Lecture 2

Fruit fly advantages

. - Fast generation time/life cycle (10 days) - Many offspring - Many Genetic tools available to the fly - turn on/off genes (control of genes)

Fruit fly disadvantages

not as similar to human genes as other organisms (mammary glands for example)

Zebrafish advantages

. - Transparent - helps you follow developmental processes in real time - can use dyes for example - Can watch eggs develop outside of mother

Zebrafish disadvantages

. - slower generation time (months), and may need to backcross to gain wanted offspring - Genetic tools not the best

Mouse advantages

. - Most similar to human genome and development (example

Mouse disadvantages

Mom keeps embryo in womb, cannot manipulate or see it as easily (in vitro development)

Frog advantages

Frog disadvantages

. - Genome is messy, has 8 copies of every gene making genetics hard to understand the function of a single gene due to the other homologs which may mask or act redundantly - Too much redundancy - very little genetics done on frog

Lineage Tracing

How we determine the developmental fate of cells

Developmental fate of cells

how we know what early developments cells will become later on

Totipotent

cell can become any type of cell

Direct Observation

Initially take advantage of hints given by organism to understand cells future function

Direct observation disadvantages

require unique/readily identifiable traits of a cell that are conserved across time (luck based)

8-cell stage of tunicate embryos

two of the cells contain yellow cytoplasm

Trunk muscles

only cells that contain yellow cytoplasm

Fate mapping with dyes

introduce dye into a group of cells early, then determine which cells are stained later in development

Marked cell

allows you to follow what happens to the cells

Requirements for fate mapping using dyes

semi-transparent embryo, or dissect embryo and look inside

Breaks in membrane/ injection into cell

introduction of dye into cell

Fate mapping using dyes disadvantage

as the cell proliferates/divides, the dye can dilute, making it difficult/impossible to track cells eventually as development progresses

Fate map of the zebrafish brain

constructed using fluorescent dyes that were injected early in development

Fluorescent dyes disadvantage

dilutes overtime

Genetic Markers as cell lineage tracers

Part of early embryo transplanted to another embryo - quail vs chicken, similar enough that transplantation is tolerated without rejection

Genetic markers as cell lineage tracers

dilution not a problem

Marking in genetic markers as cell lineage tracers

due to differences in cell morphology and characteristics like feather colour

Requirements for genetic markers as cell lineage tracers

Fate Mapping with transgenic DNA

uses endogenous GFP expression instead of similarity between animals cells

Transgenic GFP

controlled by promoter to make cells continuously express GFP in all cells; can do transplantation between GFP and non GFP animals/embryos

Embryo segment of GFP positive

transplanted into normal embryo (no GFP) and followed over development

Fate mapping with transgenic DNA advantages

dilution not a problem as cells constantly produce GFP

Transgenic Memory Cassettes

permanently labelling cells using transgenic genetic techniques; cells have multiple transgenes in an embryo and a promoter

Direct observation, dye marking (vital + fluorescent), genetic-marker-based transplantation, transgenic memory cassettes

ways to trace cell lineage (fate mapping)

In situ hybridization

allows you to visualize RNA expression pattern in whole embryo and tells you where the RNA is expressed but not the amount of RNA

In situ hybridization requirements

fixed embryos processed with a probe

Immunohistochemistry

allows you to visualize protein expression pattern in whole embryo; tells you where the protein is expressed but does not tell you the amount of protein

Post transcriptional modification

difference between immunohistochemistry and in situ hybridization

Genetics

how you interpret relationship between genotype and phenotype

Reverse Genetics

start with genotype and determine phenotype

Forward Genetics

Use genetic screen to randomly mutate genes and allow embryos to develop and see which fail to develop a trait like the heart, you know which genes were hit and correlate genes; start with phenotype, go to genotype

Loss of function

Mutated gene to lose function - important for development, remove something to see what happens to understand its relationship for a developmental process

Target mRNA to degrade (RNAi)

creates loss of function as there is no expression

Gain of Function

If gene is expressed at higher level or mis expressed/ectopically expressed somewhere it shouldn't be, what is the function, will an additional normal function occur; looking at sufficiency of gene, can it alone do something

Ways to create loss of function

. - RNAi - Null Mutant via CRISPR - Dominant Negative

Deep Sequencing RNA-Seq

provides a list of genes expressed in cells of a specific stage, tissue or mutant background; can compare lists from your tissue/cells of interest and a related tissue/cell

Transcriptional profile

unique in every cell; some genes are expressed that may not be expressed in other tissues; differentially expressed

Deep sequencing process

sequence mRNA that is actively expressed and compare to another part of the embryo

Bioinformatics

if you have a sequence of a gene, you can infer what it does; analyze shortlist of candidates via loss/gain of function experiments

Different genes

key targets/candidates to follow up on

Eye development

take tissue at diff stages and dissect out of animal; do RNA sequencing on samples to see what genes are expressed and how they change over time

Genes only expressed at certain times

may only be expressed at these stages in development

Transcription factors

proteins with DNA-binding domains

Identify Transcription factors for ICM development

use bioinformatics to determine which of the differentially expressed genes encode for transcription factors

Genes expressed in tissues inside the cell but not outside the cell

determine what type of tissue cells form as transcription factors turn on/off genes by binding DNA

Remove TF

you can see which genes care if the TF is there or not

Remove yap

Any genes that go down in yap loss of function, implication is that gene is activated by yap in WT by comparing if it is missing in the mutant

Enhancers

control where genes are expressed; Regulatory DNA around gene that encodes whether the gene is on/off

Modular

enhancers can act on their own if put upstream of another gene

Brain specific

only in cells with proper TF present causes looping of enhancer to start site to allow for transcription

Limb enhancer

recruits transcriptional complex to start transcription

Enhancers in DNA specific sequence

recognized by TFs only present at these stages in development

Transgenic constructs

scientists can make recombinant DNA in the lab in which an enhancer is cloned upstream of another gene using PCR and restriction enzymes; enhancer cloned upstream of another gene, can exploit cell specific enhancers

Transgene

recombinant DNA is integrated into an animal

Transgenic

animal with a transgene, every cell of the animal contains a copy of the transgene

LacZ

not found in mouse; clone enhancer upstream of mouse gene, where expressed is where enhancer normally activated

How to make a transgenic organism

. - Male and female mate create fertilized egg - 2 pronuclei merge, and inject transgene into egg - Gene randomly inserts into genome - Let egg develop further - Implant into a pseudo pregnant mouse - F1 progeny mouse is transgenic

After embryo starts to divide

each cell has transgene in organism

Pseudo pregnant mouse

has hormonal environment to bring embryo to term

Male and female embryo

too small and will burst; so let it develop a little

Mosaic

some cells have the condition and others do not; each adult must be observed to determine which ones got the transgene

1 cell embryo stage

male and female pronucleus have not yet fused

Male pronucleus

DNA injected for integration because it is larger

Future germ cells

segregated in tail of embryo

Inject transgenic DNA

into tail (germline of embryo)

Enhancer upstream of recombinase and memory cassette

transgenes always in organism to do lineage tracing

Reporter

something you can follow that is not native; downstream of Constituent promoter

Constituent promoter

always firing at all times

RNA polymerase

cannot get through STOP cassette

Cre recombinase

will recognize sites, cut out stop cassettes, and allow permanent expression of GFP

1st trans gene

cre (recombinase) downstream of promoter of interest

2nd transgenic construct

GFP or other reporter downstream of constituent promoter

Stop cassette

prevents polymerase from going through

Cre recombinase binding sites

allows cre to remove whatever is between loxp sites

Brainbow

other cassette is stop and memory but you can do it with multiple memory cassettes - Have enhancer/promoter for cell types of interest upstream of Cre - Multiple stop cassettes - Cre can excise 1,2, or all of the stop cassettes producing the many colours

Issues with cre in brainbow

. - less efficient at excising stop cassette ○ Can mess w amino acid composition in promoter ○ Or can tamper the loxp sites

Multiple colours

important in brain as lineage tracing has to consider many different cells of the brain

CRISPR

tool you can use to edit the genome, make a mutation, or modify in any context

Defense in bacteria against viruses

original use of CRISPR