Drosophila antennapedia (ant) gene. Transcription factor gene that encodes a transcription factor normally expressed in the developing thorax Plays a role in formation of fly legs Ectopic expression in head transforms antennae into legs Loss of expression in thorax results in legs forming to look instead like antennae Antennapedia acts in the thorax to repress genes that expressed head fate Homologues is called HoxA6, B6, C6, or D6 in vertebrates  Fly Oocytes are Preloaded with Goodies and Antero-Posterior Axis GSC proliferate create 16 cell cystocyles d connected by pole- plasm cytoplasmic cells ridge After Fuller, and Spradling. Science 2007: 316(5823), pp. 402-404 Fusosome: cytoplasmic bridge between cells that starts as an annular actin constriction. Unidirectional microtubules are oriented to run through it pump in proteins zygotic transcription , holoblastic cleavage , mRNAs  Fusosome i microtenes(same cystocytes orientation a ·  Maternal Effect Genes Maternal Effect Genes Result in products deposited by the mother into an oocyte (before fertilization) during oogenesis Either mRNA or protein There are several, but we will only discuss: 1. Nanos 2. Hunchback 3. Caudal 4. Bicoid Fruitfly Larval Morphology produce reproductive parts hugeparta Some easy morphological markers missing > - ENDS define end of comb of embryo nanos and Fig 2.10, p. 48 bicoid bicoid ↓ express bicoid ↳ deposited anteriou by cystosomes at fusosomes take cytoplasm genetic rescue nanos extremely posteriorized ↳ hunchbach - present as MRNA Antero-posterior gradients of bicoid/nanos ↳ Only nanos where is not Both Nanos and hunchback present as maternal transcripts Nanos relegated to posterior Nanos protein inhibits translation of hunchback Hunchback becomes excluded from posterior regions Nanos promotes caudal Fig 2.13, p. 51  e universally Hunchback Inhibits Caudal distributed boundry between head and Both bicoid and posterior caudal present as maternal transcripts bicoid relegated to anterior bicoid protein inhibits translation of caudal Caudal becomes excluded from anterior regions Fig 2.8, p. 46  Bicoid - anterior Putting it together Nanos - posterior Hunchback a inhibited Caudal a inhibited by Nanos by Bicoid L - restricted restricted to anterior to posterior Bicoid and nanos activate and constrain expression of hunchback and caudal to the anterior and posterior ends of the embryo respectively. These and other maternal effect genes then activate specific suites of gap genes Bicoid threshold determines hunchback domain Bicoid expression domain defines a gradient At a specific threshold, hunchback RNA is translated Gap Genes Giant: Basic leucine zipper transcription factor Activated by bicoid, caudal At a specific threshold of hunchback and caudal Krüppel: Zinc finger transcription factor Activated by bicoid Turned on by hunchback inhibits off hunchback Turns on knirps Inhibits giant Knirps: Steroid family transcription factor (zinc finger) Activated by bicoid, caudal Inhibits giant Inhibits krüppel Tailless: Steroid family transcription factor (zinc finger) Activated by bicoid Inhibits giant Inhibits krüppel  * EXAM # giveofexample Gap Gees All Together maternal effect gene > - interact > - turn off / on Pair Rule Genes embryo more becoming refined some genes activated At max and some genes activated at minimum ↳ even shipped Pair Rule and Segment Polarity Similar to information on page 77 engrailed is expressed at the anterior of margin each stripe & and delimits the anterior border of each parasegment. Boundaries Fig 2.38, p 76 get sharper and Straighter  Segment Selector Genes spatial co-linearity ↳ order of chromosome is the Antennapedia/bithorax complex same as they are the HOM complex in expressed embryo Hox complex & in cluster in specific order 1. Genes are clustered on single chromosome 2. Gene expression domain (anterior to posterior) reflects order on chromosome = 3’ 5’ colinear 3. Genes arose from ancestral duplication posterior 4. 3’ S Anterior genes dominate 5’ = ③ posterior ones in terms of anterior = setting developmental agenda = rule of posterior prevalence posterior genes dominate  preserve function in change motor enhancer turn on /off in all as one is toolkit reemployed continuously long as present Hox Complex (mammals)adult after · metamorphosis a lot Hox genes of homologs duplicated > - SIMILAR Clusters duplicated 3’ genes turn on first > - define anterior 5’ genes turn on last end > - define 3’ genes define posterior end anterior domains 5’genes define posterior domains = temporal and spatial colinearity not just where they turn on but not has when they turn every cluster every gene un duplicated genes evolution disposes of tools don't need