Early vertebrate development & developmental genetics (lectures 22&23) Flashcards
when does patterning occur in early development?
in the first 3 weeks
what is morphogenesis?
the emergence of form
what is axis formation?
occurs in a number of parts of the developing embryo
what is body plan?
the map of an organism
what are the 3 crucial axis that embryos must develop?
anterior-posterior - runs from head to tail
dorso-ventral - runs from back to belly
left-right - between 2 lateral sides of the body
dorsal
back
ventral
body
anterior
front
head
rostral
cranial
posterior
back
tail
caudral
components of cell signalling
release of signal by source cell
reception of signal by target cell
transaction of signal
cellular response
what are morphogens?
substance that controls positions of specialised cell types during morphogenesis
pattern the embryo
form gradients which activate different genes at different concentrations
influence cell fate
how do morphogens achieve long range signalling?
growth factors are often morphogens different mechanisms diffusion over long distances relay from cell to cell cellular extensions not mutually exclusive
what is the first step in establishing A-P polarity in flies?
a gradient of bicoid
fly egg us a single cell so free diffusion possible
low affinity binding sites at high concentrations of bicoid activate orthodenticle and hunchback mRNA
high affinity binding sites at low concentrations of bicoid repress caudal protein
signalling centres in early mammalian embryos
anterior visceral endoderm (AVE)
node (‘organiser’)
what is the anterior visceral endoderm (AVE)?
appears first and patterns only the anterior part of the embryo
what is the node (‘organiser’)?
patterns the whole embryo, working cooperatively with the AVE at the anterior end of the embryo
loss of the organiser genes expression affects the body plan of the embryo
left-right axis formation
vertebrate body is not bilaterally symmetrical
left is different from the right - especially organs
• heart, stomach and spleen on left
• liver on right
• lungs have different number of nodes on the left and the right
breaking of symmetry first takes place at the node
morphogens resustin the activation of a specific signalling pathway only on the left side
left-right signalling pathway
1) initiated at the node
2) nodal (a TGF-beta family morphogen) signalling activated on the left side of the embryo
3) nodal activates Pitx2 (a homeobox containing gene) which regulates downstream gene expression
4) an organ specific process, dependent on dosage of Pitx2, governs asymmetric organ development
right side is the default
if pathway isn’t activated, left side would be the same as the right side
experimental technique that taught as abut left-right pattering
invert and transplant node
genetic technique that taught as abut left-right pattering
knockout lefty-1 gene
right-left determination factor 1
the A-P axis is determined by a number of morphogens
Wnt signals specify the anterior region
RA patterns the midbrain, hindbrain and trunk
FGF gradient pattens the caudal region
hox genes
hox genes in mammals and flies
hox genes are homologous to homeotic selector genes in flies
4 hox clusters in mammals, 1 in flies
specific identity of AP patterned structures is defined by homeobox containing genes
how is the limb patterned?
in several axis
patterns in the proximal-distal axis
if you disrupt the pattern you disrupt the limb
D-V patterning
neural tube is patterned
• BMP from dorsal region
• Shh from ventral region
opposing gradients of the BMP and Shh specify neuronal subtypes
activates expression of homeobox genes
essential for normal development of the nervous system
what are congenital malformations?
those already present at birth
usually occur during embryogenesis
congenital anomalies result from disruption of normal development
can have genetic or environmental causes
what is embryogenesis?
first trimester in humans
genetic causes of congenital malformations
chromosomal defects
syndromes
single genes
multi-gene interactions
environmental causes of congenital malformations
maternal diabetes fever prescription drugs recreational drugs pollutants dietary deficiencies/excesses
what are we trying to achieve by studying congenital malformations?
identify cause of malformation
predict whether it is likely to happen again
understand why specific factors lead to malformations
devise strategies for preventing malformations
anatomical approach to studying development
study of naturally occurring mutations in human and animal models
utilised histological techniques to study how the defective structures formed and how they ended up
key methodologies for anatomical approach to studying development
gross morphology - dissection
histology
embryos/tissues dehydrated
embedded in paraffin wax
thin slices cut
different tissues/cell types visualised
physical manipulation approach to studying development
understanding development tells us about normal development
manipulation of developing embryos are used to ask specific questions and test hypotheses
key methodologies for physical manipulation approach to studying development
removal of part of the embryo and looking at the consequences
replacing one part of an embryo with another
using a drug to interfere with a developmental process
followed by anatomical analysis
genetic approach to studying development
congenital abnormalities are frequently caused by defects in genes
have a lot of genetic information from studying families with congenital malformations
basic science research has revealed many new genes that act in pathways
key methodologies for genetic approach to studying development
visualisation of gene/protein expression
measurement of levels of gene/protein expression
disruption of gene function
• total knockout - in every cell of the embryo
• conditional knockout - in cell types of choice
ectopic or ‘extra’ gene expression - knockins and transgenes
genetic expression analysis
powerful tool for showing where genes are active
closely linked to anatomical approach
gives spacial and temporal information
crucial for linking genes to specific cell types
mRNA and protein localisation is not always the same
methods for analysing gene expression
in situ hybridisation - mRNA
immunohistochemistry - protein
linkage of gene regulatory elements to a reporter gene - transgenesis
what are knockout mice?
creating a mouse with an inactive copy of the gene of interest
1) manipulate the DNA
2) transfer mutated DNA into mouse embryonic stem cells by transgenesis
3) incorporate transgenic ES cells into normal embryo
4) breed mice to obtain ‘knockout’ offspring
creating a traditional knockout mouse
modified ES cell and host embryo have different coat colours
1) clone gene and put in bacteria
2) manipulate gene so its malfunctional
3) transfer into embryonic stem cells
4) transfer into an early embryo to be incorporated into the inner cell mass
5) transfer new embryo into a new mother which has a different coat colour to the original
6) allow offspring to be born
7) select embryos that have mixed coat colour
8) breed until you get offspring that are only the coat colour of the ES cells - these are the knockout mice
takes 1-2 years
what is CRISPR/Cas9?
- uses synthetic RNA to guide enzyme (Cas9) to correct place in the genome
- inject RNA and nucleases into fertilised embryos
- cuts both DNA strands at the same time
- can target multiple sites in the genome
can generate transgenic/mutant twice in < 1 month
cheap and efficient
what is the effect of knocking out a gene?
can knockout gene of choice
see what effects it has on the embryo
complemented by anatomical analysis
what is gastrulation?
the process where 3 germ layers are formed
occurs in early development
key process is formation of the embryo
why do lp mice get neural tube defects?
lp mouse first described in 1948
gross morphology shows us that embryos are short and that neural tube closure is initiated
histology revealed a broadened floor plane
anatomical approach tells us what the main abnormalities are
1/1000 live births generally
hypothesis - broad floor plane prevents neural folds coming together
will tying together the neural tube will lead to closure?
ties threat around neural tube and Brough neural folds together
followed by histological analysis
did not result in neural tube closure
lp gene in Vangl2
shown to be a component of a signalling pathway - Wnt dependent pathway
regulates convergence-extension movements at gastrulation in vertebrates
PCP signalling regulates convergent extension movements
- take place at gastrulation
- occur by a process of cell intercalation
- result in the narrowing and lengthening of the embryo
- disruption leads to short fat embryos
- disruption of PCP signalling in mice also causes neural tube defects
human patients with neural tube defects
1/1000 babies have neural tube defects
now have identified mutations in PCP genes in human families with neural tube defects • Vangl1 • Scrib • Celsr1 • Prickle
allows genetic counselling
new therapies fro NTDs?
folic acid (B vitamin) can prevent some NTD • NTD seen in lp are not prevented by folic acid
inositol (another B vitamin) can prevent NTD in mice
inositol is now in clinical trial for prevention of NTD in humans
