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Week 9: Axes of Development (Concepts (Embryological development (Most…
Week 9: Axes of Development
Concepts
Deep homology
Homologous structures
Shared by common ancestor, e.g. human limbs and bird wings
Often arise through homology at gene level
Development differs between lineage but uses evolutionary ancient genes within lineages
Embryological development
Most decision-making involves transcription
Cell position identified
Cell fate assigned
E.g. establishing dorsal-ventral axes
Subdivide into segments
Subdivide into germ layers
Produce various tissues/organs
Human eggs
Contain large stores of
ribosomes
and
nutritive proteins
poised for rapid initiation of cell division
Unequal distribution of mRNA/proteins provides basis for cell
polarity
, setting up for development (maternal morphogenetic factors)
Has protective qualities e.g. UV filters, DNA repair enzymes
Cytoskeleton
Highly organised, polarised protein rods and fibres:
microfilaments
,
intermediate filaments, microtubules
Serves as highway system for
intracellular transport
and exhibits asymmetry of cytoskeleton elements that plays fundamental role via directed transport of molecules
Kinesin
Myosin
Dynein
Organisation permits
localisation of mRNA
encoding transcription factors that provide positional information
Translation of mRNA
and
diffusion of proteins
leads to formation of transcription factor gradient with high concentration at site of mRNA localisation
Asymmetric distribution
of mRNA during cell division leads to daughter cells inheriting different amounts of regulator and hence following different pathways
Germline
During formation of
Drosophila
egg, polarised microtubules form and extend from the oocyte nucleus toward the posterior cytoplasm
Oskar mRNA
binds adaptor proteins that interact with microtubules and transport RNA to posterior
These
pole cells
form the germline; germline cells set aside from soma early in development
Saturation mutagenesis
Generates mutants that impact body pattern, i.e. genotype of mother determines phenotype of progeny (NOT to be confused with maternal inheritance)
Maternal effect genes
Produce RNAs/proteins located in the egg with function in early development and direct influence on phenotype
Patterns form in oocyte while developing in oocyte of mother
Maternal expression of genes responsible for creating asymmetries and chemical gradients that zygote/embryo use
Hence, proteins already present at fertilisation
Bicoid
ME gene whose protein concentration patterns the anterior-posterior axis during Drosophila embryogenesis (along with HB-M)
Anterior:
bcd mRNA
All progeny of same mother have same phenotype, but may have different genotypes
Posterior:
nos mRNA
Zygotically-acting genes
Genes expressed in zygote as sole source of gene product, in contrast to maternal effect genes
Morphogen gradients
Morphogens
: secreted diffusible molecules that determine cell fate in a concentration-dependent manner
French Flag Model
Each cell has potential to develop as blue, white, or red
Position of each cell defined by concentration of morphogen
Cells interpret position value and differentiate to form a pattern (blue/white/red like French flag)
Note that gradient is high to low, need not be linear
Threshold-dependent
determination of cell fate yielding varied levels of activated transcription factor and different gene set expression
Developmental strategy
Mother deposits material that creates
asymmetries
in egg at poles (also influenced by cytoskeleton)
These set up gradients that create chemical longitude and latitude system
Germline cells develop, and morphogen gradients laid down on A-P/D-V axes
Territory is subdivided into broad
domains
(basic body plan map)
Gene interaction subdivides these areas and cells differentiate; committed to memory
Anterior-posterior axis
Sequential expression of gene classes
Maternal effect
genes, then zygotic
Gap
genes divide embryo into broad regions
Pair-rule
genes divide embryo into stripes, defining segment borders
Segmentation polarity
genes divide segments into anterior and posterior halves
Homeotic selector genes
specify the identity of each segment
E.g. abdominal-A
E.g. engrailed
E.g. even-skipped and fushi tarazu
E.g. hunchback
E.g. bicoid
Drosophila
Axis determined by gradients of Bicoid (product of bicoid) and HB-M (product of hunchback)
mRNA maternally deposited in the egg
Bicoid gradient determines axis (alongside HB-M gradient)
bcd mRNA tethered to negative ends of microtubules via 3'UTR
HB-M protein gradient depends on Nanos protein
nanos mRNA tethered to positive end of microtubule via 3'UTR
Nanos protein gradient blocks translation of hb-m mRNA, resulting in HB-M gradient
Morphogens
Caudal
Maternal product crucial in posterior end axis formation
Only translated after fertilisation
Caudal mRNA evenly distributed
Bicoid (non-TF) binds 3'UTR of caudal mRNA
Caudal protein synthesis inhibited and A-P gradient formed
Bicoid
Regulates expression of genes responsible for anterior structures
Expressed in ovary
Stimulates hunchback
Maternal bicoid mRNA packaged into head of developing embryo
Nanos
Inhibits translation of hunchback mRNA
Causes deadenylation when bound to Pumilio (instead of adenylation)
Hence no hunchback translation, no Hunchback protein produced
Abdominal formation is allowed (instead of anterior structures)
Expressed in ovary
Regulates expression of genes responsible for posterior structures
Maternal Nanos mRNA packaged into tail of developing embryo
Hunchback-maternal
Expressed in embryo
Regulates transcription of genes responsible for anterior structures
mRNA/protein level changes
Oocyte mRNA levels
High bicoid at anterior
High nanos at posterior
Consistent high levels of hunchback and caudal
Early embryonic protein levels (denoted anterior to posterior)
Decreasing levels of Bicoid
Flat then spike and decrease of Hunchback
Increasing levels of Caudal (flattening)
Increasing levels of Nanos (exponential)
Positional information
bicoid
bcd null
mutants develop without head or thorax
Bicoid gene mutation is lethal
Overexpression in mother (transgenic)
Normally: cells forming
cephalic brow
determined by specific Bicoid concentration
With increasing maternal dosage of
bcd+ gene
, brow is present more and more posteriorly
Anterior segments are pushed further down embryo
Research
PAX
Paired box
: genes encode transcription factors important in early development and specification
PAX 6
orthologous gene required for eye development
Mouse Pax6 expressed in Drosophila does not give mouse eye, but activates Drosophila eye development circuitry
Eye with crude connections to CNS
Eye formed in wrong place e.g. leg
Demonstrates conservation of Pax6 role in eye development
Eyes in insects and vertebrates are homologous structures
Hox genes
Important gene in development that is used across diverse species such as flies and men (deep homology)
Parallels between insects and mammals
Hox genes arranged and expressed in same order and direction
E.g.
Hom/Hox
genes
Insects express
1 Hom-C cluster
Mammals express
4 Hox clusters
Experiments
Find Hox genes by
hybridisation
to look for homologies with homeotic genes, since orthologous developmental genes usually specify
equivalent structures
Knockout mice
Exhibit similar phenotype to null flies
Homeotic mouse mutant: KO of
Hox C8
resulted in enlarged vertebra, extra rib on L1 vertebrate, possibly clenched fingers
Transformed segmental identity to more anterior, same as nulls in flies e.g.
Ubx
mutant
Hence, developmental strategies in animals are ancient and highly conserved
Gene expression
Visualise in developing embryos via two methods
In situ hybridisation for visualising mRNA transcriptions
Transcribe
cDNA
clone of gene to obtain single-stranded RNA probe with modified nucleotides, complementary to mRNA sequence
Incubate fixed embryo/dissected tissue with
RNA probe
(which hybridises to mRNA)
Wash away unbound probe
Add
enzyme-conjugated antibody
to specific modified nucleotide
Wash away unbound antibody
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Immunolocalisation of protein expression
Express
cDNA
clone of gene in bacteria
Inject into vertebrate host
Extract antibodies (
IgG
) to protein
Incubate fixed embryo/dissected tissue with antibody, which binds to protein
Wash away unbound antibody
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OCT4
Pluripotent transcription factor
Gene is first transcribed in 4-8 cell stage of human embryo
Expression can be silenced via
CRISPR
deletion of
POU5F1
gene
Drosophila melanogaster
Diploid animal used as development model (NB: different embryonic development compared to vertebrates)
Beneficial traits
Exhibits pleiotropy
Exhibits redundancy
Crosses easy to do
Many progeny produced
Amenable to
mutagenesis
Many developmental mutants; easily studied by inspecting embryos
Drosophila Complete Genome
sequence available
Use in research
Many developmental genes in vertebrates (e.g. Hox) first identified in Drosophila
Demonstrates many general principles of development conserved across animals (e.g. morphogen gradients; pattern formation)
Experimentally very powerful (e.g. mutagenesis; transgenics)
Much less complex than mammalian development
Study method
Mutagenise with EMS to give point mutations
Screen for female sterile, recessive mutations
Clone genes and study gene products
No eggs
Eggs with defective embryos
Ethylmethanesulfonate
is an alkylating agent that adds ethyl and causes specific mispairing
Conserved pathway
Regulation of nuclear transport of transcription factors
Drosophila
Spatzle activates toll
Activates tube
Activates pelle
Activates DIF
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Mammals
IL-1 activates IL-1R
Activates myd88
Activates IRAK
Activates TRAF-6
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Dorsal-ventral axis
Dorsal
Function
Transcription factor with mRNA/protein evenly distributed throughout the embryo
Dorsal gene (dI) encodes D-V morphogen 'dorsal'
Active ventrally in nucleus; inactive dorsally in cytoplasm
Active protein expressed highly in cells of ventral midline
Cytoplasmic activation
Spatzle
binds
TOLL
receptor on embryo cell surface
Cactus
is phosphorylated and releases
Dorsal
(i.e. normally bound and inactivated) to move to ventral nucleus
Transcribes zygotic ventral patterning genes
Not lateral/dorsal; D-V pattern
Dorsal remains in cytoplasm on dorsal surface; dorsal cell genes not turned on
Transcription factor gradient
Activated before establishing gradient from dorsal region (low) to ventral surface (high)
Spatzle and Toll gene products deposited in oogenesis and released during embryogenesis
Interaction leads to Spatzle-Toll complex triggering signal transduction pathways in cells
Cells release Dorsal, migrate to nucleus, and activates genes for ventral fates
Drosophila
Amnioserosa
Extraembryonic epithelial tissue that covers dorsal side of embryo
From lateral view, ventral to dorsal:
mesoderm, neurogenic ectoderm, lateral ectoderm, dorsal ectoderm, amnioserosa
Relation to vertebrates
Inverted
Arthropod
: Circulatory system dorsal and nerve cord ventral
Vertebrate
: nerve cord dorsal and circulatory system ventral
Homology of signals
Dpp
(Drosophila) to
BMP-4
(Xenopus)
Sog
(Drosophila) to
Chordin
(Xenopus)
Gene classes
Gap genes
Sequence of events
Mother sets up gradient of
bicoid
mRNA
Activates
hunchback-zygotic
expression
Activates other gap genes:
giant, Kruppel, knirps
Have distinct expression regions
Kruppel
has concentration-dependent response to Hunchback
Intermediate
levels
activate Kruppel
Low levels
fail to activate Kruppel
High levels
of Hb in anterior suppress Kruppel transcription
First zygotic
A-P genes
Mutant
phenotype
Large, contiguous section missing (gap)
Kruppel/knirps promoters
have differential sensitivity to maternally derived transcription factors (
Bicoid, HB-M, Caudal
)
Targets of gap gene TFs bifurcate
One branch to establish correct number of segments
One branch to assign proper identity to each segment
Expressed in adjacent domains
Pair-rules genes
Expressed in alternating bands resulting in zebra pattern that divides embryo into 15 subunits
Three primary genes
Even-skipped (eve)
Hairy
Runt
.
Five secondary genes
Incl.
fushi tarazu
In situ hybridisation
mRNA expression patterns of two pair-rule genes: even-skipped, fushi tarazu
Segmentation polarity genes
Wingless
(wg),
engrailed
(en)
Mutations
Affect wing development in adult, although both are expressed in many places/times
Wingless gene encodes constituent of the
Hedgehog
and
Wnt
signalling pathways
Receptor-mediated pathways that promote cell differentiation via signalling to the nucleus
wn encodes diffusible signalling protein
Homeotic selector genes
Regulated by interaction between
pair-rule
and
gap
genes
Homeosis is development of one body part with the phenotype of another
E.g. Antennapaedia mutant
All contain homeobox domain (
homeodomain
)
Allows transcription factors to bind DNA
May be activators or repressors of transcription
In humans, these are homeobox (
hox
) genes
Note that not all homeobox-containing genes are homeotic genes associated with development
Encode TFs and control
segment identity
E.g. Deletion of
ultrabithorax
turns third thoracic segment into second segment
Results in complete identity change: fly has four wings instead of two