Transcript PPT

BioSci 145A Lectures 18 - Gradients, cascades, and
signaling pathways regulate development I
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last lecture we talked about how gene regulation
influences cancer
this week we will try and put together the types of
techniques and experiments we have been talking about
to solve problems of transcriptional regulation in
development
– three interesting problems in transcription
• development (includes cancer)
• metabolism
• homeostasis (includes hormonal signaling)
– some of the material overlaps with Bio108 and Larry
Marsh’s genetics course but we will emphasize the
role of transcriptional regulation in the patterning
process
BioSci 145A lecture 18 (Blumberg) page 1
©copyright
Bruce Blumberg 2000. All rights reserved
Fundamentals of patterning
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How to pattern an embryo
– begin with a small asymmetry
– create cell diversity with short range inductive events
between cells
– send positional signals between cells and continue
inductive events
– differential cellular responses to these signals will
lead to the elaboration of pattern
Multiple axes need to be considered
– anterior/posterior
– dorsal/ventral
– proximal/distal (limbs)
– left/right (vertebrates)
As we will see, the signaling mechanisms along these
axes are largely conserved across evolution
Central assumption about the molecular basis of
development - each cell type may be characterized by its
pattern of gene expression
– since gene expression is controlled primarily at the
level of transcription it follows that transcription
factors will be important developmental regulators
– these will include activators and repressors that act in
various ways to modulate transcription
Important principle - activation and repression are
equally important developmental mechanisms
BioSci 145A lecture 18 (Blumberg) page 2
©copyright
Bruce Blumberg 2000. All rights reserved
Transcription factor cascades in development
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groups of transcription factors form regulatory circuits
– interactions are responsible for patterning
– known to be true for all multicellular organisms
– currently, details are only well worked out in
Drosophila and C. elegans
Since gene expression regulates development, we will
explore in some detail
– interaction between transcription factors
– interaction between and among signaling pathways
– widespread conservation of these pathways
many of the genes that control development are also
involved in the development of cancer
– in many ways cancer is a disease of development
• tumor cells share properties of embryonic cells
– capacity for division
– ability to migrate throughout the body
– secretion of bioactive substances
– understanding regulatory circuits that pattern the
embryo will aid in understanding how cancers develop
What do we want to understand?
– begin with an initial asymmetry
– asymmetry triggers positional differences
– these are translated into differential gene expression
– regions of the embryo acquire specific properties
BioSci 145A lecture 18 (Blumberg) page 3
©copyright
Bruce Blumberg 2000. All rights reserved
Transcription factor cascades in development (contd)
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How is asymmetry translated into gene expression?
– mechanisms differ among the various systems BUT
• the types are conserved among all animals
– may involve localization of
• mRNAs
• proteins
• regulatory factors (RNA binding proteins, inactive
dimerization partners)
– end result is always the same
• localized control of gene expression
After asymmetrical beginning, the body begins to be
subdivided
– regional specification - areas are specified (determined)
-> particular tissues or structures
– genes that regulate this process were identified by
mutations that cause body parts to be absent, duplicated
or formed in inappropriate places
• famous Drosophila screen by Christianne NussleinVolhardt and Eric Wieschaus (Nature, 1980 287,
795-801)
• led to the identification of the major classes of
patterning genes
– opened up the field, Nobel prize
– combined with molecular techniques, these led
to the elucidation of regulatory pathways
BioSci 145A lecture 18 (Blumberg) page 4
©copyright
Bruce Blumberg 2000. All rights reserved
Transcription factor cascades in development (contd)
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subdivision (contd)
– these genes are strong candidates to function as
genetic switches
• typically act on themselves and other regulatory
genes to create a hierarchical cascade of gene
expression
• but they must also act on downstream target
genes that actually form the structures involved
– can’t only have regulatory proteins, must
also have structural proteins and enzymes
to build tissues, organs and body parts
– one should not lose sight of the importance
of these “effector genes” that actually do
the work of development.
– most of these switch proteins act by binding to DNA
sequences in the promoters of target genes
• can be studied using all of the techniques we
have been talking about for the last few weeks
• a small number are RNA binding proteins that
modulate the localization or stability of mRNAs
• a few are proteases that modulate the activity of
other factors
• a very few regulate the transport of proteins into
the nucleus in particular parts of the embryo
BioSci 145A lecture 18 (Blumberg) page 5
©copyright
Bruce Blumberg 2000. All rights reserved
Transcription factor cascades in development (contd)
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Developmental pathways are predetermined in the egg
– the program is usually sequential and interactive
• cells can be moved around
• some plasticity in outcome
– program may be invariant in time and space (C. eleg)
– despite widely different appearances, the regulatory
mechanisms that control development are very highly
conserved in vertebrates and invertebrates
• this justifies the use of weird model organisms such
as Drosophila and C. elegans
• we use these because they have particular
advantages that allow questions to be asked and
answered precisely
many mutations in developmental regulatory genes are
lethal early in development
– single mutations that cause gross changes in
development are particularly interesting
– three classes in Drosophila -> act successively
• maternal genes - these broadly specify regions in
the embryo -> loss causes absence of large regions
• segmentation genes - lead to changes in segment
number, size or polarity
• homeotic selector genes - control the identity of
particular segments but not the number or size
BioSci 145A lecture 18 (Blumberg) page 6
©copyright
Bruce Blumberg 2000. All rights reserved
Drosophila and gradients
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BioSci 145A lecture 18 (Blumberg) page 7
©copyright
early embryo is patterned
by molecular gradients
along major body axes
– anteroposterior (A/P)
• anterior ->head
• posterior -> tail
– dorsoventral (D/V)
• dorsal -> top
(except for
Xenopus)
• ventral -> bottom
(except for
Xenopus)
A/P axis is already
determined in the
Drosophila egg
– determined after
fertilization in
amphibians and fishes
D/V axis is determined
during zygotic
development in Drosophila
– concurrently with A/P
in amphibians and
fishes
Bruce Blumberg 2000. All rights reserved
Drosophila and gradients (contd)
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identification
– Drosophila embryo is composed of segments
(parasegments)
• all invertebrates are overtly segmented
– segment boundaries are respected
• vertebrates have some vestiges of segmentation
– boundaries may not be respected
– Drosophila has very clear markers for each segment shape and number of denticles (hairs) varies between
segments and between dorsal and ventral side
• absolutely key for identifying the functions of
genes
• frequently, mutations are evaluated at an early
larval stage rather than in the adult fly
• although larval segments do not contribute to
adult structures, they reflect what will happen in
the adult
– adult fly has head, thorax and abdomen
• 3 thoracic
• 8 abdominal segments
• fly is a very specialized insect and the details of
its development are not characteristic of insects
or invertebrates as a whole
– but the molecular mechanisms are virtually
identical
BioSci 145A lecture 18 (Blumberg) page 8
©copyright
Bruce Blumberg 2000. All rights reserved
Drosophila and gradients (contd)
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BioSci 145A lecture 18 (Blumberg) page 9
©copyright
Drosophila has unique
early development
– early embryo
develops as a
syncytium (no cell
membranes)
– first asymmetry is
pole plasm vs
cytoplasm
– after 13 nuclear
divisions, the nuclei
are positioned at the
periphery
– cell membranes
then form - cellular
blastoderm
– at about this stage,
the first
determination
events occur
– nuclei do not move
in predetermined
patterns
– behave according to
position in A/P and
D/V gradients
Bruce Blumberg 2000. All rights reserved
Drosophila and gradients (contd)
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What are the gradients that specify position in the early
embryo?
– many components are put into the oocyte by the nurse
cells during oogenesis, (proteins, RNAs)
– mutations in maternal genes important for early
development -> female sterile mutations (no effect on
mother)
• females have no progeny
• embryos may have cuticular defects
Critical components of four developmental pathways are laid
down in the oocyte by follicle cells
– anterior
– posterior
– terminal (both ends)
– dorsoventral
BioSci 145A lecture 18 (Blumberg) page 10
©copyright
Bruce Blumberg 2000. All rights reserved
Drosophila and gradients (contd)
• Each pathway has a morphogen
– morphogens are diffusible substances that form
concentration gradients that pattern the embryo - many
definitions, loosely applied
• Gradient model was popularized by Child, Huxley
and De Beer
– activity gradients -> formation of specialized
regions in the embryo
• in its strictest definition, a morphogen patterns tissues
directly depending on its concentration
– Classic paper in the field is Turing, A.M. (1952)
The chemical basis of morphogenesis. Phil.
Trans. R. Soc. Lond. B 237, 37-72.
– Alan Turing was a British mathematician whose
claim to fame was the solving of the German
wartime code called “enigma”
» enabled the allied to eavesdrop on military
transmissions and win the war
• Louis Wolpert -> many theoretical treatments of
morphogenesis
– Wolpert, L. (1989). Positional information
revisited. Development 107s, 3-12.
• Francis Crick provided legitimacy to the model with a
quantitative treatment of the activity of diffusible
substances - worked over small distances 0.1-1mm
– Crick, F. (1970). Diffusion in embryogenesis.
Nature 225, 420-422.
BioSci 145A lecture 18 (Blumberg) page 11
©copyright
Bruce Blumberg 2000. All rights reserved
Drosophila and gradients (contd)
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morphogens (contd)
– most common definition of morphogen is a diffusible
substance responsible for patterning the embryo
important components of each pathway are localized in
the oocyte before fertilization
– anterior system patterns the head and thorax.
Products in the maternal germline are required to
localize the bicoid mRNA. bicoid protein is the
morphogen
– posterior system patterns the abdominal segments.
many components act to cause the localization of the
nanos gene.
– terminal system is responsible for the specialized
structures in unsegmented termini of the head and
tail. morphogen acts through the transmembrane
receptor torso
– dorsoventral system is responsible for patterning the
d/v aspect of the embryo. Patterning occurs through a
gradient of the dorsal protein in the nucleus
key feature is that these pathways all use somewhat
different mechanisms to generate pattern.
– many of these mechanisms are widely used in other
organisms and in transcriptional regulation in general
BioSci 145A lecture 18 (Blumberg) page 12
©copyright
Bruce Blumberg 2000. All rights reserved
Gradients in the oocyte
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summary of the four systems
– multiple levels at which the pathways can be affected
– ~30 maternal genes may be grouped by location, and
activity
– with the exception of dpp, all of the zygotic targets of
the patterning systems are transcription factors
• many are repressors
– anterior and posterior systems interact, d/v and terminal
act independently of others
BioSci 145A lecture 18 (Blumberg) page 13
©copyright
Bruce Blumberg 2000. All rights reserved
Gradients in the oocyte (contd)
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BioSci 145A lecture 18 (Blumberg) page 14
©copyright
How does one go about
testing whether gene
products are required?
– inject cytoplasm or
purified components
from wt embryos into
mutants and assay for
rescue
– can use this method to
map where mutants
are in the pathway (but
very tedious)
– only the morphogen
itself can elicit
localized,
concentrationdependent rescue
– rescue is sufficient to
conclude that the
mutation causes a
deficiency of some
material in the
embryo.
Bruce Blumberg 2000. All rights reserved
A/P patterning uses localized regulators
• Bicoid is an anterior morphogen in the strictest sense
– bicoid protein or wt cytoplasm rescues bicoid
embryos in a concentration-dependent manner with a
high point at the site of injection
• this implies that the targets are ubiquitous
– bicoid mRNA is localized to the very anterior of the
embryo
– bicoid protein diffuses after translation forming a
concentration gradient
BioSci 145A lecture 18 (Blumberg) page 15
©copyright
Bruce Blumberg 2000. All rights reserved
A/P patterning uses localized regulators (contd)
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– bicoid is a homeodomain protein that directly
specifies anterior -> instructive
• bicoid is a positive regulator of hunchback ->
band of hb expression where bcd is expressed
Posterior development involves many genes
– females mutant for any of these genes produce
progeny that lack abdominal structures
– apparently, each of these gene products is required for
localizing the next one in the pathway -> all required
to localize the morphogen nanos (can rescue all)
– reason for all of these components may be the
requirement for transport the entire length of the egg
BioSci 145A lecture 18 (Blumberg) page 16
©copyright
Bruce Blumberg 2000. All rights reserved
A/P patterning uses localized regulators (contd)
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Posterior development (contd)
– posterior gene cascade illustrates many levels of
gene regulatory control
– cappuccino, and spire are required to localize staufen
protein at the pole
– staufen protein localizes oskar mRNA
– oskar/staufen localize vasa
• target of vasa is not known
– vasa is a RNA binding protein similar to translation
initiation factors (RNA helicase)
• vasa protein found in germ cells of most species
– valois has not been cloned
– tudor appears to be a RNA binding protein that is
similar to a EBV coactivator protein
• required both for abdominal development and
germ cell formation
– unknown how vasa, valois and tudor affect nanos
but they do
– actually, it is fairly remarkable that the functions of
these genes is not known after so many years of
intensive study.
BioSci 145A lecture 18 (Blumberg) page 17
©copyright
Bruce Blumberg 2000. All rights reserved
A/P patterning uses localized regulators (contd)
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anterior vs posterior
– both have a localized
mRNA that produces a
diffusible protein that acts
as a morphogen by
injection experiments
– bicoid and nanos both
function by regulating the
expression of hunchback
BUT
– nanos is required to
repress hunchback
• it is permissive rather
than instructive
– localization of the
posterior components is
much more complicated
than anterior
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bicoid is required to transcribe hunchback mRNA
– hunchback protein is a repressor
– nanos represses translation of hunchback protein in
the posterior
• it represses a repressor - common in development
– net effect is to have hunchback protein in the anterior
half of the embryo only
BioSci 145A lecture 18 (Blumberg) page 18
©copyright
Bruce Blumberg 2000. All rights reserved
Dorsoventral patterning
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BioSci 145A lecture 18 (Blumberg) page 19
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D/V patterning differs
from A/P patterning
– occurs after
cellularization has
occurred
– therefore, it uses
localized
receptor/ligand
interactions
– much more typical
developmental
pathways since most
organisms never
have syncytial state
as does Drosophila
complex interaction
between oocyte and
follicle cells
– process begins again
with the localization
of a mRNA - gurken
– multistep process details are not
critical here.
– cap and spire are
also required for
gurken localization
Bruce Blumberg 2000. All rights reserved
Dorsoventral patterning (contd)
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dorsalizing pathway looks like this
– gurken encodes a protein resembling TGF
• TGF is a growth factor related to EGF
– torpedo is downstream and encodes the Drosophila
EGF-receptor
• probably gurken receptor
– activation of torpedo (receptor tyrosine kinase) leads
to the activation of a typical MAP kinase signaling
pathway
– ultimate result is to prevent activation of the ventral
pathway on the dorsal side of the embryo
– a very similar pathway is used in cell differentiation
in the eye
– illustrates common modus operandi - activates its
own dorsal specifying genes (dpp) while repressing
the activity of genes of the opposite (ventral)
pathway
BioSci 145A lecture 18 (Blumberg) page 20
©copyright
Bruce Blumberg 2000. All rights reserved
Dorsoventral patterning (contd)
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BioSci 145A lecture 18 (Blumberg) page 21
©copyright
Ventral pathway (dorsal
group mutants) is
required for the
formation of a variety
of structures
– dorsal midline
– dorsal ectoderm
– neural ectoderm
– mesdoderm
– ventral midline
gene products produce
ventral phenotype
– mutants produce
dorsalized embryos
(hence dorsal
pathway)
– mutations in any of
the dorsal group
gene prevent the
formation of
ventral and
intermediate
phenotypes
– ventral determinant
can rescue
Bruce Blumberg 2000. All rights reserved
Dorsoventral patterning (contd)
BioSci 145A lecture 18 (Blumberg) page 22
©copyright
Bruce Blumberg 2000. All rights reserved
Dorsoventral patterning (contd)
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pathway in summary
– initial signal is not known
– signal leads to a protease cascade
snake -> easter -> spatzle
– spatzle is the ligand for toll - a critical step in the
cascade
• toll - embryos have no ventral structures
• toll injection rescues ventral structures
• paradoxically, any cytoplasm from a wt embryo
will rescue a toll mutant
– toll activity is found in all parts of a wild
type embryo
– toll is ubiquitously expressed but only activated in
the ventral part of the embryo
• but the ligand is in the perivitelline space and
can presumably diffuse
• spatzle either can not diffuse far or cleavage
does not occur unless toll is in proximity
– toll is similar to the interleukin 1 receptor and the
entire pathway from spatzle -> dorsal is completely
homologous
• IL-1 pathway is required for activation of helper
T lymphocytes and antibody-mediated immunity
• binding of spatzle (IL-1) is sufficient to trigger
the whole ventral-determining pathway
BioSci 145A lecture 18 (Blumberg) page 23
©copyright
Bruce Blumberg 2000. All rights reserved
Dorsoventral patterning (contd)
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BioSci 145A lecture 18 (Blumberg) page 24
©copyright
comparison of IL-1 and
spatzle -> dorsal signaling
pathways
– similarity is actually
rather remarkable
– nothing is new in
evolution!
– also remarkable that
other genes in the
pathway have not been
identified yet
– in lymphoid cells,
activation of the
pathway results in the
release of NF-B from
I- B and its transport
to the nucleus
– cactus appears to have
the same function as IB
– remember that NF- B
is also a viral oncogene
called v-rel
– this should reinforce
the link in your minds
between development
and cancer
Bruce Blumberg 2000. All rights reserved
Dorsoventral patterning (contd)
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BioSci 145A lecture 18 (Blumberg) page 25
©copyright
dorsal (NF- B) is
uniformly distributed
throughout the embryo
– it is specifically
transported to nuclei
in ventral cells where
the spatzle->toll
pathway is activated
– amount of dorsal
protein in the nucleus
is associated with the
degree of
ventralization
– dorsal is a
transcriptional
activator that turns on
twist and snail
• these are
required for
ventral structures
– it also represses
decapentaplegic and
zerknült that are
required for dorsal
structures
Bruce Blumberg 2000. All rights reserved
Dorsoventral patterning (contd)
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D/V patterning requires three localized systems to work
properly
– gurken->torpedo-->represses spatzle dorsally
– snake->easter->spatzle-->dorsal induces ventral cells
• represses dorsal pathway locally
– dpp mediates dorsal development via several members of
the TGF-/BMB signaling pathway that we will talk about
next time.
BioSci 145A lecture 18 (Blumberg) page 26
©copyright
Bruce Blumberg 2000. All rights reserved
Terminal system
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terminal system is not dissimilar to the D/V system but
the downstream genes are quite different
– torso is the key player and it is another receptor
tyrosine kinase
– its activation ultimately leads to the production of
two transcription factors
• tailless - a nuclear receptor that probably
functions as a constitutive repressor
• huckebein - a zinc finger protein that can both
activate and repress transcription
– tailless is required for the formation of terminal
structures
• mutations lead to loss of specialized terminal
structures
– huckebein is required for mesoderm and endoderm
patterning
• it represses the formation of ventral mesoderm in
the ends of the embryo
• it activates genes that cause invagination of gut
primordia at both ends of the embryo
• also regulates wingless and engrailed expression
in the head
BioSci 145A lecture 18 (Blumberg) page 27
©copyright
Bruce Blumberg 2000. All rights reserved
Axis determination in summary
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maternal patterning genes act to broadly partition the embryo
into large, exclusive domains
– anterior system leads to hunchback anterior
– posterior system represses hunchback and promotes knirps
and giant
– dorsal/ventral system leads to decapentaplegic and
zerknült dorsally, twist and snail ventrally
the presence of these proteins, and the boundaries that they
establish are interpreted by the next set of genes to refine
patterning along the axes.
BioSci 145A lecture 18 (Blumberg) page 28
©copyright
Bruce Blumberg 2000. All rights reserved
Next lecture
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patterning of the embryo by zygotically-expressed genes
final comments
BioSci 145A lecture 18 (Blumberg) page 29
©copyright
Bruce Blumberg 2000. All rights reserved