Patterning Drosophila Embryo Flashcards
Patterning
Differences between cells getting established
Drosophila early embryo basic
Egg formed in mother ovary
Fertilised
Laid
First just a single nucleus
Then just nuclear division
Within 60 minutes a pop of nuclei has formed
Then by 90 mins they have migrated out to the edge
Then at 150 mins - the syncytial blastoderm with the nuclei lining the outer edge of the syncytium
Then the beginning of cell formation by infolding of the membrane
Pole cells towards posterior form first
Go on to form germ cells
Cell layer forms around the surface
Opaque yolk in the centre
Different cells along ap axis form diff segments
What part of embryo gastrula yes
Strip on the ventral side gastrula yes and forms the inner mesoderm
Most of rest makes ectoderm
Except for gut ectoderm at each end (A and P)
Strip along dorsal makes serosa
Segment identities are already established by 3hrs
AP axis patterning mutants
2 diff classes of mutants
Zygotic effects
Mutant embryo is abnormal
Genes involved are needed to be functional in the embryos cells
Maternal effect genes
Needed functional in mother to produce normal embryo
Mother with mutation makes affected embryo even if embryo has functional copy
Gene function needed during oogenesis
Classes of maternal effect gene mutations
Anterior end affected
Posterior end affected
OSKAR and NANOS mutants
Mother with these mutations
Make egg that gives embryo with no posterior patterning
Needed for posterior patterning in embryo
BICOID mutant
Bicoid mutant mother gives embryo with no anterior patterning
Bicoid functional needed from mother to pattern anterior patterning
Patterning cells in drosophila oogenesis
Ovary in mothers abdomen
Consists of parallel ovariole tubes
Each ovariole is independent of neighbouring ones
In tube oocyte safe produced sequentially
Mature as they move along tube and are laid when mature
Oocyte is not alone
It is accompanied by other cells
-stem cells at proximal end
-produce cells called egg chambers
-each egg chamber consists of large oocyte surrounded by layer of much smaller follicle cells
-at proximal end of egg chamber there is 15 supporting nurse cells
-as egg natures follicle cells produce shell and nurse cells die
Patterning of the oocyte
Occurs by interaction of adjacent egg chambers in tube
Produced sequentially so each chamber has an older sister in front of it
At particular stage the older sister produces a signal
Received by follicle cells on nearest side of younger sister (the egg oocyte end of chamber)
Changes their expression
At later stage a reciprocal signal comes from these follicular cells
This signalling causes MTs to be arranged correctly in the oocyte
These two phases of signalling are necessary for MT arrangement to happen in oocyte
AP axis patterning is dependent in it
Differences between the three cell pops in the egg chamber
Oocyte is big
Stuffed w yolk and maternal gene products
Oocyte is inert and doesn’t transcribe it’s own genes just receives material produced by cells around it
Follicle cells put yolk in the oocyte
Nurse cells are polyploid and polythene - for producing gene products to be put into oocyte
Follicle and nurse cells contribute to oocyte development and die off
MT polarisation in oocyte and it’s patterning contributions
Plus end (where additional subunits are added) is oriented away from nurse cells - pointed towards older sister direction
Minus ends towards nurse cells
Parallel orientation
Kinesics travel to plus end
Dyenins to minus
maternal Gene products given to the oocyte travel along them
OSKAR RNA is transcribed in nurse cell
Carried to plus end by kinesin and builds up at this future POSTERIOR end of the oocyte
OSKAR RNA localisation consequences
Comes into oocyte early
Carried by kinesin to plus end
OSKAR protein translated
Binds NANOS RNA when it comes in
Sets up future posterior end of embryo
BICOID RNA in oocyte
After OSKAR stuff
BICOID RNA from nurse cell binds dyenin
Transported to and enriched at plus end
So is shunted back to minus end nearest nurse cells
Sets up future development of anterior end
Patterning AP of early embryo - ANTERIOR
BICOID rna is localised to this end
It is tethered by cytoskeleton components
Then at about 60mins after laid
Begins to be translated into bicoid protein
RNA tightly tethered but protein is not
Protein produced locally but can diffuse through syncytium common cytoplasm (has only been nuclear division)
This sets up a gradient of bicoid protein conc across ap axis
High conc at ant
Fades into background by about halfway along axis
-because bicoid protein is unstable
- it is produced locally at anterior
-diffusing away
-gradually breaks down as it moves away
-so get stable gradient of bicoid as go along axis
Bicoid properties
Protein is a TF
Controls set of genes
Maternal product TF
Need a lot of Bicoid to turn on Otd
So gradient causes Otd to turn on in anterior half of embryo
The. At lower bicoid conc hunchback is turned on partway through axis? Still in anterior half?
Is still a syncytium so these zygotic products can diffuse out and create gradients in common cytoplasm
Bicoid had consequences on spatial localisation of products
Turns on many targets along axis
This is beginnings of process that subdivides the ap axis of segment identity
Bicoid protein interaction w maternal RNA (caudal eg)
Is able to bind maternal RNA molecules
Maternal Caudal TF RNA binds to bicoid protein where bicoid protein is
And is degraded when it binds
So bicoid protein presence causes degradation of maternal caudal rna
So caudal can’t be present anteriorly
Only posterior?
Patterning embryo posterior - OSKAR and NANOS
OSKAR RNA localised to post of egg
OSKAR protein binds NANOS RNA
Localised NANOS RNA to the posterior
About an hour where NANOS protein is translated and diffuses from anterior
Binds posterior maternal hunchback and causes it’s degradation
Hunchback RNA IS DISTRIBUTED UNIFORMLY
But since NANOS protein in posterior
It is degraded there
Two hunchback RNA stages
Maternal hunchback RNA comes into oocyte and is degraded in posterior by NANOS protein
Then is turned on in anterior (zygotically) by Bicoid protein activity
Gradients in patterning - syncytium vs multicellular environment
In the syncytium
Eg with bicoid
Have bicoid maternal rna localised in anterior
Then protein is only produced in that localised area
Is unstable so diffuses out and breaks down as it moves ooht
Sets up a gradient in the anterior of the oocyte
Has consequences in transcription along the axis
Typically it is different in multicellular environments
Secretion of signal
Causes gradient in extracellular space across the cell surfaces
Different response thresholds in diff cells
So cells respond differently across the gradient
AP axis divisions
Segments
Repeated anatomical units, developmental modules
Simple way of building body complexity
Segments in larva not massively different
But in adult there are many
Segment grouping
Tagmata
Eg
Head
Thorax
Abdomen
Head segments fuse together in drosophila adult and lose a lot of distinct look
when is drosophila body plan determined?
in development of egg
already halfway through embryogenesis (~10hrs) embryo looks v segmented
so body plan already somewhat determined before this
adult structures origin basic
adult doesnt form directly from larva (ie larva doesnt just sprout legs/wings and many larval segments dont correspond to adult ones
adult built somewhat from scratch
larval
larval skin becomes pupa outside
parts of larval tissue in each segment bud off and retain fate becoming the imaginal discs
stay proliferating in larva until it pupates and become adult structures within the pupa
adult segments are given their identity during this stage (stage 13 11hrs in)
gap gene regulation basic
done by morphogen gradients
protein gradients in early embryo revision
bicoid rna anterior side of oocyte
posterior - nanos RNA
hunchback and caudal generally distributed
bicoid and nanos mRNA translated into protein at respective ends
proteins form gradients across AP axis
maternal caudal mRNA translation inhibited by bicoid protein in anterior - so gradient of nanos and caudal from posterior
other way around with hunchback - mRNA translation inhibited by nanos in posteior - forms gradient from anterior
these gradients give the nuclei in the syncytial blastoderm info about their position along the AP axis
act as morphogens and affect zygotic expression of nuclei differently across the AP axis
bicoid maternal gradients to zygotic gene activation revision
bicoid (in anterior) activates Otd and Hunchback zygotic expression (different from maternal hunchback from earlier)
Otd has a higher threshold so is expressed in nuclei only in the VERY anterior end
Hunchback has a lower threshold for bicoid levels so is activated further along the AP axis from anterior
now there is already a difference between very anterior and further less anterior (Otd+Hb and just Hb further down)
gap genes basic
their expression results in patterning of the embryo
from A to P
knirps
hb
giant
kruppel
knirps
giant
hb
tailless
gap gene expression in cells of embryo reflect their AP position “positional info”
gap gene mutants
mutating a gap gene causes a “gap” in the body plan along the AP axis - the gap gene domain is missing
kruppel necessity
is required for thorax segments
mutant gives gap in thorax - head stuck onto abdomen
knirps necessity
required for but further posterior - mutant gives large chunk of abdomen missing
Kruppel and hunchback interaction
kruppel inhibited at high Hb conc
is activated at a lower one
hence its expression in future thorax region
Knirps gap gene regulation
is activated by caudal coming from posterior
inhibited by hunchback from anterior
is active just behind kruppel
cant activate further posterior even tho caudal is higher there as - Tail-less inhibits its expression there
so only activates just where caudal is high enough but its repressors from posterior are low enough
gap gene refinement - cross-regulation
early on a lot of the gap gene domains overlap
but they cross regulate - try to inhibit each other
ends up with dynamic change from fuzzy broad domains to v precise domains of expression
gap gene summary basic
divide ap axis into domains
next step - gap gene products are TF factors that regulate
-segment formation
-segment identity
what genes do gap genes regulate?
pair-rule genes
pair-rule gene expression patterns
each pair-rule gene is expressed in 7 stripes corresponding to alternate PARASEGMENTS (hence 14 parasegments in total)
parasegments are forerunners of the final segments
-even-skipped (eve) is expressed every ODD parasegment
-fushi tarazu (ftz) expressed in even parasegments
-other pair-rule genes (hairy, paired, runt, sloppy paired, odd-skipped, odd-paired etc…) are expressed in the parasegments but are out of sync with them
7 stripe expression pattern of pair rule genes necessity
are important for development
if eve is missing - gives phenotype - just even number parasegments stuck together
ftz mutant just gives the odd number parasegments stuck together
mutations in pair-rule genes affect cells in every alternate parasegment
eve gene regulation
regulation is modular
each stripe regulated independently by a separate enhancer
-put a reporter on one of these enhancers and it will show just ONE parasegement
nuclei in different stripes activate the same eve gene with a DIFF enhancer
eve gene regulation - stripe 2 enhancer
eve expression stripe 2 (parasegment 3 ig)
the enhancer for this one location is regulated by combiantion of maternal gene products and zygotic gap gene expression
hunchback and bicoid (bicoid is maternal) (anterior) gradients activate it
Giant gap gene product inhibits stripe 2 on anterior side
Krupppel inhibits expression on posterior side
mutate giant(or the enhancetr’s giant binding site) - stripe 2 enhancer activation spreads much further anterior than it should
same for posterior but kruppel mutation
mutate bicoid binding site on the enhancer - no stripe 2 at all)
hair an runt pair-rule genes (and eve)
are primary pair-rule genes
similar regulation as eve for all 3 of these
the rest of the pair-rule genes (incl Ftz) dont respond to gap genes
but instead to the primary pair rule genes
called secondary pair rule genes
secondary pair-rule gene regulation
eve in the odd stripes represses ftz expression - so ftz is only active in the even stripes
lots of dynamic interactions between the secondary pair-rule genes similarly to what happens with the gap gene domains cleans up the expression boundaries
order of things affecting ap patterning so far
maternal gradients
gap genes
pair rule genes
next steps after pair-rule gene division into 14 parasegments
- activation of genes that defin parasegmental borders and then patterning detail WITHIN segments
- assignment of segmental identity so that each develops with its characteristic anatomy
segmentation genes
define the parasegmental borders
and then pattern within the segments
pair-rule genes degine the POSITIONS of the parasegments
expression of pair-rule genes is transient (unstable proteins)
so the next genes they activate (segmentation genes) establish the segments permanently
pair-rule genes and Engrailed
engrailed is the key segmentation gene
en initiation is controlled by pair-rule gene combinations
have 14 stripes of engrailed
because ftz and eve combine to activate engrailed
ftz in even no. segments
eve in odd no. segments
engrailed is only activated in certain region within the segments die to other pair rule genes whose 7 stripes are on slightly out of phase with eve and ftz - offset by a few cells
opa (odd paired) prevents activation by ftz in even segments
causes engrailed to be activated only in the anterior of the parasegments
same in odd numbered segments where runt prevents eve activating en except in just anterior due to few cell offset
future body segments relation to parasegments
parasegments are defined by anterior en expression
but future body segments are defined by posterior expression of en
so parasegment and segment borders dint line up
important change at stage where segment genes are activated
after 3.5hrs cell membranes form and CELLULAR blastoderm is formed
TFs can no longer diffuse directly
nuclei in cells need to communicate via secreted signals
eg Hedgehog, Wingless
what does engrailed expression define in segment
the POSTERIOR COMPARTMENT
TF expresed in posterior of each segment
activates signals which stabilise the boundary
posterior and anterior compartment cells differ in adhesion so A and P cells do not mix together and stay separate
stabilising the parasegment boundary
engrailed in the posterior compartment of segment
activates expression of the Hedgehog secreted signalling molecule
hedgehog then diffuses forward (anteriorly) to stimulate
hedgehog activates wingless expression in these cells
hedgehog and wingless form positive feedback loop (wingless activates En?? expression by diffusing to these more posterior cells too)
this keeps expression of En, Hh, and Wg stable even after the pair-rule genes stop their expression - which was necessary for the set up but are no longer needed)
Patterning within segments
cascade of further segmentation genes
Wg expression in the posterior compartment prevents production of Denticles - an anterior feature of future segments
Serrate signalling molecule is activated in the gaps between Hh and Wg signals
where diffused Hh and Serrate mix - they activate Rho
Serrate +Rho mixing casues formation of denticles
Wg suppresses this elsewhere - naked cuticle
diagram in notes
engrailed and looking ahead to adult segments
adult segments also have A and P compartments regulated by Engrailed
to regulate this - Engrailed is expressed in the imaginal discs
Pt2 - assignment of segment identity
Done by Hox genes
segmetns initially all similar in embryo
follow different developmental programmes to attain different segmetnal anatomies and to form tagmata
-eg thorax = legs, abdomem - no legs
pair rule genes (individual segment AP) and gap genes (overall segments) information combines to give Hox gene activation to give segment identity based on segment location A-P
larval segment identity:
somewhat similar early on
later on begin to look diff (head, thorax, abdomen)
later even - segment differences
-head relatively small
-thorax segments have different muscle layout to abdomen - also has vestigial legs
Hox gene complexes:
sets of genes related to each other in complexes
in drosophila the Hox gene complex has been split in two
but can be looked at as one
order of genes on chromosome responds directly to the order of the segemetns on AP axis to which they give identity
fewer genes in the abdominal segment as they are v similar to each other so need less to differentiate them
posterior dominance
expression domains of Hox genes overlap
the most posterior gene’s function dominates
eg antennapedia function is repressed by Ubx presence
3 most posterior hox gene mutant
Ubx, abd-A, Abd-B triple mutant
Head, T1, and T2 are all fine
but segmetns behind that ALL have T2 identity - even habe the vestigial leg indicating thoracic identity
A1-A8 segments transformed
A9 is fine somehow tho
in the absence of these posterior Hox genes
they still express Antennipedia
but now the more posterior ones arent there to dominate over it
antennipedia ends up defining the identity of these segmetns too
homeotic genes
Hox genes are examples
homeotic mutations definmes by characteristic phenotypes
something being transformed to be like another feature
v dofferent to gap gene mutations where mutated part is missing instead of transformed
embryo to adult segment identity basic
larval segments arise from hox gene expression
the identities of the adult structures come from the imaginal discs
wings come from imaginal discs set aside in T2
T3 haltere imaginal discs
etc
adult segment features
head segments seen cleareer in biting mouthpart insects eg locusts
diff segments have diff mouthparts ig
head anf thorax segments have appendages forming diverse structures (legs, mouthparts, etc)
drosophila is dipteran so abdominal segments generally lack appendage
Ubx, Abd-A mutant adult
this would not make it to adult stage
but prediction
internally in larva there will be many wing and leg imaginal discs in segments past T2
fine up to T1
then T2 onwards is transformed to T2 - A9-10 are fine
Hox gene mutations giving viable adult flies
regulatory mutations tend to more likely give viable adult flies
affect the expression patterns of Hox genes
Ubx regulatory mutant
prevents its expression in T3
is just expressed in the abdomen
so it cant dominate over antennipedia in T3 as it normally would
so antennipedia gives T3 T2 identity
T3 now trasnformed to habve T2 wings and legs instead of T3 legs and Halteres
Regulatory mutation in antennipedia
gain of function where antennipedia is mutated in an enhancer allowing it to be expressed firther anteriorly
gives T2 identity yo head segments
- transforms antennae into legs
weak expression so not full transform but thoracic identitiy partially given to head
identity of the 3 thoracic segments
the combos of Hox genes expressed in thoracic segments gives them their different identities
Scr (sex combs reduced) + Antp -> T1
Antp alone -> T2
Antp+Ubx -> T3
Scr mutant - sex combs are T1 leg identity - Scr mutated - T1 legs become more T2 like - so sex combs are hence reduced
evolution of Hox genes
v high conserved amongst bilateria
v ancient function in AP axis patterning (so it ancestrally in all bilateria - some have lost this tho but still ancestral to them)
Hox gene complexes arose by ancient gene duplication events and divergence
-new gene duplicates
-new duplicate diverges - helps define AP axis detail even more
-repeat this to make AP axis of more and more complex body plans
how did diff body parts arise in diff species
thought before that diff hox genes arising by duplication in diff lineages gave diff body plans
but this is wrong
arthropods have diverse body plans
but their hox genes dont differ much at all
same hox gene product between two species is more similar to diff hox gene product within the same species????????????? (in arthropods at least)
differences in body plans arises from how the segments RESPOND to hox genes
animal ancestor inferred to have complete Hox gene set alreadt - due to animals sharing Hox stuff
mouse Hox genes
4 gene complexes
due to whole genome duplication event during vertebrate evolution
position on chromosome corresponds to position of patterning on AP axis
mouse hox genes and AP diversity of spinal column
mesodermal somites
vertebrae arise from somites
diff identities of vertebrae due to hox fgene patterning
mutate lumbar identity Hox genes - causes them to gain thoracic identity and heve ribs
imaginal discs beginnings
start as small groups of ectodermal cells
bud off embryonic ectoderm (mostly head and thorax)
remain undifferentiated epithelial cell and proliferate
gain their segment identity from wherever they are formed in the embryo
one for each structure
-leg
-wing
-haltere
-mouthpart
-…
patterning the wing imaginal disc
buds off in mid embryo from T2 segment
cells proliferate and grow during larval development to about 50,000 cells
wing disc goes on to form 1/2 of thorax (top and part of side) and the wing:
-grows and folds out to form wing - coming together of dorsal and ventral surfaces to form the wing blade
-has DV and AP axes
-can see that within fate map of imaginal disc
-concentrate on AP
AP tissue patterning in the wing
characteristic wing vein pattern from A to P
distinct A and P compartments
P compartment expresses Engrailed - gives post identity
no vein along The p border
Engrailed expression set up in wing imaginal disc
imaginal disc inherits this compartment identity by virtue of where it arises in the embryonic segments - their origin in the embryo
When imaginal disc cells bud off they bud off from cells at the PARASEGMENT boundary (offset from segment one remember)
so this gives the difference in En expression in A and P of ID
En is a SELECTOR gene for posterior identity
engrailed mutant wing (en as posterior selector)
anterior features (vein 1,2,3) not affected
posterior compartment partially transformed to anterior compartment identity (formation of vein 1,2,3 somewhat)
en allows the posterior cells to gain posterior identity instead of the anterior DEFAULT
En second role in wing patterning
sets up gradient for patterning wihtin the A and P compartments
almost the same as what happens in the embryo segments
-boundary of En/No En acts as part of set up of Hedgehog and Decapentaplegic (Dpp)
-En activates Hh expression
-Hh diffuses a short distance to anterior cells
-activates dpp expression in anterior cells at the AP compartment boundary
-the AP boundary now becomes an ORGANISER: dpp diffuses to form concentration gradients that pattern the details of the A and P compartments (eg wing veins)
get dpp morphogen gradients on either side
diffuses out in both directions forming pattering gradients from the boundary
diff organiser to the embryo
dpp target activation
dpp is long range organising signal
target genes ativated according to dpp conc
> Spalt at high conc - closest to AP border
Omb responds to lower levels so further from border in wider domain
Brinker only expressed if they are not exposed to a moderate level of dpp - normally inhibited by it in cells nearer dpp boundary
completely symmetrical pattern in A and P directions
Rho gene expressed at boundaries of these above genes
Rho expression corresponds to vein formation on wing
in ant - wing vein 3 formed at highest dpp
1 at furthest lowest dpp
same for posterior side BUT it is interpreted differently due to posterior identity given by presence of engrailed
Dpp mutant wing
loss of function
would predict no dpp patterning gradient so veinless wing
INSTEAD end up with v reduced wing (possible loss of veins but hard to tell)
whole wing stricture is changed
so Dpp doesnt just activate wing vein formation genes but it also regulates growth of the wings by activating Vestigial (Vg) gene
Dpp gain of function
cant see wing veins on Dpp loss of function
instead need to to gain of function
constitutively activate dpp in cline of cells in A compartment
sets up new patterning gradient - dpp constitutive cells act as new organiser
get whole new wing structure formed
can interpret dpp function:
>Dpp forms new organiser in anterior
>diffuses out in both directions from clone cells
>activates vg in cells nearby - get outgrowth of wing
>but also get the patterning elements in that new part of the wing
>-it is summetrical - because there is no engrailed expression to differentiate posterior identity - just anterior mirror
>-only get the further elements - veins 1 and 2 as expression of dpp in clone cells is weaker so the gradients are lower in conc so dont get the closer elements from the highest conc
evidence for both dpp functions
Dpp and Vg mechanism
Dpp activates Vg in wing blade cells in the wing pouch
this promotes proliferation in wing pouch to give enough cells to form wing blade
so dpp has 2 functions
growth of wing
patternign of wing veins
Haltere imaginal disc
a modified wing
ancestral insect form has 2 pairs of sinmilar wings - on T2 and T3
in diptera T3 hindwing highly modifies to haltere structure
starts same as wing within embryo development
identical at early stage
but in larval stage where they proliferate - dramatic difference in size of discs appears - halteres much smaller
but has the same positional patterning components - en, hh, dpp (from the parasegmental border in the embryo)
engrailed
regulating hh
regulating dpp
same as wing but they go down diff pathway because of differing hox genes in the T2 and T3 segments
wing vs haltere patternign differences
Ubx present in T3
but not T2
so same positional values and compartments but different development because of diff hox context
see from ubx mutant
Ubx effect on haltere development
modifies haltere disc growth
Dpp activation of Vg is repressed by Ubx
end up with little expansion of this area
haltere remains small
allows haltere development instead
so hox genes alloew for differences in fore and hindwings in insects
see in other orders too
bees have smaller hindwing
beetles have elytra as forewing
Leg imaginal disc
has AP and Proximo-distal axes
forms by outgrowth of the middle part of concentric ring looking pattern to form the distal part of leg structure
different pattern elements for different leg parts
leg patterning AP axis
similar to wing and haltere -some differences
en in post
hh diffuses towards ant
activates BOTH dpp and wingless (in the ventral half of AP compartment border with dpp in dorsal half of the border)
these signals regulate ap axis patterning
diagram in notes
PD axis leg patterning
Dpp and Wg signalling involved but not as gradients
the signals are initial trigger for a sequential gene regulatory cascade
three key TFs (sometimes known as - Leg Gap Genes)
-Dll - distalless
-hth - homothorax
-dac - Dachshund
their combinatorial action leads to 5 domains along the PD axis
intrinsic temporal gene regulatory sequence resulting in spatial sequencing of leg gap genes
wg+dpp acitvate Dll and inhibit hth and Dac
as cells proliferate onef from the centre get pushed out
pushed out cells leave the dpp+wg overlap region
cells from the middle that have had dll expression activated have not yet turned it off - but since they have left the wg+dpp region - dac is no linger inhibited so Dll can activate it
-then after another period of time they move out more dac begins to inhibit dll
(hth expressed furthest out idk but think it must be expressed once dac represses dll - so dac no longer activated - so hth can come on maybe?? not on the regulatory chart)
so much diff method of patterning - the wg+dll for setting up initial expression - then intrinsic temporal gene regulatory sequence results in spatial patterning
Dll and Dac mutations
partial loss of function Dll:
loss of distal part of leg
Dac loss of function - loss of middle part of leg
eye-antennal imaginal disc
one for each eye and antenna
two discs in one form eye and antenna
antennal disc - similar to leg disc in patterning
A and P compartment
Dpp in ventral part
wg in dorsal
gives rise to dll, dac, hth
antenna and legs both modified from more general appendage - different due to hox gene context
