set aside early in devel, not arise from particular germ layer

How cells become diff: Gastrulation: 3 germ layers

Find fate of cell: cell lineage tracing, fate mapping. Dye injections can be used for fate mapping. To derive fate map, multiple experiments required Decisions cell fate most part irreversible Determination: Cells committed irreversibly to particular devel fate Differentiation expressing cell-type specific characters. Determination typically precedes overt differentiation Standard test: Cells determined if transplantation from normal to new position in embryo no ∆ normal fate

fate mapping early stage embryos

fate map: single cells /groups cells marked at particular time pt in devel, & at later time pts, location & identity of labeled cells noted. require many individual experiments to see range of fates that can be produced by particular cell. Cell fate specification: Bc determined (or committed) cells don’t look diff from non-determined cells, distinguishing 2 is based on experimentation (operational definition). cell determined, transplanting to diff region embryo (usually host embryo of same species & stage) not affect devel fate. cell not yet determined, develop according to new environ.

Cell /devel fate: for particular/group cells. Tracing cell lineages requires cells transparent, division patterns reproducible animal to animal (cells always divide same # times, same orientations, have same fates

cell lineage nematode C. elegans complete known, powerful tool devel analysis. most animals, esp vert,

cond tracing lineages high resolution not met.

fate mapping later stage embryos

Frog blastula

frog fate map at beginning of gastrulation

forms toes

Mlcs in cytoplasm (cytoplasmic determinants)Influence fate of cells receive them

Cytoplasmic determinants become asymm localized

Cell division gives 1 daughter most cytoplasmic determinants

commitment to particular fate progressive: cells have memory C. H. Waddington’s epigenetic landscape

Mech determined Cell autonomous (intrinsic): cells acquire fates independent of interactions w neighboring cells. asymmetric division events in cell in question. mother cell prod cytoplasmic determinants become localized unequally along 1 axis of mother cell. When cell divides across axis, determinants unequally distributed to daughter cells where influence cell fate Conditional (extrinsic): cells acquire fates based on interactions w neighboring cells

Conditional (extrinsic) determinationInduction, morphogenic gradients, pos feedback

induction

asymm division. Cond determination, cells acquire fates based on sigs from neighboring cells. Cell sig determines cell fates: induction. Sequential induction can generate progressively more complicated patterns

Morphogen gradients: Morphogen: diffusable mlc secreted at distance from target. Cells spec

fates of target cells. graded distribution, cells respond directly to diff amts mlc & become spec

to diff fates. ∆ gradient ∆ pattern

Specialized induction mediated by morphogen gradients. Morphogens: high conc near source, lower at greater distances. Cells w approp devel history can respond to morphogen in conc dependent way; high conc will induce different cell fates than low or intermediate conc of morphogen. pos feedback/lateral inhibition, system starts off homogeneous & symm can pattern itself spont, even where no organized external sig

OFF

Threshold reached

ON

1 S

IGN

AL,

1

OU

TCO

ME

Source of signal

diff threshold levels for diff devel decisions

threshold 2 reached1 S

IGN

AL,

MU

LT

OU

TCO

MES

threshold 1 reachedFATE CFATE A FATE B

Source of signal

FRENCH FLAG MODEL Wolpert ‘60s

morphogen gradient outcomes

positive feedback aka lateral inhibition

threshold 2reached

threshold 1reached

DIGIT 4 DIGIT 3 DIGIT 2

1 signal, multiple outcomes

threshold level for devel decision

Group equiv cells

cells compete by inhib neighbors

Some predominate

normal fly

Fly w patch of cells mutant for Delta

example: Notch-mediated pos feedback

intestinal epithelial cells

neurons

all cells in embryo/adult: same genomic DNA seq. not all cells within embryo or adult express same set genes. cells become different bc express diff genes. proteins confer cells ability to be diff (morphologically & functionally)

Same genome, diff cells: reg gene expression = cell specification

components differential gene expression nature switches that turn genes on/off (post)transcriptional controls events turn particular switches on /off devel events: asymm divisions, cell sig

gene expression can be controlled at many levels

Most often used control pt in devel

Promoter contains seq recognized & bound by RNA pol, ~50 nuc upstream of transcription start site

“upstream” “downstream”

promoters vary in strength of binding RNA pol. promoters w strong bind reg by transcriptional repressors. w weak binding reg by transcriptional activators. activators & repressors can act in concert to provide highly sensitive transcriptional reg

of enz that make tryptophan

constitutivelypresent

prok genes can be reg via repressor proteins

negative feedback loop

Tryptophan low

Tryptophan high

Biosynth genes ONBiosynth genes OFF

RepressorOFF

Repressor ON

prok genes can be reg via activator proteins

activators enhancer RNA pol binding promoter

Prok transcriptional reg

E. coli use gluc as E source but if no gluc available, can get by breaking down lactose. genes Lac operon allow E. coli to utilize lactose. E. coli wants to express Lac genes when: lactose is in the medium, glucose is not around. Lac genes: gluc only OFF lactose only ON gluc & lactose OFF neither OFF

repressors and activators can work together to reg Lac operon

inactive repressor

Lac repressor inhib

transcription when lactose not present

allolactose

Allolactose: metabolite of lactose; levels reflect lactose levels

inactive

CAP activates transcription when

gluc not present

cAMP

Gluc low, cAMP ^

inactive repressor

inactive

Lac operon is controlled by

2 signals

euk cells contain 3 distinct RNA pol: I, II, III (II transcribes mRNAs), euk RNA pol require assistance from general transcription factors, euk contain large amnts untranscribed DNA bw genes, euk DNA packaged into complex chromatin structures can prevent access to RNA pol

euk RNA pol II requires set of GENERAL TRANSCRIPTION

FACTORS

But this is not enough!

euk activators & repressors: euk transcriptional regulators: can activate /repress transcription, bind reg elements enhancers, bind enhancers thousands of base pairs away from gene they reg, bind enhancers up/ downstream of gene they reg

transcription regulators contain variety DNA binding motifs transcription regulators usually bind major groove DNA

transcription regulators work together as committee to control gene expression

combinatorial control transcriptional status of any gene result of combo transcriptional reg

Transcription initiation: RNA Pol II can only bind at euk promoters w help of large group GTFs. Activation large complexes promoted by transcription activators, prevented by transcription repressors, bind to reg seq at large distances from promoter

example of single contact bw

transcription reg & 1 base pair DNA

small # of transcription factors acting in combo can specify variety

of cell fates

single gene can be turned on by diff transcriptional reg

Transcribed region

Transcribed region

Cell A

Cell B

transcriptional reg: possible to express genes where/when we want

GFP expressed under control of gamma-crystallin reg seq

single gene can organize entire organ thru cascade transcriptional reg

single transcription regulator can coord expression many diff genes

Each gene has region DNA adjacent promoter where RNA pol binds. Genes controlled by proteins (transcriptional regulators) bind to reg DNA seq via noncov bonding to nuc within the major groove of DNA. Repressors function by blocking binding RNA pol to promoter. Activators function by facilitating binding RNA pol to promoter. repressor & activator combine to reg Lac operon in response to C source availability. Euk transcriptional regulation: specialized RNA pol for mRNA (RNA pol II); much more non-coding DNA bw genes; DNA packaged in chromatin. RNA pol II doesn’t bind directly promoter region. promoter contains seq (most commonly, TATA box) that bind to one or more of the general transcription factors that recruit RNA polymerase and associate with it to facilitate transcription and its proper regulation. complex RNA pol w general transcription factors, not sufficient activate transcription. Specific activators /repressors bind to enhancer sequences (enhancers) that can be quite far from the start site of transcription. activators can facilitate looping out of the DNA to allow the activator or repressor to interact w transcriptional machinery. Enhancers can occur up/downstream of euk genes. Each gene reg by combo of enhancer seq, & whether given gene on /off result of combinatorial action of multiple transcriptional reg. single transcriptional reg (or particular combo of reg) can activate many genes. single transcriptional regulator can simult activate some genes repress others. difference is combo of transcription factors that bind diff genes.