1Developmental Biology - Biology 4361Cell Fate, Potency, and DeterminationSeptember 27, October 4, 2005Fate Mapping - fate - sum of all structures cell will form in later stages of normal development - fate map = diagram - embryonic regions with distinct fate = primordium or rudiment - cells multiply & move – fate maps change - fate mapping methods - label cells - watch over time- fluorescent tags- label donor cells - transplant - organisms w/ variable cleavage- different groups of cells can contribute to fateClonal Analysis cell clone - all surviving descendants of single founder cell- clonal analysis methods:- fluorescent labeling (resembles fate mapping)- somatic crossover - generates genetic label - signal not diluted over time- positioned randomly throughout tissue- somatic crossover analysis-outline = cell mixing- growth kinetics (if daughters = 1/n of organ, then original = n/2)- e.g. insect epidermis has restriction lines - clones do not cross= compartments- compartments filled with several clones = polyclone- polyclones are unit of size and shape regulation- polyclones are units of gene regulation- each compartment has unique combination of active & inactiveselector genes (binary zip code; i.e. sets of genes on or off)- selector genes - control expression of other genesPotency of Embryonic Cellspotency - widest range of capabilities of cell= total of all structures a cell or its descendants can form if placed in the appropriateenvironment- experimental of potency established by operational definitions- methods: - isolation - cell (or region) removed from influence of embryo - in vitro- heterotopic transplantation - cell or region moved to different region2- cell or region influenced by cells other than normal neighbors- “potency of region always includes its fate” (p. 129)- pluripotent - if cell or region can form more structures than their fate- totipotent - cell or region can give rise to complete individual- community effect- single cells or small number of cells - depart from fate - blend with new region- larger groups retain original fate- community effect depends on intracellular communicationDetermination- as development proceeds, range of cell fates diminishes- when potency of cell restricted to its fate, it is considered determined- process by which fate & potency becomes identical = determination- determination methods:- isolation- heterotopic transplantation- clonal analysis - clone must be restricted to fateNOTE - clonal analysis used as a negative criterion only; i.e. ifclone differentiates into multiple fates, the cell or tissue was notdetermined- determination proceeds in a stepwise manner- preceded by bias (preliminary determination)- methods: - clonal analysis- transplantation- isolation- cells are “born” with a bias- bias confirmed or modified through cell-cell interactions - determination examples: Drosophila gastrula (see below for notes on gastrulation)- within prospective thorax and abdomen, dorsal ectoderm forms only epidermis; ventralectoderm forms both epidermal and neural cells- dorsal epidermal analge (DEA) - forms epidermal cells- ventral neurogenic region (VNR) - forms epidermal and neural- experiments:- isolation experiment: - all DEA cells form epidermis (in accord with fate)- VNR cells tended to form neurons - (not in accord with fate: mixtureexpected)- transplant - switch regions - DEA to DEA (controls) - mostly epidermal; no neurons3- DEA to VNR - strong community effect; transplanted cell groups formedepidermis (original fate); single cell transplants - formed neural ormixed clones- VNR to DEA - single cells neural or epidermal in proportion tohomotopic transplantation; VNR transplanted to other regions =mostly neural; no epidermal- results indicates that original vias of VNR cells to form neuronscounteracted more strongly in DEA than in other regions- bias can be acquired by uneven distributions of cytoplasmic or membrane-boundactivities- e.g. Drosophila embryo - biases of VNR and DEA traced back to regulatoryprotein that accumulates in nuclei of ventral but not dorsal blastodermcells- modification of biases1. Community effect - exchange among equivalent cells of signals that stabilize thesame determined state for all of them2. Lateral inhibition (opposite of community effect): equivalent cells competewith one another to attain a preferred fate; one cell inhibits neighbors fromattaining same fate3. Embryonic induction - interaction between nonequivalent cells (note Speeman’seye induction experiment)NOTE - neural plate cell determination at gastrulation will be covered in a later lecture Mouse embryonic cell determination- most, if not all mouse blastomeres up to 8-cell stage are pluripotent- test by dissociating blastocysts then adding blastomeres back to host blastulasand following fate - genetic markers used to follow contribution of blastomeres- at 16-cell stage, some blastomeres pluripotent, others starting determination process- mammalian cells from morula through at least 16-cell stage are biased, but notaltogether determinedProperties of the Determined State- pattern formation - patterns set up from earliest point- e.g. many eggs have unequal distribution of material - e.g. animal-vegetaldistribution sets up dorsal-ventral pattern- patterns change with time and developmental age- pattern formation precedes cell differentiation4- cells determined before reaching terminal differentiated state- determination assessed by operational criteria - i.e. tested by isolation, transplantation, etc., not by morphological orbiochemical methods- e.g. mammalian morula cells: inside and outside morphologically distinct, butnot necessarily determined (i.e. can be interchangeable)- determination multi-faceted, stepwise process; negative and positive- loss of potency (potential) = negative or subtractive process- instruction (i.e. cells need additional instruction (gene activity) in order to followalternate pathway) = positive or additive process- e.g. prospective neural plate would form epidermis in the absence offurther instruction- instructional mechanisms: - localized cytoplasmic determinants - cause blastomeres to be determined (orbiased) to form cell lineages or major body regions- e.g. pole plasm (insects) - form germ cells (pole cells; primordial germcells)- cell-cell communication (intercellular signaling) - community effect- induction- lateral inhibition- hierarchy of determinative events- time sequence of