L21-23: Developmental Genetics Flashcards
What is developmental biology?
The study of the process by which organisms grow and develop
Focuses on growth, differentiation and morphogenesis
Why is developmental biology important?
To understand congenital malformations and diseases and understanding regeneration, repair, aging and cancer
What is development in mammals?
The period between fertilisation and birth
What is the developing organism known as?
An embryo
What does the study of developmental biology focus on?
The initiation and construction
Where does most organogenesis occur?
During embryogenesis
Where do most congenital anomalies take place?
In the first 8 weeks
What are the 3 general approaches to studying development?
Anatomical
Physical
Genetic
What is a model organism?
A well established experimental biological system
Which gene in the eye can be studied to find functionality?
The Pax6 gene
What is the oldest know bilaterian?
The Ikaria wariootia
Which genes are focused on in Drosophila?
Homeotic selector genes (Homeobox genes)
What is temporo-spatial collinearity?
When genes are expressed in chronological order
Why is Drosophila useful?
-Genome sequenced
-Genes have some homologues in mammals
-Many mutants available
-Short life cycle
-Easily accessed larvae
-Allowed the understanding of basic gene networks that regulate early body plan
How can we find what a gene does?
By removing it
Why are zebrafish useful?
-Genome sequenced
-Fundamental developmental processes similar to mammals
-Easy to manipulate genetically
-Easily to manipulate experimentally
-Regenerates body parts
-Transparent embryos
Why are amphibians useful?
-Have large embryos to be easily manipulated
-More similar anatomically to mammals than files/fish
-Capable of regenerating body parts
Why are birds useful?
-Large accessible eggs
-Easy to manipulate and image
-Anatomically similar to mammals
What is a disadvantage of using birds as a model?
They have complex genetics
Why are mice useful?
-Genome sequenced
-Similar to human
-Large number of mutants available
-Possible to manipulate genome
-Relatively rapid life cycle
-Model of choice for most developmental genetics
What are the disadvantages of using mice as a model?
-Difficult to physically manipulate
-Relatively affordable
What genetic tools are available for use?
-Morpholinos (siRNA)
-Chemical mutagenesis
-Transgenesis (transient)
-Transgenesis (germline)
-Single gene knockout and knockins
-Conditional gene knockouts
-CRISPR
Why are animal models essential?
-Cannot experiment on humans
-Cannot model disease processes in cell culture
-Cannot test toxicity of new drugs/ disease treatment in cell culture
-Cannot model complex development/ aging processes in cell culture
What would be advantages to using humans as a model?
-Have genetic disease kindreds
-Many tools available for identifying disease genes
What are disadvantages to using humans as models?
-Cannot genetically manipulate (ethical!!)
-Complex genetics
-Limited access to human embryos
What are organoids?
Self-organising three-dimensional cell cultures
Where were organoids derived from?
Pluripotent stem cells
What are the main functions for organoids?
They are used to recapitulate early stages of development
What are the key characteristics for a good model for developmental genetics research?
-Sequenced genome
-Anatomically resembles human
-Rapid rate of development to maturity
-Large number of offspring
-Easily manipulated and genetically tracible
-Readily available
When does patterning occur?
Early in development
What is morphogenesis?
The emergence of form, the process by which an organism/structure beings to develop 3D form
What are the 3 fundamental processes in developmental biology?
Morphogenesis
Differentiation
Growth
What is axis formation?
The establishment of body plan
What is a body plan?
The map of an organism
How many axis are crucial to the foundation of the body?
3
What are the 3 crucial axes of the body?
-Antero-posterior
-Dorso-ventral
Left-right
What is body axis dependent on?
Different parts of the embryo becoming distinct
What are examples of long range signalling molecules required in embryos?
Hedgehog, Wnt and TGFbeta
What is an example of a short range signalling molecule required in embryos?
Notch
What determines genes being activated in long range signalling?
The type of signal and how strongly it binds
What are the 4 components to cell signalling?
-Release and transmission of a signal by source cell
-Reception of the signal by the target cell
-Transduction of the signal
-Cellular response - requires activation/repression of gene expression
What is used to pattern the embryo?
Growth factors (morphogens)
How do morphogens work?
Form gradients as they diffuse which activates different genes at different concentrations and determines specific characteristics
What is the first step to establish Anterior-posterior polarity in flies?
A gradient of bicoid
How do morphogens achieve long range signalling in cellularised tissues?
-Different mechanisms suggested
-Diffusion over long distances
-Relay from cell to cell
-Cellular extensions
-Not mutually exclusive (different morphogens, different mechanisms)
What is the sonic hedgehog signalling?
It is a crucial vertebrate morphogen
What are the signalling centres in early mammalian embryos?
-Anterior visceral endoderm (AVE) - first, patterns anterior part of embryo
-Node - patterns whole embryo, works cooperatively with AVE at anterior
Where does the breaking of symmetry first take place?
At the node
Which organs differ from left to right?
Left - heart, stomach, spleen
Right - liver
Lungs have different number of nodes
What is the L-R signalling pathway?
-Initiated at the node
-Morphogens secreted
-Nodal signalling activated on left side of embryo
-Nodal activates Pitx2 regulating downstream gene expression
-organ specific process when enough Pitx2 governing asymmetric organ development
What is the function of the Pitx2 gene?
Regulates downstream gene expression
What does the L-R signalling pathway suggest?
That the right side is default
How does Shh (sonic hedgehog signalling) act in L-R signalling?
it acts at the midline barrier to stop left signals getting to the right
What tools were used to teach L-R patterning?
Physical tools: inverting and transplanting the node
Genetic tools: Knockout Lefty-1/ Shh gene
What are the consequences to the Nodal being on the left side?
The thorax becomes inverted (heart and lungs switch)
What are the consequences to the nodal being on the right side?
The organs in the abdomen switch sides or both organs in the abdomen and in the thoracic cavity are switched
What are the consequences to nodal being on both sides?
Left isomerism the organs on the left are mirrored in the right side
What are the consequences of nodal being on neither side?
Right isomerism the organs on the right are mirrored in the left side
Which morphogens pattern the AP axis?
-Wnt signals specify the anterior
-RA patterns the midbrain, hindbrain and trunk
-FGF gradient patterns the caudal region
Which genes pattern the AP axis?
Hox
How many Hox genes are there in mammals?
4
How are the specific identities of AP patterned structures defined?
By homeobox-containing genes
What happens to embryos when Hox6 is activated in ribless regions?
Extra ribs are found
What happens when Hox10 is expressed in the regions ribs are located?
No ribs develop in the embryo
What happens when the Hox10 gene is inactive?
Ribs become everywhere
Which signals are contained in D-V patterning when neural tube is patterned?
BMP dorsal region
Shh ventral region
Why is D-V patterning essential?
For normal development of the nervous system
Where are limbs patterned?
In the proximal-distal axis
Which structure is required for limb outgrowth?
Apical ectodermal ridge (AER)
What is the result of disrupting Hox genes?
Digit patterning defects
How does Thalidomide impact patterning?
It disrupts limb patterning by stopping the production of blood vessels which blocks proliferation and altering the expression of Shh, FGF and Hox genes
Which transcription factor is degraded from Thalidomide?
Sall4
What is the impact of Sall4 mutations?
Can cause Duane Radial Ray Syndrome
What does Sall4 regulate?
Key growth factors and Hox genes in developing limbs
What are congenital malformations?
Malformations that are already present at birthW
hen do congenital malformations take place?
During embryogenesis
Why do congenital anomalies take place?
As a result of disruption of normal development
What environmental factors cause congenital malformations?
-Radiation
-Maternal diabetes
-Fever
-Prescription drugs
-Recreational drugs
-Pollutants
-Dietary deficiencies/ excesses
What genetic factors cause congenital malformations?
-Chromosomal defects
-Syndromes
-Single genes
-Multi-gene interactions
What is the anatomical approach?
The study of naturally occurring mutations in humans and animal models
Use histological techniques to study how defective structures are formed and how they end up
What are the key methodologies for anatomy?
-Gross morphology
-Histology
What is the process of histology?
-Embryo/tissues dehydrated
-Embedded in paraffin wax
-Thin slices cut
-Different tissues/cell types visualised
What is the physical manipulation approach?
-It is based on the principle that understanding abnormal development tells us about normal development
-Manipulation of developing embryos used to ask specific questions and test hypotheses
-Common methodologies are physical and chemical
What are the key methodologies for the physical manipulation approach?
-Removal of part of the embryo
-Replacing one part of an embryo with another
-Using a drug to interfere with a developmental process
What is the genetic approach?
Studying the genes of the families with congenital malformations
What are the key developmental genetic techniques?
-Visualisation of gene/protein expression
-Transcriptomic sequencing
-Measurement of levels of gene/protein expression
-Disruption of gene function (total and conditional knockout)
-Ectopic or “extra” gene expression (knockins, transgenes)
What is gene expression analysis?
Shows when genes are active
Gives spatial and temporal information
Links genes to specific cell types, tissues/ organs
What are methods for analysing gene expression?
-In situ hybridisation
-Immunohistochemistry (uses antibodies)
-Linkage of gene regulatory elements to a reporter gene
What is the effect of knocking out a gene?
Shows the effect it has on the embryo
What is an example of a knockout mouse?
CRISPR-Cas9
What is the benefit of the CRISPR-Cas9 mouse?
Generates mutants faster, with higher efficiency and at a lower cost
What are the different genetic manipulations that are possible?
Knockout of genes (shows what happens when function is deleted)
Single amino acid-substitution (allows reproduction of human mutations)
Floxed mice (allow knockout in defined spatio-temporal manner)
What is gastrulation?
Where 3 germ layers are formed
When does gastrulation take place?
In early development
What is involved in gastrulation?
Cell signalling and cell movements
What are the 3 different cell types developed during gastrulation?
Zygote, blastocyte and gastrula
How can gastrulation be replicated?
During differentiation of stem cells
What are the factors of neural tube defects (NTDs)?
-One of the most common congenital malformations
-~2/1000 live births
-300,000 babies born with NTD each year
-Multifactorial
-Caused by abnormalities in the normal process of neurulation
What is the Lp mouse?
It is a model of severe neural tube defects
What happens during NTDs?
Embryos are short and neural tube closure is not initiated, and there is a broadened floor plate
How was it tested that the broad floor plate prevents the neural folds coming together?
The neural tube was tied together bringing the folds together which resulted in no neural tube closure
What is the Lp gene known as in Drosophila?
Vangl2
What is Vangl2 involved in?
It is a component of a signalling pathway (Wnt dependent pathway)
In flies, the pathway regulates position of cells in cuticle and in eye
Regulates the convergence-extension movements and gastrulation in vertebrates
What is PCP signalling?
It takes place at gastrulation
By a process of cell intercalation
Results in the narrowing and lengthening of the embryo
What is the result of defects in CE?
Prevents neural tube closure
What are therapies of NTDs?
-Folic acid (not NTDs seen in Lps)
-B vitamin - inositol
