Developmental biology lecture 3 (cell differentiation - time and space)

possible methods for detecting gene expression through mRNA

1. …

2. … amplification from mRNA

3. in situ …

1. transcriptomics

2. RT PCR

3. hybridisation

… amplification from mRNA works by fist converting the target mRNA to … by … … followed by standard … amplification

RT PCR, cDNA, reverse transcriptase, PCR

transcriptomics for detection of gene expression works by sequencing the whole … of … at a specific time and under specific conditions

transcriptome, RNA

in situ hybridisation works by adding a labelled complementary nucleic acid … to visualise where a gene … is located

probe, product

the probe used for in situ hybridisation can be visualised by a … or can be made …

stain, fluorescent

alongside in situ hybridisation another approach is the use of … genes, for example by inserting the … gene into the gene of interest

reporter, GFP

cells in development become more and more … in their fate until eventually a single … has become irreversibly determined

restricted, fate

the fate of a cell is determined when it can no longer respond to changing … signals and change its … pathway

environmental, differentiation

… cell fate is a reversible stage where a cell commits to a pathway but can still be influenced by its … to change

specified, environment

… cell fate is the subsequent irreversible stage where a cell it locked into its fate regardless of the surrounding … or …

determined, signals, relocation

… … is a means of ensuring that all cells in a field of similar cells exposed to the same signals and the same neighbouring cells do not undergo the same … pathway at the same …

lateral inhibition, differentiation, time

lateral inhibition is often the result of differentiating cells producing an … molecule that acts … to prevent the neighbouring cells differentiating the same way

inhibitory, locally

NEUROGENESIS AND LATERAL INHIBITION

1. at first neighbouring neural plate cells all express …, … and …

2. with time one cell expresses more …, which inhibits expression of … in the neighbouring cell

3. the cell which expresses more … develops into a … and inhibits the neighbouring cell from following the same fate

1. delta, notch and neurogenin

2. delta, neurogenin

3. delta, neuroblast

the delta-notch pathway represses expression of … and other … genes as a core part of lateral inhibition related to neurogenesis - it spaces out the differentiation of … in the developing … system

neurogenin, proneural, neurons, nervous

The signalling molecule produced by the anchor cell is … which binds to the … receptor on the inner vulval cell and stimulates the … pathway.

The inner vulval cell then produces a … family ligand signalling molecule which binds to … receptors on outer vulval cells to stimulate the … pathway

LIN3, LET23, EGF

DSL, LIN12, Notch

cells are traced through development through a technique called cell … … or cell … …

lineage analysis, fate mapping

for the chick-quail … technique … tissue was transplanted into the … embryo, and since quail cells are more … they were used to map the … pathway and fate of … … cells

chimeras, quail, chick, dense, migration, neural crest

nowadays we don’t use techniques such as the chick-quail chimeras to establish cell lineage instead we use analysis through … tags

genetic

… are produced when each neuron in the brain is tagged with a different colour and this enables us to examine cell … and … (neurons and targets)

brainbows, connections and interactions

to generate … mice embryos genetic engineering modified the … protein to produce different colours (RFPs, BFPs, YFPs etc) and multiple copies are then inserted into the genome which can … within individual cells

confetti, GFP, recombine

… is a maternal effect gene in Drosophila which acts as a morphogen and has a role in … establishment

bicoid, axis

the effect of the biophysical environment is mediated through … signals

mechanical

reduced gravity in spaceflight, which leads to low … stimulation, has been associates with increased bone … and decreased … absorption

mechanical, resorption, calcium

reduced human foetal movement leads to …, thin fragile … and … …

fractures, bones and joint dysplasia

when foetal movement is reduced …, … formation and … are abnormal

joints, bone, shape

the hippo pathway acts as a … of a cell’s …

sensor, environment

the hippo gene (Hpo) encodes an … … of a signalling pathway

intracellular component

what is the mammalian ortholog of the Hpo protein

Mst1/2

when the hippo pathway in inactive is gene expression on or off?

on

when the hippo pathway is active is gene expression on or off?

off

is the hippo pathway active or inactive when Hpo (Mst1/2), Lats1/2 and Yap/Taz are phosphorylated?

active

is the hippo pathway active or inactive when Hpo (Mst1/2), Lats1/2 and Yap/Taz are unphosphorylated?

inactive

HIPPO SIGNALLING PATHWAY

1. when … (… in mammals) and … are unphosphorylated … can enter the nucleus and enable transcription

2. … is bound to Lats1/2

3. when … (…) and … are phosphorylated … (coactivator) is also phosphorylated and is unable to enter the … and enable gene expression

4. when hippo pathway is active cell … is interrupted

1. Hpo, Mst1/2, Lats1/2, Yap/Taz

2. Mob1

3. Hpo, Mst1/2, Lats1/2, Yap/Taz, nucleus

4. proliferation

the hippo pathway can be triggered by the … …

physical environment

the transcriptional coactivators … and … in the hippo pathway are … and … of mechanical inputs and then bind to TFs such as …

Yap, Taz, sensors, mediators, TEADs

Engler’s experiment in 2006 showed that the soft matrix was …, the intermediate matrix was … and the stiff matrix was … when stem cells were grown

neurogenic, myogenic, osteogenic

localisation of … activity in developing skeletal … is responsive to … stimulation

Yap, rudiments, mechanical

when there is foetal movement and muscle forms, … protein is elevated in regions of a developing humerus where … change is occuring, but when there is no movement and no muscle the localised … protein is lost and shape is …

Yap, shape, Yap, altered

a … could emerge in the embryo by cells receiving a … … through a set of … … unique to that … (write out the whole sentance)

a pattern could emerge in the embryo by cells receiving a positional address through a set of molecular signals unique to that position

4 ways cells receive positional information

1. localisation of cytoplasmic determinants

2. induction

3. morphogen concept

4. physical environment

localisation of … … is a mechanism to make daughter cells … from each other and to ensure daughter cells have different … to each other

cytoplasmic determinants, different, properties

a … is a signal (chemical/molecule) involved in … … where cells have a … response depending on the … of the signal they are exposed to

morphogen, pattern formation, graded, concentration

the french flag model suggests that a cell at any point along the … can interpret the local … and respond in a manner appropriate to the …

gradient, concentration, position

Drosophila development shows the importance of cytoplasmic determinants, morphogens and cell signalling. Which of these do the following types of genes harness for development?

1. maternal effect genes

2. segmentation genes

3. homeotic genes (do not really use any of these)

1. cytoplasmic determinants and morphogens

2. cell signalling

positional info cells receive refers to the … … and … cells experience appropriate to their relative … within the embryo, this influences cell … and …

molecular signals, cues, position, differentiation and morphogenesis

besides molecular cues for positional info which can be localised through cytoplasmic determinants and cell-cell signalling, cues might also be generated by the … … and we refer to these as … cues

physical environment, biophysical

if movement in the chick or mouse foetuses is reduced then:

1. … … is abnormal

2. … are abnormal

3. … of joints/bones is altered

4. at a molecular level … … are altered - including … and

1. bone formation

2. joints

3. shape

4. signalling, Wnt

… forces can influence cell … … which is shown in embryos which develop without muscle contrations where their cells of the developing … do not … correctly and their … … is altered

physical, signalling pathways, skeleton, differentiate, gene expression

important destinctions to remember

1. … forces can influence cell … …

2. cell … can be influenced by the … or … environment of the cell

1. physical forces can infulence cell signalling pathways

2. cell differentiation can be infuenced by the biophysical or mechanical environment