Download PPT - Bruce Blumberg

Bio 108 - 3/15/2000
Molecular Genetics of Pattern Formation II
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Contact information
office hours W/F 3-4
phone 824-8573
[email protected] (preferred contact mode)
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Lectures posted at
http://blumberg-serv.bio.uci.edu/bio108-w2000/index.htm
http://blumberg.bio.uci.edu/labtemp/bio108-w2000/index.htm
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Exam update
– Dr. Cho and I will write the exam.
– Questions will be approximately equally distributed
between the sections each of us taught
– The examination will not be identical to any given in
previous years
BioSci 108 lecture 27 (Blumberg) page 1
©copyright
Bruce Blumberg 2000. All rights reserved
Anteroposterior patterning (contd)
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anterior system involves only a small number of genes
– most of these are responsible for localizing bicoid
mRNA
– mutations in the bicoid gene cause the loss of
anterior structures
– increase in the number of copies of the bicoid gene
cause increases in the extent of the anterior
bicoid protein is the active anterior morphogen (fig 2159)
– concentration of bicoid protein, or the number of
bicoid genes directly influences patterning
– bicoid is a homeodomain protein that functions by
directly activating the expression of its target gene
hunchback
– wherever bicoid is injected into an embryo, head
structures form (supp figure)
• this proves that it is a true morphogen that acts
directly to pattern the anterior
– a vertebrate relative of bicoid exists, called
goosecoid
• goosecoid is involved in patterning the anterior
mesodermal tissues in the vertebrate embryo
• works by repressing transcription of target
genes, rather than activating
– can’t diffuse either since vertebrate
embryos always have cells
BioSci 108 lecture 27 (Blumberg) page 2
©copyright
Bruce Blumberg 2000. All rights reserved
Segmentation genes subdivide the embryo
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The maternal genes have broadly divided the embryo into
anterior, posterior, dorsal and ventral
– this pattern of gene expression is interpreted by the
next group of genes to act
– the zygotically-expressed segmentation genes
segmentation genes begin to be transcribed when zygotic
transcription begins at the cellular blastoderm stage
– segmentation genes control the number of segments
and their polarity
– segmentation genes do not influence the overall
polarity of the embryo
segmentation genes can be subdivided into three classes
(Fig. 21-60)
– criteria for classification
• phenotypes of the resulting embryos
• time that the genes act
– gap genes - act first, interpret the maternal gradients
and subdivide the embryos into broad domains
• begin to be transcribed in syncytial blastoderm
• mutations in a single gene eliminate one or more
adjacent segments
• mutations in a different gap gene cause different
but partially overlapping defects
• Krüppel mutations cause the loss of eight
segments T1-A5
BioSci 108 lecture 27 (Blumberg) page 3
©copyright
Bruce Blumberg 2000. All rights reserved
Segmentation genes (contd)
– Pair rule genes act next
• interpret the information specified by the gap
genes and divide the embryos into 14 segments
• mutations cause deletion of alternating segments
• about eight genes have been identified
• all pair rule genes are expressed in two segment
periodicity
– but expression borders relative to segment
boundaries differ among genes
• even-skipped (eve) mutants lack the whole of
even-numbered parasegments
• fushi tarazu (ftz) mutants lack the whole of odd
numbered parasegments
• hairy mutants lack periodic regions of similar
width that are out of register with the
parasegmental units
– segment polarity genes are the last to act
• mutations produce larvae with a normal number
of segments
• but part of each segment is deleted and replaced
with a mirror image duplication of the region
that is present
• in gooseberry mutants, the posterior half of each
segment is replaced by a mirror image
duplication of the anterior half segment
BioSci 108 lecture 27 (Blumberg) page 4
©copyright
Bruce Blumberg 2000. All rights reserved
Hierarchical regulation of segmentation genes
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Segmentation genes have all been cloned and the
majority are transcription factors
– can directly regulate the expression of other genes
– one can compare how they act on one another and on
other genes by comparing gene expression in normal
and mutant embryos
Molecular markers are a key concept in developmental
biology
– one can determine what has happened to a structure
by observing the expression of genes that normally
mark its presence
– can actually deduce the regulatory circuits this way
– example 1:
• even numbered parasegments are marked by eve
expression.
• if eve expression is lost in a mutant, one can
presume that even numbered parasegments are
lost
– example 2:
• in Xenopus, the organizer is marked by
goosecoid expression.
• if gsc expression is lost, one can presume that
the organizer has been lost
– It is not possible to publish a paper today describing
the morphological effects of a mutation - molecular
markers are required
BioSci 108 lecture 27 (Blumberg) page 5
©copyright
Bruce Blumberg 2000. All rights reserved
Hierarchical regulation of segmentation genes (contd)
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phenotypes from mutations in gap genes do not precisely
correspond to the regions where the mRNA for the gene
is expressed
– implies that the protein can diffuse beyond the
boundaries of where the mRNA is expressed
• not possible after cellular blastoderm stage
– example (Fig 21-61)
• both Kruppel and hunchback mRNAs show nonoverlapping expression domains
• however, domains lost in mutants do overlap
with each other significantly
– implication
• gap genes regulate each other’s expression
• most are transcriptional repressors
• boundaries established between fields of gene
expression are very important for subsequent
patterning events
expression of pair rule genes corresponds closely to the
regions that are deleted in loss-of-function mutations (Fig
21-62).
– products of some early acting genes can diffuse a bit
from their site of synthesis
– others (e.g. ftz) are expressed only in the structures
that would be lost
expression of segment polarity genes corresponds
precisely with regions lost in loss-of-function mutations
BioSci 108 lecture 27 (Blumberg) page 6
©copyright
Bruce Blumberg 2000. All rights reserved
Hierarchical regulation of segmentation genes (contd)
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developing hierarchy
– egg polarity genes provide global signals
– these cause gap genes to be expressed in particular
regions of the embryo
– products of gap genes produce a second tier of
positional signals that refine the pattern by regulating
the pair rule genes
– pair rule genes interact combinatorially to regulate
the segment polarity genes
ultimate aim is to precisely specify pattern while using a
minimum number of components
– this is accomplished by sequentially refining a broad
pattern created by only a few genes
– mechanism is not so different from intercalary
regeneration that we discussed a few lectures back
– two possible ways to interpret a gradient to generate
pattern (Fig 21-63)
• cells can respond to morphogen gradient directly
by adopting many states (analog model)
– requires very sensitive discrimination
• cells can respond to morphogen gradient by
adopting one state or another (digital model)
– this sets up two new local signals (or
positional confrontations) that can generate
a new state in between
– no sensitive discrimination required
BioSci 108 lecture 27 (Blumberg) page 7
©copyright
Bruce Blumberg 2000. All rights reserved
Hierarchical regulation of segmentation genes (contd)
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how does one map the hierarchy?
– test expression of positional markers in mutant
backgrount
– experiment:
• test expression of ftz in Kruppel background
• observation
– ftz expression is lost but only in the region
where Kruppel is normally expressed
• conclusion
– kruppel directly or indirectly regulates the
expression of ftz in that part of the embryo
– experiment:
• test expression of Kruppel in ftz background
• observation
– no effect on Kruppel expression
• conclusion
– ftz is downstream of kruppel
– ftz product does not feed back to regulate
kruppel
combinatorial regulation of segmentation genes
– interactions between genes in the same tier of the
hierarchy are also important (e.g. gap genes)
– Kruppel and hunchback are expressed with a sharp
boundary between
• it turns out that these repress each other
BioSci 108 lecture 27 (Blumberg) page 8
©copyright
Bruce Blumberg 2000. All rights reserved
Hierarchical regulation of segmentation genes (contd)
•
combinatorial regulation of segmentation genes
– interactions between genes in the same tier of the
hierarchy are also important (e.g. gap genes)
– Kruppel and hunchback are expressed with a sharp
boundary between
• it turns out that these repress each other
• also occurs with other gap genes, most of which
are transcriptional repressors
• this mutual repression helps to establish the very
sharp borders of expression and precisely
position the expression of downstream genes
– similar mechanisms aid in establishing the sharp
expression boundaries of the pair rule genes (Fig 2164)
• these genes interpret information from gap genes
to yield a reproducible expression pattern of
mutual exclusions.
• combination of early expressed pair rule genes
and gap genes helps to position the later pair rule
and segment polarity genes
• each cell becomes distinguishable by a pattern of
pair rule gene expression (~4 cells wide) (Figure
21-65)
– combinations of pair rule genes can specify
regions for segment polarity gene
expression even down to the single cell
level (e.g. engrailed) (other pix)
BioSci 108 lecture 27 (Blumberg) page 9
©copyright
Bruce Blumberg 2000. All rights reserved
Hierarchical regulation of segmentation genes (contd)
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entire process of regulating zygotic gene expression and
the interactions among patterning genes occurs at the
level of transcriptional regulation
– promoters of the gap, pair rule and segment polarity
genes contain multiple binding sites for other
members of the families
– many of these factors compete for the same binding
sites
• allows precise control of spatial expression
• same sites can be used by different proteins that
may bind to the same site but are expressed in
different compartments
– many homeodomain proteins bind to the
same targets
BioSci 108 lecture 27 (Blumberg) page 10
©copyright
Bruce Blumberg 2000. All rights reserved
Combined action of maternal and segmentation genes
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Observation is that genes are first expressed in patterns
that only approximate the final picture (Fig 21-65)
– the pattern is later refined so that domains are
precisely delineated
– pattern is transient
• gastrulation movements disturb the spatial
patterns of the gap and pair-rule genes
• but these have already imposed positional values
onto the cells
– these labels must be remembered for patterning
process to continue
• expression patterns of segment polarity genes
and homeotic selector genes are responsible for
maintaining the cellular memory of early
patterning events
segment polarity genes mark the parts of each
parasegment
– expressed in repeated patterns
– each parasegment is divided into three regions by the
concerted action of the pair rule genes
– this leads to specific localization of segment polarity
genes
– many of these genes are extracellular receptors and
their ligands (e.g. wingless)
– these rapidly set up autoregulatory loops to maintain
expression but keep it localized
BioSci 108 lecture 27 (Blumberg) page 11
©copyright
Bruce Blumberg 2000. All rights reserved
Combined action of maternal and segmentation genes
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segment polarity genes mark (contd)
– the chemical distinction created by segment polarity
genes may persist into adulthood
– e.g. engrailed is first expressed in on row of cells
from each parasegment (Fig. 21-66)
• anterior border of each parasegment
• posterior border of segment
• it persists in the posterior part of each segment in
the adult
in addition to regulating the segment polarity genes, the
product of the pair rule, gap and maternal genes act
together to cause localized regulation of homeotic
selector genes that we will talk about next lecture
BioSci 108 lecture 27 (Blumberg) page 12
©copyright
Bruce Blumberg 2000. All rights reserved