Drosophila Development

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Drosophila Development
blastoderm cells are NOT totipotent(able to
develop into all types of cells) even right
after cellularization, damage to an area of the
embryo leads to defective later development in
that part of the animal stages in the
development of regional specificity are under
genetic control earliest steps in development
are coded for by mRNAs made by the mom maternal
effect genes genes required in the mother for
the proper development of the embryo zygotic
genes developmental genes that function in the
embryo proper most organisms have a number of
maternal effect genes that regulate the
earliest stages of cellular development
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Drosophila Development
maternal effect genes are easy to spot using
reciprocal crosses m/m females X / males
m/ 100 abnormal heterozygous embryos note
that the females do NOT have to have a
phenotype-- the gene functions in the
mother, not in the embryo / females X m/m
males m/ 100 normal heterozygous
embryos bicoid maternal effect mRNA localized
in the front of the fly embryo protein forms a
gradient higher in the anterior half, lower in
posterior nanos maternal effect mRNA localized
at the posterior of the fly embryo protein
forms a gradient higher in posterior, lower in
the anterior gradients seen before
cellularization (formation of cell membranes)
and continues through blastulation and into
segmentation
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Drosophila Development
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Drosophila Development
at the larval stage, larva are composed of a
series of segments which will each make up
specific structures in the adult
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Drosophila Development
segmentation genes series of genes which
generates the segments of fruit fly larvae
that typically operate at 1 of 4 levels 1)
coordinate genes determine the
anterior-posterior axis bicoid and nanos are
the most obvious, but there are others 2) gap
genes proteins expressed in a group of
continuous segments along the embryo-- gaps
appear in the normal segment pattern just like
bicoid and nanos, the gap genes affect
transcription of other genes and work together
to turn them on and off
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Drosophila Development
hunchback in green kruppel in red both in yellow
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Drosophila Development
3) pair rule genes are the next level of
organization of the segments typically affect
alternating segments in the embryo where they
are/ aren't expressed
fushi tarazu (ftz) gene
hairy gene
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Drosophila Development
regulation of gene expression even at this stage
is complex
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Drosophila Development
4) segment polarity genes show a difference in
the anterior or posterior part of a segment--
engrailed is the most common example segment
polarity genes are repeated for each different
segment note that the larva is growing while
the new patterns are being created wingless is
another segment polarity gene-- expressed
opposite of engrailed
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Drosophila Development
4 levels of genes for controlling
segmentation 1) coordinate genes bicoid,
nanos 2) gap proteins hunchback, kruppel 3)
pair rule genes fushi tarazu, hairy 4)
segment polarity genes wingless, engrailed
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Drosophila Development
inside of each segment, there are about 20
different imaginal disks imaginal disk
embryonic tissue that is specialized to become
the principal structures in the adult-- eyes,
legs, wings, etc
interaction of a number of different proteins are
required to generate the complex pattern of
tissue found in the adult
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Drosophila Development
gap genes, pair rule genes, and segment polarity
genes cooperate to control the segment
identity genes imaginal disks from different
segments usually become different
structures-- ie. eyes vs antenna vs legs vs
wings homeotic mutation mutation that
transforms one segment into another ie. body
parts that normally appear from imaginal disks
appear in the wrong location most homeotic
genes contain a similar DNA binding motif, a
helix turn helix
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Terminal Specification
bicoid and nanos mRNAs are localized to the
anterior and posterior poles specifically by
binding to the cytoskeleton the egg receives
information and mRNA from the nurse
cells follicle cells at the other end orient
microtubules so that the '' end is always at
the posterior (growing end) bicoid mRNA bind 2
proteins (exuperantia and swallow) that link to
dynein, a protein which anchors microtubules at
the '-' anterior end nanos mRNA binds oskar, a
protein that links to the microtubule motor
kinesin that moves to the '' end (ie.
posterior) localizing the mRNA for bicoid and
nanos by binding to the microtubules generates
mRNA gradients leading to protein gradients in
the cell
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Terminal Specification
oskar mRNA is NOT localized in the cell itself--
it is found throughout oskar is only translated
in the posterior of the syncitium because
another protein, staufen, binds to kinesin I
(moves to the '' end) and regulates the
translation of the stufen protein (ie. like
P-granules control that) bicoid itself inhibits
the translation of the caudal protein nanos
inhibits the translation of the hunchback protein
(anterior gap gene) translation regulation is
very common before zygotic genes are
transcribed translational regulation occurs in
numerous other systems as well very important
for regulating the proteins present in a cell
particularly during unequal cell divisions or
syncitial divisions- transcriptional control
by itself is insufficient
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Terminal Specification
coordinate genes regulate the zyotic
transcription and translation of gap
genes bicoid represses the translation of caudal
mRNA-- inhibits posterior gene also activates
transcription of hunchback bicoid and hunchback
work together to transcribe buttonhead, empty
spiracles and orthodenticle genes essential for
head structures
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Terminal Specification
torso tyrosine kinase receptor required for the
formation of both anterior and posterior
structures mRNA and protein is found throughout
the cell localized activation of the receptor
at the ends is what's important activated by the
'torso-like' gene-- same phenotype as torso,
different gene only expressed in nurse and
follicle cells torso signaling inactivates
groucho, a transcriptional inhibitor of tailless
and huckebein, gap genes responsible for
posterior and anterior termini
huckebeintailless telson (posterior)
huckebeintailless bicoid acron (anterior) 3
sets of genes required to specify the
anterior/posterior axis anterior, posterior,
and termini 4 types of regulation seen mRNA
localization, transcriptional, translational,
and localized signaling
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Gap Genes
gap gene expression is dynamic-- there is a low
level throughout the fly which becomes localized
to a few high level areas gap genes are
initiated by the earliest genes bicoid and
hunchback activate hunchback and giant and
inhibit knirps nanos and caudal activate knirps
and giant, inhibiting others giant has
enhancers for both an anterior and a posterior
expression band gap genes then regulate each
other kruppel is inhibited by hunchback
giant as well as knirps and tailless
kruppel inhibits both giant and hunchback
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Pair-Rule Genes
8 different genes divide the embryo into
alternating 'zebra-stripe' patterns gap genes
overlap a bit-- pair rule genes can't
hunchbackgreen, kruppelred, bothyellow
(immuno) different enhancers are required for
different bands in the embryo-- multiple genes
can turn on the pair rule genes
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Pair-Rule Genes
primary pair-rule genes are directly regulated by
the gap genes secondary pair rule genes are
regulated by 1) gap genes 2) primary pair
rule genes 3) themselves fushi-tarazu (ftz)
binds its own enhancer to activate its own
transcription self-fullfilling-- once turned
on, continues to turn itself on 7 stripes on, 7
stripes off 14 parasegments 2 half-
parasegments (1 on, 1 off) make up a segment of
the embryo 1 parasegment is posterior half of
one segment and anterior half of next
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Segment Polarity Genes
only get initiated after cellularization-- cannot
happen in the syncitium performs 2 major
functions 1) reinforce parasegment boundaries
2) establishes cell fates within a segment each
parasegment uses the Wnt and hedgehog signaling
pathways (ie. same signaling pathway is used
in different regions of the embryo) each segment
has multiple rows of nuclei making up 1/2 of
parasegments only 1 row of nuclei expresses
wingless (wg), a wnt protein homolog only 1 row
of proteins express engrailed/hedgehog next to
the wg cells engrailed is activated by
even-skipped, ftz, and paired engrailed is
repressed by odd-skipped, runt, and
sloppy-paired gives 14 bands of expression 1
nucleus thick along A-P axis always marks the
anterior boundary of a parasegment posterior
boundary of a segment
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Segment Polarity Genes
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Segment Polarity Genes
wingless and hedgehog are synthesized in
neighboring cells-- wingless activates
hedgehog expression hedgehog activates
wingless expression 2 cells signal each other
to be different-- maintains expression
pattern engrailed is the transcription factor
activated by frizzled receptor pathway through
disheveled, zeste-white3 (aka GSK-3), and
armadillo (b-catenin) turns on expression of
the hedgehog protein amount of signal each cell
receives (ie. hedgehog and wnt) determines its
cell fate-- ie. morphogen concentration
gradients also regulates the amount of
frizzled or patched receptors expressed ectopic
expression of wnt or hedgehog (ie. increased
amounts or general expression) changes fates
of cells within the segments
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Homeotic Genes
originally identified in two clusters,
antennipedia and bithorax clusters when
sequenced, clusters were next to each other on
the chromosome homeotic mutants genes which
alter the body plan of the embryo ie. wings
become legs or vice versa-- segments change their
identity order of genes along the chromosome
mimics order of expression along the
anterior-posterior axis expression of hox genes
is regulated by gap and pair rule genes and
are refined by segment polarity genes
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Homeotic Genes
homeobox genes are initially expressed starting
at the anterior limit of the normal expression
and extending along posterior parasegments more
posterior homeobox genes downregulate expression
of the anterior ones, giving a band of high
expression that covers roughly 1 segment
posterior
anterior
deformed sex combs reduced antennapedia
initial pattern
deformed sex combs reduced antennapedia
final pattern
length of the A-P axis expressing a given
homeobox gene can vary between genes in one
organism and between organisms (ie. giraffe)
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Homeotic Genes
for homeotic genes, patterns are started using
transient expression patterns (ie. gap and
pair rule genes) two families of proteins
regulate the chromatin structure of the hox
locus to allow the cells to remember their
fates polycomb group genes remember which hox
genes are repressed in a given
segment trithorax group genes remember which
parts of chromatin should be transcriptionally
active the homeobox genes are transcription
factors which regulate segment specific
proteins which actively give the segments
different properties realisator gene gene which
gives a segment or imaginal disk a potential
function ie. eyeless (pax6 homolog) is essential
for eye formation
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Homeotic Genes
all of the homeotic genes in this cluster contain
the 'homeobox' a conserved helix turn helix
motif that is very similar between family
members all recognize a core sequence of TAAT
specific amino acids control outer
specificity other homeobox containing genes
from outside of the hox cluster can work with
the hox genes to regulate segment
identity other regions of the hox proteins also
bind to DNA and interact with proteins ie.
different motifs within transcription factors
regulate functionality
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Hox Genes in Other Organisms
the homeotic gene cluster in flies is found in
essentially every animal studied, from
tunicates and sea urchins on up, vertebrate and
invertebrate gene order along the chromosome and
anterior-posterior expression pattern are also
conserved-- ancient mechanism of evolutionary
value genes that regulate chromatin of the hox
cluster are also conserved to make things more
complicated, there are 4 similar hox gene
clusters in mammals-- duplication of a
duplication somewhen in the past paralogous
group hox genes with similar anterior-posterior
expression patterns and same chromosomal
location within the cluster mammalian hox genes
are numbered 1-13, some only at the very
posterior not every hox cluster has a paralog
to flies, but order is still conserved
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Hox Genes in Other Organisms
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Hox Genes in Other Organisms
gene knockouts for one hox paralog generally give
a mild phenotype hoxD3 gives a mild cervical
vertebra phenotype hoxA3 gives different
malformations in the segment, but not the bone
double mutants make both phenotypes more severe
paralogs work together to determine segment
identity, so NO orthologs retinoic acid analog
of vitamin A that is a morphogen in many tissues
anterior hox genes are very sensitive to
retinoic acid posterior hox genes are
relatively insensitive to retinoic acid extra
RA can cause extra posterior structures to form
and fewer anterior acts as a teratogen to alter
segment identity-- causes bones to fuse and
limbs to be malformed
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Hox Genes in Other Organisms
organisms all use the hox genes to generate the
anterior-posterior differences, but they fine
tune the expression pattern of the genes to
suit their needs ie. there are 5 types of
vertebrae cervical, thoracic, lumbar, sacral,
caudal chickens and mice have different
numbers of each type of vertebrae by altering
anterior limits of hox gene expression, they can
chance the characteristics of those vertebrae
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Amphibian Axis Formation
unlike flies and C elegans, vertebrate
blastomeres are identical and expressing the
same genes-- conditional specification neighborin
g cells must tell the blastomeres what to become
(ie. induced) Hans Spemann tied eggs in the same
plane as the division and perpendicular to
it-- tied in same plane gave twins, perpendicular
gave tissue deformed in one plane, normal
embryo in other
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Amphibian Axis Formation
upper 'grey crescent' contained information
essential for development give rise to the
dorsal lip of the blastopore- likely involves
gastrulation early gastrula epithelial tissue
donors could become epithelial or neural
tissue later gastrula could not change their
fate any more-- committed to a fate cells
remember the location from which they came
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Amphibian Axis Formation
in early gastrula, dorsal blastopore lip ('grey
crescent') is only tissuethat is differentiated--
able to initiate gastrulation when
transplanted used donor tissue of different
colors host/donor tissue could be
identified both donor and host tissue could
move in through the blastopore second
gastrulation gives a second embryo face to
face-- joined at transplant organizer dorsal
lip that induces dorsal development-- organizes
the embryo first induction primary embryonic
induction
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Amphibian Axis Formation
key step-- ectoderm is induced to form mesoderm
from endoderm signals isolated ectoderm or
endoderm tissues cannot form mesoderm cells
vegetal pole cells induce animal cap to form
mesoderm sperm entry induces ventral polarity--
sends out signals for others Nieuwkoop center
dorsal-most vegetal cells of the blastula near
sperm entry site that induces all other
differences between cells brachyury key gene
induced to form mesodermal tissue activated by
nodal or activin-- TGFb superfamily members
b-catenin factor most likely to generate the
Nieuwkoop center accumulates in dorsal region
of the egg right after fertilization more b
catenin signal in dorsal nuclei-- key for forming
dorsal-ventral axis-- dorsal cell fates
inhibited by GSK-3 kinase (ie. same as with Wnt)
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Amphibian Axis Formation
dominant negative GSK-3 proteins cause 2nd
axis disheveled-like protein also moves to
dorsal sites moves along microtubules (ala
nanos in flies) dorsal side b-catenin stable
ventral side GSK inactivated, b-catenin
degraded b-cateninTcf3 activate siamois gene
ectopic siamois initiates second axis
formation works with TGFb family (Vg1/nodal)
for maximum effect
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Amphibian Axis Formation
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Amphibian Axis Formation
Nieuwkoop center induces mesoderm formation but
remains endodermal organizer becomes
mesodermal and migrates through blastopore
mesoderm induces ectoderm to form neural
tissue 4 major functions of the organizer 1)
self-differentiates into dorsal mesoderm--
autonomous development first of the induced
tissues outside of nieuwkoop center 2) ability
to dorsalize surrounding mesoderm (to paraxial
somite forming mesoderm) from ventral
mesoderm 3) dorsalize ectoderm, including
neural tube formation 4) initiates movements
during gastrulation in vertebrates,
dorsal-ventral axis forms first, right after
fertilization movement into the gastrula defines
the anterior-posterior axis first mesoderm
migrating in is anterior, lateral migration is
posterior
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Amphibian Axis Formation
goosecoid transcription factor characteristic of
the organizer region Li ions (GSK-3
inhibitors) expand goosecoid expresssion UV
light inhibits goosecoid expresssion and
organizer formation goosecoid expression also
induces secondary axis formation by itself can
form an organizer- autonomous specification agent
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Amphibian Axis Formation
goosecoid turns on genes- activates morphogen
transcription in notochord paracrine factors
from notochord are sufficient to induce neural
tissue BMPs are the inducing agent, but
induces ectoderm to make epidermis neural
tissue is the default state and mesoderm blocks
the induction noggin (TGFb superfamily) is
sufficient to induce dorsal structures first
expressed in dorsal blastopore lip, then later
the notochord works in UV irradiated
(ventralized) embryos to make normal embryos
blocks the functions of BMP-2 and BMP-4 chordin
second gene localized in dorsal blastopore lip
and notochord functions like noggin to block
BMP factor interaction with receptors block
receptor binding, blocks smad phosphorylation/acti
vation
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Amphibian Axis Formation
BMP-4 induces epidermal formation from the
ectoderm blocks organizer region-- dominant
negatives gives only neural tissue ectopic
BMP-4 prevents gastrulation and involution (no
mesoderm) transcription is inhibited in the
organizer region BMP-4 binding activates
transcription of epidermal transcription factors
Xvent1, Xmsx1, Vox and Xom- also supresses
neural gene transcription low BMP-4 gives
muscles intermediate gives kidney
classic morphogen gradient high BMP-4
gives blood cell most anterior mesoderm isn't
notochord but pharyngeal endoderm pharyngeal
endoderm inhibits BMP-4 as well as Wnts to make
forebrain cerberus, dickkopf, and frzb bind and
inhibit Wnt signaling cerberus binds Wnt-8,
frzb is a truncated, secreted frizzled receptor
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Amphibian Axis Formation
several different inducers act at different
points along the axis initiates at the
neiuwkoop center determined by sperm entry
organizer region sets up dorsal blastopore lip
and allows gastrulation mesoderm moves in,
anterior first followed by more posterior mesoderm
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Amphibian Axis Formation
neurogenin key gene in converting ectoderm into
neural tissue transcription repressed by
binding BMP-4 neurogenin induces expression of
neuroD- directly activates neural genes when
Wnts are repressed by cerberus or noggin, an
addional factor Xbf2 is produced, which further
represses epidermal genes anterior most region
forms the brain w/ thicker neural
plate regionalization different organs/tissue
being formed along an axis transplanted cells
from different regions of gastrula form correct
tissues for their location
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Amphibian Axis Formation
transplantation of different organs could induce
structures in embryos mature liver could
induce forebrain bone marrow induces spinal
cord signals could come from other organisms
same signals are used note mature tissues
still use many of the same signals as embryos 2
different gradients are used to set up the embryo
axes posterior is determined by Wnt signaling
(Wnt-8) induces/related to b-catenin signaling
gradient as well ectopic Wnt-8 causes spinal
cord to be found more anteriorly Wnt-8 is
inhibited anteriorly by Frzb, cereberus, and
dickkopf FGFs and retinoic acid are also present
more at the posterior end retinoic acid
posteriorizes neural tube and patterns the
hindbrain regulates hox gene expression in
cell culture depending on concentration
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Amphibian Axis Formation
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Amphibian Axis Formation
insulin-like growth factors provide an anterior
gradient required for anterior neural
tube ectopic IGF caused formation of ectopic
heads IGF inhibitors block formation of anterior
structures anterior markers like Otx2 are
almost totally silenced anterior/posterior
position is independent of BMPs
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Amphibian Axis Formation
embryos are also asymmetric internally-- ie.
heart is on the left side gut coiling is
counterclockwise or clockwise, etc left-right
axis is important too nodal family protein is
expressed on the left side of the tadpole
microtubules are required-- inhibitors block
left-right axis formation Vg1 (another TGFb
superfamily member) is expressed evenly
left-right only processed fully on the
left pitx2 is a transcription factor important
in Vg1 and nodal left side induction sonic
hedgehog is involved as well- can equalize
halves