Techn. Done - MCB104 - Lecture 17: Developmental Genetics & Personalized Genomics

What is Developmental genetics?

• The study of genetic control of organismal development

  • Many genes that control development are highly conserved throughout evolution

  • Genes involved in key developmental decisions often occur in hierarchies

  • Ex: How does single cell of fertilized egg differentiate into hundreds of cell types?

How is all life related?

• Cells of many eukaryotes have structural features in common: for example nuclei and mitochondria

• Metabolic pathways are virtually identical in all organisms

• Almost all cells use same genetic code

• Many homologous proteins have highly conserved amino acid sequences

• Means information acquired from a model organism can often be applied to other organisms

What does the Pax6 gene do?

• Drosophilia, mice, and humans are mutant alleles of the eyeless/Pax-6/AN Gene

What are some different developmental schemes?

• Disparate strategies are sometimes used by different organisms to accomplish the same developmental goal

• Ex: cell fate in 2 cell embryos of C. elegans and humans

  • Mosaic determination in C. Elegans

    • Each cell has been assigned a developmental fate

    • Abnormal development if one cell is removed

  • Regulative determination in humans

    • Cells can alter their development fates according to environment

    • Twins result if 2 cells are separated

What’s the C. elegans lineage?

• The location of the sperm fusing with the oocyte determined Anterior/Posterior polarity

• The first cell division produces an anterior AB cell and posterior P1 cell

• Each division has a different fate based on neighboring cells

• Invariant lineage for all 959 somatic cells in hermaphrodite (1031 in males)

Screen Example: Drosophila Eye Development

• Eye development is easy to study in flies

  • Visible, non-lethal

• Ommatidium: Each optical unit of compound eye

  • In Drososphila there are 8 photoreceptors per ommatidium

How do you primary screen for an ommatidium defects

• Mutagenized flies

• Identified mutants where ommatidium lack 7th photoreceptor

• Performed complementation tests and found 2 complementation groups

How do you identify mutations?

• Mutagenesis method determines how you identify mutations

  • Transposon tagging allows inverse PCR to sequence flanking sequence

  • Positional Cloning narrows location on chromosome

    • Typically sequence candidate genes to validate

  • Genome sequencing identifies all polymorphisms

    • Compare sequences within complementation groups to find shared mutated genes

  • Verifying gene requires you either

    • Rescue with wild-type transgene

    • Recapitulate phenotype by introducing mutation into wild type strain

What is sevenless and bride of sevenless

• The 2 genes identified in the screen for R7 loss are sev and boss

• Structure gives clue for function

  • sev is an RTK

  • boss is an orphan GPCR (no known ligand)

• boss is the ligand that activates sev activating a signaling cascade

Secondary Screen - What are Pleiotropy and Redundancy?

• Some components of genetic pathways are missed in primary genetic screens

  • Pleiotropy: genes may be involved in several developmental pathways, including viability

  • Redundancy: 2 or more genes may perform the same function, a mutation in one will NOT result in a mutant phenotype

Secondary Screen- Why use Modifier Screen may identify pleitropic genes

• Organism used for mutagenesis has a hypomorphic mutation that results in a sensitized background

• Heterozygous mutations in a pleiotropic gene may modify phenotype caused by hypomorphic mutation

What’s a Modifier Screen for Pleiotropy?

• Using a hypomorph, can screen for enhancing and suppressing mutations

  • Hypomorph is more sensitive to mutations than wild-type

What are transgenes for sensitized backgrounds

• Can use transgenes to generate gain of function mutations

  • With appropriate regulatory sequences, can be limited to specific cells

• Overexpression of Ras from transgene results in slightly rough eye

• Additional mutations that enhance or suppress the phenotype were identified

What is involved in the Sevenless pathway?

• DOS: daughter of sevenless

  • Not shown, scaffold protein

• DRK: Downstream of Receptor Kinase

  • Enhancer of sevenless

• SOS: Son of Sevenless (if hypomorph of SOS, it would be fewer R7, if knock it down, pathway lowers further, enhancement of SEV phenotype, but it wouldn’t affect the Ras, it’s always open)

  • Gas-GEF

• Ras Pathway:

  • Ras: Monomeric GTPase

  • Res-GAP: Ras GTPase activating protein

  • RAF: MAPKKK

  • MEK: MAPKK

  • MAPK: Mitogen activated protein kinase

• TFs

  • PNT: pointed, activator of R7 genes

  • Yan: anterior open, Antagonist of R7 genes

Show the pathway of Sevenless

What are some key points of the sevenless pathway?

• Can you find:

  • Monomeric GTPase regulation

  • Kinase Cascade

  • Positive Regulation

  • Negative Regulation

  • Double Negative Regulation

• For each protein can you predict whether it was identified as an enhancer or suppressor of

  • sevenless hypomorph

  • Ras gain of function

How can you do systematic screening of mutants

• Mutagens alter genome at random, making traditional mutagenesis screens inefficient and incomplete

• For model organisms, centralized centers maintain thousands of stocks, each with specific mutation

• Many model organism have consortia whose goal is to generate knockouts of every ORF

• Can be combined with nonmutagenic methods like RNAi or CRISPRi

  • Knockdown function in screen, acquire existing null mutant, assess phenotype

How can you analyze Developmental Pathways

• Characterize action of each gene in pathway

  • Location and timing of gene expression

    • During development, where and when is mRNA found

  • Location of protein product

    • During development, where and when is protein found?

  • Developmental phenotype

    • What cells or tissues are affected by loss of function

How can you track RNA expression with RNA in situ hybridization

• RNA in situ hybridization

  • Label cDNA of gene of interest

  • Use as probe to detect mRNA in organism

  • If Fluorescent probe, called Fluorescent in situ hybridization (FISH)

How can you track RNA expression with RNA seq

• Determine sequences of all mRNAs in cell

• Frequency of sequence relates to abundance in cell

How can you track protein localization?

• Immunostraining

  • Use antibodies against specific protein

  • Specimen must be fixed

• Translational reporter

  • Directly tag protein

  • Need to be certain tag doesn’t disrupt protein

How can you determine required location and timing? Example recombinant system?

• Genetic mosaics - individuals composed of cells of more than one genotype

• FLP/FRT recombination system

  • Similar to Cre/LoxP

  • Found naturally in yeast

  • FLP protein catalyzes reciprocal crossing-over at DNA targets called FRTs

  • FLP can be expressed from a transgene in certain cells

  • FLP originally not thermostable for mammals, but enhanced FLP (FLPe) is functional

What is FLP for Mosaics?

• Used FLP FRT to induce mitotic recombination in dihybrid for gene of interest and linked marker gene

• Cells lacking marker gene and lack gene of interest

• Can characterize which cells require target gene to have wt phenotype

  • In sev this is R7

  • In boss this is R8

How can you determine timing

• Temperature sensitive alleles can be activated/inactivated by changing temperature

• Some other methods:

  • Chemical agonist/antagonists

  • Photoinactivation with tagged genes

How can you determine Order

• If loss or overactivity of gene A function affects expression of gene B and not the converse, then gene A functions upstream of gene B in a genetic pathway

• Ex:

  • Expression of prospero is activated by Ras, but Ras isn’t activated by prospero

  • Therefore, Ras is upstream of prospero

How can you determine Epistasis, Developmental Pathway Edition

• Start with mutations in Gene A and B

• Phenotype of double mutant resembles one of single mutants

  • Usually defines earlier acting step in pathway

• Useful in investigating switch/regulation pathways (on/off)

• Required Conditions

  • Phenotypes of single mutants must be different

  • Mutant alleles must be null or constitutive

Epistasis and Sevenless - describe Ras?

• Ras gain of function transgene masks sev^- phenotype

• Ras is downstream of sev

Describe eyeless/Pax6

• Identified in mutagenesis screen

• Phenotype - no eyes

• In humans, mice and flies, loss of Pax6 leads to defects in eye formation

How do Expression of Pax6 work

• In situ hybridization probe for Pax6 mRNA in human fetal tissues

• Expression is in areas that will eventually, produce eyes

• Suggests potential conserved role

How can you identify expression patterns of regulatory sequences?

• Isolate segments of DNA around gene of interest

• Insert segments of vector containing minimal promoter and reporter gene

• Insert constructs into organism

• Observe expression pattern of reporter

• Often done by sequentially narrowing down fragment size

• Sometimes identified via CHIP-seq data

How does Regulation of Pax6 occur?

• Many enhancers regulate expression of Pax6

  • Note retina enhancer is intron

• Many of transcription factors targeting Pax6 enhancers are homeobox genes

What are Pax6 Regulatory Targets

• Can use CHIP-seq to identify specfic enhancers that Pax6 binds

• Different Tissues have different TFs, leading to different expression patterns

What is GAL4/UAS for Ectopic Expression

• GAL4 is a yeast transcription factor and very strong activator of gene expression

• Upstream Activation Sequence (UAS) is the enhancer sequence GAL4 binds

• Conditional Expression lines can be made using 2 constructs

  • GAL4 with a tissue specific regulatory sequence

  • Desired gene with UAS

• Modular system can simplify experimental setups

Is Pax6 sufficient for eye formation?

• Yes

• Expressing Pax6 ectopically using different Gal4 constructs to activate UAS driven Pax6 transcription leads to formation of eyes

What is Evolutionary connection and Pax6

• Cavefish often lose vision over evolutionary time

• One mechanism is loss of Pax6 expression in their optic buds

What is personalized Genomics

Genetics can influence a wide range of medically relevant characteristics

• Disease risk

• Drug Responses

• Metabolic Processes

• Immune Function

Personalized Genomics is the study of individual’s genome in order to make predictions about range of traits

• Often applied to medical purposes

What is Prenatal and Neonatal Genetic Testing

• Most widespread example of personalized genetics, samples are taken from embryos/fetuses and checked for various genetic diseases

• includes

  • testing for various aneuplodies and chromosomal rearrangements

  • identifying known mendelian disease alleles

Describe Cancer Genomics/Precision Oncology

• Variants of specific genes are associated with increased risks of cancer

• Sequencing tumors can help make predictions as to tumor behavior

• Combining individual and tumor WGS can guide therapeutic strategies

What Infectious Disease Genomics

• Sequencing can be used to identify specific variants of an infectious agent, serving as diagnostic tool

• Specific genetic variants influence susceptibility to different pathogens

  • Heterozygotes for the sick cell allele are more resistant to malaria

  • Homozygotes for the CCR5 -Δ32 allele to resist HIV infection but are most susceptible to west wile virus

• Identifying those variants allows patients to better estimate risks

What is Pharmacogenomics

• Pharmacogenomics is the study of gene/drug interactions

• Drug metabolism includes various functions including

  • Uptake

  • Release

  • Breakdown

  • Products

• Each of these functions involves various genes

• Alterations in function of these genes can alter effectiveness, optimal dosage, and side effects of drugs

• Ideally, one could sequence a patient’s genome and use the results to identify which drugs and at which dosage would be the best

What is Warfarin

• Widely prescribed anticoagulant drug used to prevent blood clotting related diseases in patients with deep vein thrombosis, atrial fibrillation, recurrent strokes or heart valve prosthesis

• High inter-individual variance in optimal dosing combined with narrow therapeutic range making prescribing it challenging

  • Dose too low: blood clotting

  • Dose too high: internal bleeding

• Need to identify sources of variation to predict ideal dosage

What is Warfarin Mechanism of Action

• Warfarin is an inhibitor of vitamin K epoxide reductase complex subunit 1 (VKORC1)

• Inhibition of VKORC1 reduces amount of Vitamin K available as cofactor of clotting proteins

• Variation of VKORC1 accounts for ~30% of variation in warfarin dose

What are Warfarin Pharmacokinetics

• Warfarin is a mixture of R and S stereoisomers

• S stereoisomer is primarily metabolized via CYP2C9

• Variation of CYP2C9 and VKORC1 together account for ~40% of variant in dose

• Adding age, weight, height, interacting drugs and symptoms only predicts about ~55%

How can you interpret data correctly

• Studying effects of specific variants in human genome is extremely challenging

• Literature full of associations based on

  • Very low N, including case reports

  • Single studies failed to replicate

  • In vitro/molecular/functional assays

• Generally, put more trust in connections that have been demonstrated in multiple studies