Module 5 Flashcards
in flux of calcium results in:
completion of meiosis – oocyte was arrested in meiosis I metaphase
initiation of mRNA translation
vitelline envelope is cross-linked and becomes impermeable
egg changes from flacid to turgid
single site of sperm entry
the micropyle
superficial cleavage –
large central yolk mass – rapid syncytial nuclear division (S/M cycle = 8 minutes) creates the syncytium
division cycle 9
pole cells are enclosed by membranes at extreme posterior of egg and sit outside of syncytium
syncytial blastoderm)
nuclei migrate to cortex and undergo three addition cortical divisions
energids
– specialized islands of cytoplasm around the zygotic nuclei
cellularization occurs
during interphase of division cycle 14 to create the cellular blastoderm (has approx. 6000 cells)
first zygotic gene transcription is seen
in cycle 11 and increases (it turns out that some zygotic gene transcription is required for cellularization)
cycles 1-10 time?
cycles 10-13 time?
13
synchronous- rapid
8 mins and longer
25 mins
cycle 14
cellularization starts immediately following the 13th divison (two phases – 1st microtubules, 2nd actin microfilaments)
division 14
after cellularization is asynchronous (75 to 175 minutes) – patches of cells enter mitosis together – mitotic domains (some cells never divide after cellularization)
Drosophila mid-blastula transition (MBT)
associated with:?
slowing of nuclear division
onset of cellularization
increase in new RNA transcription
Maternal-to-zygotic transition (MZT)
maternally loaded mRNAs are actively degraded and control of development is passed over to the zygotic genome
During the MZT what is degraded?
maternal transcripts are actively degraded (some – such as nanos and hsp83 - are protected in the posterior pole region)
the coordination of of MBT and MZT is controlled by:
the ratio of chromatin (nuclei) to cytoplasm
the Smaug protein (RNA binding protein) and targeted degradation of maternally loaded mRNAs
the Zelda protein (a transcription factor) and activation of zygotic genes
MBT/MZT – control of onset
(#2) activity of the protein Smaug
(#2) activity of the protein Smaug
find it :
encoded by maternal mRNA – Smaug is an RNA binding protein associated with repression of translation – in early Drosophila embryo Smaug targets maternal mRNA for degradation
lose it : Smaug maternal mutants – embryos resulting from eggs laid by these mutant mothers (lack of mRNA degradation)
do not slow down nuclear division,
do not undergo cellularization properly
do not show robust zygotic genome transcription
move it: Smaug mutant is rescued only in anterior when Smaug is expressed in a gradient from anterior (left) to posterior (right)
MBT/MZT – control of onset
(#3the Zelda protein (a transcription factor) regulates the activation of zygotic genes
)
(#3the Zelda protein (a transcription factor) regulates the activation of zygotic genes
)
encoded by maternal RNA
binds to a particular enhancer sequence found in the promoters of zygotic genes that are the first ones transcribed (some of these genes are involved in the initial activation of the pathways that establish dorsal/ventral and anterior/posterior polarity, also sex determination)
genes with highest affinity for Zelda are the first activated
MBT/MZT – control of onset
(#1) nuclear/cytoplasmic ratio
Increasing the overall amount of DNA in the same volume of cytoplasm may dilute repressors.
this was discovered using a neat trick – in Drosophila there are mutants that result in the development of haploid embryos (they die later in development)
normal diploid embryos cellularize immediately after the 13th division – but haploid embryos are delayed and cellularize during cycle 15 after an additional 14th division (a haploid embryo at cycle 14 will contain only half the total amount of genomic DNA as a diploid embryo at the same stage)
sisB
sex determination pathway
zen
(dorsal ventral polarity pathway)
sisB (sex determination pathway) and zen (dorsal ventral polarity pathway
) are both genes normally having very early zygotic expression. This expression is greatly reduced in the Zelda mutant embryos
Drosophila gastrulation
shortly after mid-blastula transition – gastrulation (segregation of presumptive ectoderm, mesoderm, endoderm)
mesoderm forms from the ventral furrow – pinches off and becomes a tube
endoderm invagination – anterior and posterior midgut invaginations
pole cells migrate into posterior midgut invagination (later they will actually squeeze through the midgut epithelium)
cephalic furrow also forms
part 2- Droso grastulation
ectoderm and mesoderm cells undergo convergence and extension – movement toward posterior and ventral midline
these cells are collectively known as the germ band
germ band extension – tail is pushed around the egg case (maybe?) and comes to lie behind head on dorsal side (Drosophila is a long germ band insect)
during germ band extension, organogenesis begins, segmentation becomes apparent and precursors of the adult fly are specified (small groups of cells that will form the imaginal discs).
nervous system forms from neurogenic ventral ectoderm
Part 3- droso gastr
about halfway through embryogenesis (total 22 hours) the tail is brought back to the posterior position of the embryo – this process is known as germ band retraction
following germ band retraction there is a hole in the dorsal epidermis that is covered by an extra-embryonic tissue known as the amnioserosa (it is extra-embryonic because it will die during development)
dorsal lateral epidermal sheets stretch and meet along the dorsal midline – process known as dorsal closure (highly homologous to mammalian wound healing).
The following all happen at the same time
which formation invagination and extension?
ventral furrow formation
anterior midgut invagination
posterior midgut invagination
germ band extension
segmental identify
specified in embryogenesis persists through larval and adult stages
Drosophila oogenesis
A cystoblast (daughter cell of stem cell) undergoes 4 specialized mitotic divisions having incomplete cytokinesis.
Within these 16 cell interconnected cells, one of the two cells with 4 connections (called ring canals) becomes the oocyte.
The other 15 cells enter an endo-cell cycle (G phase – S phase) and become polyploid nurse cells, “feeding” the developing oocyte. As a result many gene products (proteins and transcripts) are dumped into the developing oocyte.
This is important for understanding maternal effect mutations as well as establishment of anterior vs. posterior and dorsal vs. ventral axes in egg.
a RING canal.
incomplete cytokinesis – the contractile ring that normally functions in cytokinesis is arrested and remains as a stable structure
what defines the anterior-posterior and dorsal-ventral axes of the embryo.
localization of oskar, bicoid, and gurken mRNAs
Posterior localisation of oskar mRNA
Posterior localisation of oskar mRNA directs formation of the pole plasm that contains the abdominal and germline determinants.
Bicoid mRNA localizes to anterior
of the oocyte, directing where head and thorax of embryo develop.
Gurken* is translated on the dorsal side,
Gurken* is translated on the dorsal side, producing a signalling molecule that causes adjacent follicle cells to define the embryo’s dorsal-ventral axis.
initially uniform wrt fate
follicular epithelium
follicle cells have a receptor for gurken protein (gurken = epidermal growth factor (EGR) & the receptor, called
torpedo
Par-1 function?
organizes microtubules such that their plus ends are at posterior
What is PAR1
isolated by performing screens for mutants in the model genetic organism C. elegans (a nematode)
partitioning defective mutants inappropriately undergo symmetrical divisions in early embryo – this causes loss of differential cell fates (we’ll being seeing more about Par protein later in the course)
has a conserved role in cell polarity (conserved from invertebrates to mammals)
Par-1 is a serine/threonine KINASE
in Drosophila it is known to regulate the microtubule cytoskeleton
oskar mRNA is transported by what? and where?
by a kinesin to the posterior of the oocyte (kinesins are generally plus end MT directed motors)
only in the posterior oocyte can oskar mRNA be translated & Oskar protein recruits more Par-1 this does what?
reinforces the MT organization (plus ends posterior)
posterior pole is now distinctive – pole plasm why?
it contains determinants for producing abdominal (posterior) segments of the embryo as well as the germ cell determinants.
Oocyte MT polarity is the key
the network of MTs extends from the oocyte posterior through the ring canals into the nurse cells
different motor proteins (dyneins & kinesins) will move either towards the MT minus end (all dyneins) or plus end (most kinesins)
cargo tethered to specific motor proteins can, therefore, be delivered to either the anterior or posterior of the developing oocyte
using this general mechanism of MT associated motor transport, two key determinants are also set up at this time
bicoid mRNA is localized to the oocyte anterior
nanos mRNA (because it is bound by Oskar protein) is localized to the oocyte posterior (more on this mechanism later)
oogenesis & Dorsal-Ventral Polarity
what happens as oocyte grows?
As oocyte grows the oocyte nucleus moves to an anterior dorsal position (how does it move….pushed by MTs??)
gurken is again used – localized expression between the oocyte nucleus and oocyte membrane forms a gradient
follicle cells nearby are dorsalized by the gurken signal
Is gurken required by oocyte?
pole cells transplantation usage?
causes of maternal mutants?
maternal mutants of gurken cause ventralization of eggs & embryos (eggs and resulting embryos lack dorsal structures)
same is true of torpedo mutants
pole cell transplantation can be used to create germline / somatic mosaics – this was the technique used to determine where each gene is required
answer - gurken activity is required in oocyte (germline cells) & torpedo activity is required in the follicle cells (somatic cells)
Pole cell transplantation established that torpedo was not needed in the germline but?
but was required in the somatic follicle cells for proper dorsalization of egg & embryo
Pole Cell Transplantation can be used to create Germline/Somatic Mosaics
how?
The recipient of the pole cell transplant also carries a separate genetic mutation that prevents germ line development. The non-mosaic egg chambers don’t develop. The successfully developed eggs must arise from the chimeric (mosaic) egg chambers
torpedo is not required in the germline,
but it is required in the somatic follicle cells.
gurken is not required in the somatic follicle cells, but
it is required in the germline
the binding of Gurken ligand to Torpedo receptor initiates ?
activated Torpedo activates a transcription factor called Mirror, which in turn inhibits expression
a cascade of activities resulting in the creation of the dorsal/ventral axis
expression of pipe – and Pipe protein will only be found where there is no Gurken signal (i.e. ventral cells)
Pipe protein
(a heparan sulfate sulfotransferase) is secreted into the vitelline envelope
Ultimately, Pipe expression results in
the localization of protease activity in the vitelline envelope only on the ventral side of the embryo
what expressed in ventral cells of the follicular epithelium surrounding the developing oocyte.
Pipe sulfotransferase
Pipe modifies
components of the developing eggshell to produce a ventral cue embedded in the vitelline membrane.
This ventral cue is believed to promote one or more of the proteolysis steps in the perivitelline space –
a cascade of proteolytic activation.
increased Gurken results in less
sulfated vitelline envelope proeteins
A cascade of proteolytic activation
proteases are secreted into the perivitelline space in an inactive form – they must be cleaved to be activated
Gastrulation defective is bound by the sulfated vitelline membrane proteins – it cleaves & activates the 2nd protease called?
3rd called?
cleaves a protein called?
snake
easter
spatzle
Processed Spätzle
is a paracrine factor and ligand for a receptor called Toll found on the egg membrane
The receptor for the processed Spätzle ligand is known as
Toll
Toll mRNA is laid down in oocyte – and it is not translated until?
until after fertilization
__________ is present over the entire surface of the syncytial membrane
_________. ligand is only present in the perivitelline space on the ventral side of the embryo
Toll
Spatzle
trasncription factor ________ is also found throughout the embryo where it is bound to a repressor called Cactus
Dorsal
transcription factor Dorsal is also found throughout the embryo where it is bound to a repressor called
Cactus
we find activation of Toll by Spätzle when and where
around the syncytial blastoderm stage
only on the ventral side of the embryo
Dorsal is a morphogen
– it concentration within the nucleus can cause activation of different sets of genes
high dorsal >
low dorsal >
mesoderm
ectoderm
what does Dorsal actually do?
specified mesoderm invaginates in response to high levels of Dorsal
high Dorsal levels activate genes that are involved in mesoderm differentiation –including twist
Gap Genes
mutants lack large regions of the body – several contiguous segments
Gap mutants lacked large regions of the body (several contiguous segments).
Pair-rule genes:
mutants lack portions of every other segment
Segment polarity genes:
mutants have defects (deletions, duplications, polarity reversals) in every segment
Maternal effect genes:
Encode messenger RNAs that are placed in different regions of the Drosophila egg.
Pair-rule genes:
details
Drosophila zygotic genes regulated by gap gene proteins which divide the embryo into periodic units, resulting in a striped pattern of seven transverse bands perpendicular to the anterior-posterior axis. Pair-rule mutants lacked portions of every other segment.
Segment polarity genes:
Segment polarity genes: Drosophila zygotic genes activated by the proteins encoded by the pair-rule genes whose mRNA and protein products divide the embryo into 14-segment-wide units, establishing the periodicity of the embryo.
Segment polarity mutants showed defects (deletions, duplications, polarity reversals) in every segment.
Homeotic selector genes
A class of Drosophila genes regulated by the protein products of the gap, pair-rule, and segment polarity genes whose transcription determines the developmental fate of each segment.
Homeotic mutants: Result from
mutations of homeotic selector genes, in which one structure is replaced by another (as where an antenna is replaced by a leg).
Morphogen gradients
Morphogen gradients
early experiments involving ligation of embryos, or treatment with uv light, or RNAase gave some clues that insect eggs organize themselves with gradients of morphogens – one at anterior pole other at posterior pole
critical for A/P axis formation:
______ mRNA localized to anterior tip
&
______ mRNA found at posterior tip – both due to MT polarization in the developing oocyte
Bicoid
nanos
bicoid – the anterior morphogen - evidence
find lose move
find
bicoid – the anterior morphogen - evidence
lose
bicoid protein can be observed in a gradient – high in anterior and low in posterior
embryos lacking bicoid can’t make a head – embryos develop with two posterior ends (= necessity)
injection experiments
bicoid mRNA injected into the head of bicoid mutants achieved full rescue
bicoid mRNA injected back into a bicoid mutant embryo induces head structures – wherever the site of injection. Moreover – the areas nearby develop into thoracic structures. (exactly like would be expected for a concentration dependent response)
move
a large amount of bicoid mRNA injected into the posterior of wild-type results head formation at both ends (= sufficiency)
bicoid protein really is a morphogen! – further evid
the identification of morphogens, long predicted to exist, was a REALLY BIG DEAL
researchers went to great lengths to confirm this idea – further evidence could be found in embryos which had been genetically modified to vary the dosage of the bicoid gene – more gene product should change the gradient
ANTERIOR LOCALIZATION OF BICOID mRNA in OOCYTE
bicoid mRNA – 3’UTR contains sequences critical for anterior localization
sequence of bicoid 3’UTR interact with the products of two other anterior group genes – Exuperantia and Swallow – which are required to keep bicoid mRNA localized to the anterior pole
bicoid mRNA and associated proteins – once in the oocyte, are maintained at the anterior end by directed transport along the polarized microtubules
anterior end of egg contains a large MTOC – Microtubule Organizing Centre – like a centrosome it caps microtubules at their minus ends
bicoid mRNA in the oocyte is associated with a minus end directed MT motor protein – a DYNEIN motor protein
POSTERIOR LOCALIZATION OF NANOS mRNA in OOCYTE
the main posterior “organizer” is nanos
general idea is that nanos mRNA is not initially localized in the growing oocyte cytoplasm – free to diffuse
meanwhile…oskar mRNA is transported to MT plus ends by a kinesin motor protein – it takes along with it a protein called Staufen
at their destination – the posterior pole – oskar mRNA and Staufen protein bind to cortical actin filaments – and oskar mRNA is translated only at posterior pole
Oskar protein – now localized to the posterior of the oocyte can bind and “trap” nanos mRNA
any nanos mRNA that is not trapped by the Oskar-Staufen “trap” is repressed
Smaug binds to nanos 3’UTR and recruits CUP which does what?
but Oskar can dissociate CUP
repressed by association with two protein repressors – Smaug
prevents association with the ribosome (translational repression)
and this permits translation of nanos mRNA only at the posterior pole –this sets up the nanos protein gradient
hunchback and caudal
how?
two maternally provided mRNAs – encoded by the genes hunchback (hb) & caudal (cad) – are uniformly distributed throughout the egg
but protein products of these mRNAs are critical for anterior/posterior patterning and these proteins are found as gradients in the early syncytial blastoderm
ans to how?
It turns out that translation of the hb and cad mRNAs is repressed by the diffusion gradients of Nanos and Bicoid proteins, respectively.
