embryo lab 1 Flashcards
three processes involved in development of a pluricellular organism
- cell growth
- differentiation
- morphogenesis
morphogenesis of embryo
- initial flat, three layered embryonic disc undergoes morphogenesis to form 3D
- complex processes involving interacting molecules regulate these processes
- alterations in the morphogenetic processes cause birth defects
- movement of convergence and extension in gastrulation: responsible for neural tube closure
cell behaviours that control morphogenesis
- cell shape, size, position (movement), number, adhesion
- drive differential growth which in turn regulates morphogenesis
- dysmorphogenesis may result from alterations of such basic behaviours and produce birth defects
commitment vs determination
- c: engagement in a given differentiation pathway
- d: irreversible engagement in a given differentiation pathway
- may be a time dependence on induction effects
- at first a cell is committed then determined towards differentiation
experimental embryology
- investigating and understanding the processes occurring in normal development and their alterations
- aims to diagnose, prevent and cure birth defects besides basic knowledge
- basic developmental mechanisms are highly conserved among species: animal models useful
- data from animal models must always be validated in humans
characteristics of useful animal models(6)
- practical to obtain
- easy breeding
- short life cycle
- production of numerous progeny
- low cost
- non seasonal life cycle
animal models
invertebrate
- nematode worm
- common fruit fly
vertebrate
- frog
- zebrafish
- chick
- mouse
- include insects and worms
caenorhabditis elegans (nematode worm)
- life cycle 3-4 days
- 1 day from fert to hatch
- transparent
- approx 1000 cells
- well known genome and transcriptome and mutants
cellular analysis to further understand morphogenesis
fate map:
- show what a region is supposed to become:
- microinjection of fluorescence dextran
microsurgery
- cultures of explants, in vitro testing of differentiation factors
- cell tracking allows to see derivatives of groups of cells
- in vitro culture: histological analysis to study commitment and induction
- grafting: move part inside embryo: in vivo grafting
molecular analysis to further understand morphogenesis
- gene studies
- expression analysis by in situ hybridisation, immunodetection
- cells have specific transcriptome and proteome
- ISH taget RNAs, IF proteins
gain and loss of function studies
loss:
- transgenesis by homologous recombination in DNA
- antisense RNA technology interfering with the RNA
- dominant negative mutated RNA overexpression,blocking antibodies, inhibitor pharmacological treatment to protein
gain:
- transgenesis in DNA
- ectopic overexpression RNA
- grafting beads containing proteins, activators phacoloical treatment to protein
antisense RNA technology
- interfering with the RNA
- the RNA hybridises with an anti-sense oligonucleotide: double strand RNA degradation
experimental procedure showing E-cadherin role
- fertilisation in vitro
- injection of E-cadherin anti sense oligos in Xenope
- development of blastula
- results: dissassembled blastula, no cavity: suggesting loss of adhesion among blastomeres
- conclusion: E-cadherin is NECESSARY for cell junctions
experimental procedure showing B-cadherin role
- observation: beta-cadherin expression in amphibian blastula: dorsal enrichment
- injection into ventral side
- results: extra axis on ventral side
- conclusion: beta-cadherin is sufficient to induce dorsalization of the embryo
study of induction: how mesoderm formed in frogs?
- nieuwkoop experiment
- a signal from vegetal hemisphere causes equatorial cells to from mesoderm
- animal pole juxtaposed with vegetal pole from an unlabelled embryo and resulting conjugate is cultured
- fluorescent tissue differentiated as notochord and muscle rather than epidermis:
- an induction occurred: of cells derive from epidermis
