Chapter 4 Embryo

CHAPTER 4 Approaches to development: experimental embryology NORMAL DEVELOPMENT - NORMAL DEVELOPMENT refers to the course of development that the embryo follows in standard laboratory conditions - There is no experimental manipulations - Development occurs naturally - EMBRYONIC DEVELOPMENT is predictable - Mammals and birds: Development occurs at a standard physiological temperature - Free-living embryos (Xenopus or zebrafish): rate of development varies on temperature - Development can be MATERNAL (due to the components of the egg or the genome of the mother) or ZYGOTIC (due to the components newly synthesized by the embryo itself after fertilization) TERMS TO REMEMBER 1. Anterior or cranial end - front end of an animal 2. Posterior or caudal end - rear end of an animal 3. Dorsal - upper surface of an animal 4. Ventral -lower surface of an animal (chest area) MICROSCOPIC SECTIONS

5. Transverse section - Section taken across the long axis of an animal (separating the posterior and anterior region) 6. Longitudinal - parallel to the long axis 7. Sagittal VERTICAL longitudinal section that is cut at the midline 8. Parasagittal VERTICAL longitudinal section that is cut at one side of the midline 9. Frontal or coronal section - HORIZONTAL longitudinal section that separates the dorsal and ventral sides STAGE SERIES - All models used for laboratory work have published stage series - Describes the course of development as a number of standard stages (stages the embryo goes through as it develops) identified through external features using a dissecting microscope THE FATE MAP - Diagram that shows what will become of each REGION of the embryo in the course of NORMAL development 1. Where will it move? 2. How will it change shape? 3. What structures will it form? - FATE MAP changes from stage to stage due to morphogenetic movements and growth

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The precision of a fate map depends on how much random cell mixing occurs in development no mixing occurs as like in the Caenorhabditis elegans, it will be based on CELLULAR LEVEL FATE MAP does not indicate anything about developmental commitment (commitment is usually acquired through a series of intercellular interactions) ALL PARTS OF EMBRYO have a fate throughout development

MOSAIC - Experimentally isolated parts DEVELOPS according to the fate map REGULATIVE - Isolate parts form MORE STRUCTURES expected from the fate map - Depends on the double gradient system Ex: ADMP-Chordin system in Xenopus How are fate maps constructed? 1. Label SINGLE cells or regions of embryos 2. Locate the position and shape of the labeled patch at a later stage of development Labeling can be a. Application of extracellular label to a patch of cells b. Injection of an intracellular label to one cell

Ex: Injecting to two early blastomeres of a Xenopus embryo mRNA for green fluorescent protein (GFP) then locate the labeled domains at NEURULA stage c. Grafting a labeled tissue to replace an exactly equivalent piece in the host embryo CLONAL ANALYSIS - Form of fate mapping wherein a single cell is labeled by determination - Position and cell types of the progeny (of the single cell) is identified at a later stage Labeling can be a. Injection of one cell with a lineage label (used for large cells and organisms that do not grow significantly) ex: Xenopus or zebrafish or sea urchin b. Introduction into a single cell a genetic label that will persist without dilution - Can be the insertion by replication-incompetent retrovirus or genetic recombination even that yields a visible marker ex: Mouse and chick - CLONAL ANALYSIS: decide whether a cell is committed to form a particular structure or cell type at the time of labeling * a label applied early can span up to A and B but if the label is applied later, it will only span either A or B - CLONAL ANALYSIS can prove lack of commitment but not presence of commitment

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USUALLY USED in analysis Drosophila segmentation and of vertebrae hindbrain patterning COMPARTMENT: indicate a region in an embryo whose boundaries are boundaries of clonal restriction if there is a compartment, no cells may enter or no cells may leave - A compartment usually corresponds to a visible structure such as a segment or organ rudiment (organ not fully developed) and is maintained either by physical boundaries to cell migration such as basement membranes or by differential adhesion of the cells of the compartment compared to those outside Cells in the compartment stick to each other

DEVELOPMENTAL COMMITMENT - Formerly uncommitted cells become committed cells to form body parts of cell types - Commitment is the combination of transcription factors present in the cell so it can be visualized directly by observing the expression of the relevant genes using in situ hybridization 1. Specification - a cell or tissue is said to be specified to become a PARTICULAR STRUCTURE

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It will still develop into a particular structure even if the cell or tissue is isolated from the embryo Specification map * shows that the cells have been programmed to do or be at this particular developmental stage SPECIFICATION need not be the same as the fate in normal development Ex: neural plate of a Xenopus blastula may not differeniate into a neuroepithelium but into epidermis when cultured in isolation (in order to form neuroepitheliym, inductive signal from the mesoderm is needed)

2. Determination - Commitment is irreversible - It will continue to develop autonomously even if is moved to any other region of the embryo - Pathway of development is UNALTERED - Cells have lost their responsiveness or competence to the singals that originally turned on the relevant combination of transcription factors 1. Orthotopic graft - Graft to the same position of another embryo 2. Heterotopic graft - Graft to the different position in the host - Test for determination Potency - range of possible cells types or structures into which a

particular cell population may develop Embryonic stem cells - cells that can form any cell type in the body - pluripotent and tissue specific stem cells - multipotent CYTOPLASMIC DETERMINANTS - Determinant is a substance or substances located in part of an egg or blastomere - The determinant guarantees the assumption of a particular state of commitment by the cells that inherit is during cleavage (determinants for morphogenesis) - Cell division is asymmetrical and two daughter cells will follow different pathways od development - Determinants are important during the earliest stages of embryonic development: first two or three distinctly specified regions in embryo are established Ex: bicoid and nanos mRNA in the Drosophila egg PAR3,PAR6 and aPKC in the C. elegans INDUCTION - The extracellular signals are called inducing factors - Growth factors, cytokines or hormones - Competence is the ability to respond to an inductive signal - Signal transduction pathway is also coupled to

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the regulation of transcription factors Ex: Xenopus embryo the mesoderm is induced from animal hemisphere tissue in response to the nodals emitted from the vegetal region 1. Instructive Induction responding tissue has a choice before it (mesoderm or endoderm), and in normal development the interaction results in an increase in complexity of embryo There are two different types of instructive induction depends on a. regional specification: if the signaling center lies at one end of a cell sheet and is the source of a concentration of a signal substance * Threshold response to the difference concentrations – morphogen Ex of morphogens: sonic hedgehod protein in the neural tube and the limb bud, active BMP (bone morphogenic protein in Xenopus) or Dpp (Decapentaplegic protein) in Drosophila b. If the signaling center lie in one cell sheet and responding cells in another – single threshold response ex: induction of nasal, lens and otic placodes 2. Appositional induction - only one threshold response would be made

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by the responding tissue and the inducing factor would not be called a morphogen 3. Permissive induction signal is necessary for the successful self-differentiation of the responding tissue but cannot influence the developmental pathway selected important in late development ex: development of kidney INDUCTIVE SIGNALS DIFFUSE THROUGH EXTRACELLULAR SPACE CYTONEMES: transport through FINE intercellular processes

LATERAL INHIBITION - Another type of cell communication - Known in terms of the behavior of Notch-Delta system - The individual cells from a uniform population follow one pathway while the surrounding cells follow another Ex: neurogenesis, formation of endocrine cells in the epithelia of the gut - Activator and inhibitor substance - Activator substance is short ranged and only active through intercellular contacts - Inhibitor substance is long ranged, moving freely with diffusion

STOICHASTICITY IN DEVELOPMENT - there is random determination (random distribution or random pattern) - DETERMINANTS become localized at one end of the cell rather than the other - Multiple cell types appear to differentiate from a single type of progenitor - SYMMETRY BREAKING THE “EPIGENETIC LANDSCAPE” - Developmental commitment is the subject of a remarkable concept first introduced by the embryologist and geneticist C.H. WADDINGTON - WADDINGTON introduced the term EPIGENETIC as synonym of development, recognizing that a large element of development consists of the regulation of gene expression - The epigenetic landscape – portraying symmetry breaking event - Ball: cell in an embryo - Valleys: states of developmental commitment - Each Bifurcation: developmental choice Problems 1. Diagram relates only to cell states and does not capture the structure of embryo 2. Exchange of inductive signals are not represemted 3. Most developmental choices are not symmetry breaking events BUT DETERMINISTIC EVENTS

controlled by inductive signals How is EPIGENETICS DEFINED NOW? - It is the understanding of gene expression in terms of chromatin structure rather than of DNA alone - Importance of DNA methylation as a controller of gene expression and numerous chemical modifications of histones and other chromosomal proteins, which can collectively prevent or enable the activity of transcription CRITERIA FOR PROOF 1. Expression - molecule should be expressed in the right place, right stage and in a biologically active form - in situ hybridization or immunostaining - use of retinoic acid 2. Activity - molecule must have appropriate biological activity in a suitable test system

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ex: a candidate inducing factor should be able to evoke the correct responses from its target tissue ex: the candidate cytoplasmic determinant: it should be possible to inject it into another part of the cell or blastomere and cause the injected region to develop along the pathway caused by the determinant

3. Inhibition - molecule is in vivo (inside) then the process for white it is thought responsible should fail to occur - redundancy - inhibition may be achieved at the DNA level by mutation of a gene to inactivity - Ex: neurogenesis, formation of endocrine cells in the epithelia of the gut - At RNA level, using of antisense morpholinos or RNAi - At protein level, introduction of a specific inhibitor of the normal gene product