Download and mutant - McGraw Hill Higher Education

PowerPoint to accompany
Genetics: From Genes to
Genomes
Fourth Edition
Leland H. Hartwell, Leroy Hood,
Michael L. Goldberg, Ann E. Reynolds,
and Lee M. Silver
Prepared by Mary A. Bedell
University of Georgia
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Hartwell et al., 4th edition
1
PART V
How Genes Are Regulated
CHAPTER
Using Genetics to
Study Development
CHAPTER OUTLINE
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18.1 Model Organisms: Prototypes for Developmental Genetics
18.2 Using Mutations to Dissect Development
18.3 Analysis of Developmental Pathways
18.4 A Comprehensive Example: Body-Plan Development in Drosophila
18.5 How Genes Help Control Development
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How does the single cell of a fertilized egg
differentiate into thousands of cell types?
Figure 18.1
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Model organisms: Prototypes for
developmental genetics
Five model organisms:
•
•
•
•
•
Saccharomyces cerevisiae (yeast)
Arabidopsis thaliana (plant)
Drosophila melanogaster (fruit fly)
Caenorhabditis elegans (roundworm)
Mus musculus (mouse)
Advantages for research
• Easy to grow
• Rapid reproduction
• Genetic resources
 e.g. stock centers that maintain and share mutant strains
• Genome sequencing
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Two of the model organisms used in
developmental genetics
Mutations in Drosophila
genes can affect early
embryonic development
Transparency of C. elegans
facilitates study of the
worm’s development
Figure 18.3
Figure 18.2
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All living forms are related
Cells of many eukaryotes have microscopic features in
common – e.g. nuclei and mitochondria
Metabolic pathways are virtually identical in all organisms
Almost all cells use the same genetic code
Many homologous proteins have highly conserved amino
acid sequences
Many developmental strategies are conserved in
multicellular eukaryotes
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A transcription factor that is critical for eye
development in Drosophila and mouse
Drosophila with mutant
Mouse embryos with wild-type (L) and
alleles of the eyeless gene mutant (R) alleles of the Pax-6 gene
Figure 18.4b
Humans with mutations in the Pax-6
gene have aniridia (not shown)
Figure 18.4a
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Despite many biological similarities,
all species are unique
Disparate strategies are used by different organisms to
accomplish the same developmental goal
Example: cell fate differences in two-cell embryos of C.
elegans and humans
•Mosaic determination in C. elegans
 Each cell has been assigned a developmental fate
 Abnormal development if one cell is removed
•Regulative determination in humans
 Cells can alter their development fates according to the
environment
 Twins result if the two cells are separated
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Using mutations to dissect development
Identification of different alleles of the same gene can be
important to understanding developmental processes
Loss-of-function mutations - usually recessive
• Can alter the amino acid sequence – results in
diminished (or no) biochemical activity
• Can interfere with gene expression (transcription, RNA
processing, translation) – results in decreased (or no)
expression of a normal protein
Gain-of function mutations – usually dominant
• Can produce too much protein, or proteins with new
function
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Loss-of-function mutations that are recessive
Null mutations – complete loss-of-function
• Knockouts can be made by gene-targeting (Fig 18.5)
Hypomorphic mutations – partial loss-of-function
• Useful for understanding how one gene functions at
multiple times in development
 e.g. wingless gene in Drosophila is essential for viability of
embryos and for formation of wings in adults
Conditional mutations – loss-of-function only under certain
conditions
• e.g. Temperature-sensitive mutations (Fig 18.6)
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Constructing knockout mice: Creating a cell
line with a knockout allele of a gene
Use recombinant DNA
techniques to insert the
neomycin (neo) resistance
gene into the gene of interest
Embryonic stem (ES) cell line
established from early
embryos of agouti mice
Introduce disrupted gene
into ES cells and select for
neomycin resistance
Identify ES colony with
knockout allele
Figure 18.5a -c
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Constructing knockout mice: Creating mice
that carry the knockout gene
Produce blastocysts by
breeding black mice
Inject targeted ES cells into
blastocysts and put blastocysts
into uterus of another black mouse
Offspring will be chimeric
and can transmit knockout
allele to their offspring
Figure 18.5d - f
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Time-of-function analysis using a
temperature-sensitive mutation
Embryonic development in C. elegans females homozygous
for zyg-9 gene
• At permissive temperature, development is normal
• After short pulse of higher temperature, development is
normal during some windows of time and abnormal
during a critical period of time
Figure 18.6
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Loss-of-function mutations that are dominant
Haploinsufficiency
• In some genes, one wild-type allele is not sufficient for
normal development
Dominant negative mutations
• Inactive protein expressed from mutant allele reduces
the function of normal protein expressed from the wildtype allele
• Example: multimeric proteins (Fig 8.31 on p. 279), or
mutant receptor that sequesters a ligand (Fig 18.7)
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Engineering a dominant-negative mutation in
fibroblast growth factor receptor (FGFR)
Figure 18.7
Normal FGFR is on the cell
surface and interacts with
extracellular ligand
Mutant FGFR cannot localize to
cell surface
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Phenotypic effects of dominant-negative FGFR
Transgenic mice expressing mutant FGFR had several
defects, including abnormal limb development
Non-transgenic mouse limb
Transgenic mouse limb
Figure 18.7c
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RNA interference (RNAi) disrupts gene
function without mutations
RNAi targets the degradation of mRNA from specific genes
Vector constructed to synthesize double-stranded (ds) RNA
with sequence to gene of interest (Fig 18.8a)
dsRNA introduced into developing embryos
dsRNA is degraded into shorter fragments that serve as
templates for degradation of target mRNAs
Can produce a phenocopy of a loss-of-function mutation
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Synthesis of dsRNA and abnormal
development caused by RNAi in C. elegans
Wild-type vulva, no dsRNA
Figure 18.8
Abnormal vulva, dsRNA for par-1
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Gain-of-function mutations are
usually dominant
Mutations causing excessive gene activity
• Results from highly specific alterations
• Much less frequent than loss-of-function mutations
• Several mechanisms: promoter mutations that increase
transcription, mutations in receptors that increase
affinity for ligand, mutations in receptors that cause
constitutive activity
Mutations causing ectopic gene expression
• Expression of a gene in an abnormal place or time
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Achondroplastic dwarfism in the mouse is
caused by constitutive activation of FGFR3
Figure 18.9
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Ectopic expression of the eyeless gene
produces ectopic eye tissue in Drosophila
Transgenic flies with eyeless
gene under control of a heatshock promoter
Flies grown at high temperature
have high eyeless expression in
all parts of the body
Ectopic eye development in
different parts of body
Figure 18.10
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Analysis of developmental pathways
Characterize the action of each gene in a pathway
• Nature of the encoded protein
 Infer amino acid sequence from nucleotide sequence
 Computer searches to identify known motifs
• Location and timing of gene expression
 During development, where and when is the mRNA found?
• Location of the protein product
 During development, where and when is the protein found?
• Developmental phenotypes
 What cells or tissues are affected by loss-of-function?
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A motif found in many transcription factors
that regulate development
Homeodomain – DNA
binding domain
Interacts with specific
sequences in DNA
Figure 18.11
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In situ hybridization locates cells
expressing a gene of interest
Labeled cDNA for Pax-6 gene
used as a probe on sections
of human fetal tissues
Hybridization of Pax-6 probe
to the developing neural
retina and eye lens
Figure 18.12
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Using antibody tagging to follow the
localization of proteins
Synthetic gene encodes a
fusion protein, which can be
expressed in bacteria and then
used to generate specific
antibodies
Staining of Drosophila imaginal
disc with fluorescently-labeled
antibodies against several
proteins
Figure 18.13a, b
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Using GFP tagging to follow the
localization of proteins
Recombinant gene encoding a
fusion protein with green
fluorescent protein (GFP) at the
C terminus
A mouse with a GFP-tagged
transgene expressed in the skin
Figure 18.13c, d
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Using genetic mosaics to understand
developmental phenotypes in Arabidopsis
Blue tissue has a marker gene
and is AGAMOUS+
White tissue doesn't have the
marker gene and is AGAMOUS−
Signal from blue AGAMOUS+
L2 cell is needed for proper
differentiation of L1 cells
Figure 18.14
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Interactions of genes in a developmental
pathway must be determined
Genes don't work in isolation!
• Many biological processes are complicated and require
coordinated action of many genes
Analysis of effects of one gene on expression of another
gene
• Does a mutation in one gene affect the level or
distribution of mRNA or protein from another gene?
Analysis of double mutants – epistatic interactions
• Do mutations in two different genes define successive
steps in a pathway?
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In Drosophila, the wingless gene product is
required for expression of the vestigial gene
Staining of Drosophila wing imaginal disks for wingless
protein (Wg, green) and vestigial protein (Vg, red)
(top) Wild-type
• Yellow is observed in cells
expressing both Wg and Vg
(bottom) wingless mutant
• Vg protein appears only in a
very narrow band
Figure 18.15
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Genetic interactions that can be defined by
analysis of double mutants
Epistasis – usually defines the earlier acting step in the
pathway
• Phenotype of double mutant resembles one of the
single mutants
Suppressor – mutation in one gene counteracts the effects
of mutation in another gene
• Phenotype of double mutant is similar to wild-type
Enhancer – mutation in one gene exacerbates the effects of
mutation in another gene
• Phenotype of double mutant is worse than either single
mutant
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Double mutant analysis:
Epistasis in the secretion pathway
Mutant for both gene A
and gene B has phenotype
like that of mutant for gene
A only
Both proteins act in the
same pathway
Gene A protein acts earlier
than gene B − gene A is
epistatic to gene B
Figure 18.16a
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Double mutant analysis:
Epistasis in the pathway for vulva formation
In this case, MEK-2 acts at a later step in the pathway than
LET-60
But, a mutation in MEK-2 is epistatic to a mutation in LET-60
Figure 18.16b
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Interpreting the results of double
mutant analysis
Is the effect due to epistasis, a suppressor, or an enhancer?
Need more information in addition to double mutant
phenotype:
• Are the mutations loss-of-function or gain-of-function?
• Does the whole pathway have a single output or does
blocking different steps have a different outcome?
• What are the biochemical roles (e.g. transcription
factor, kinase, hormone receptor) of the encoded
proteins?
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A comprehensive example: Genetic analysis of
body-plan development in Drosophila
Clearly defined segments are formed in the embryo and
each segment forms a specific structure in the adult
• How does the developing embryo establish the proper
number of body segments?
 Early in development, the products of segmentation genes
subdivide the body into an array of identical body
segments
• How does each body segment know what kind of
structures it should form?
 Later in development, the products of homeotic genes
assign a unique identity to each segment
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Early Drosophila
development: From
fertilization to cellular
blastoderm
During the first three hours after
fertilization:
Syncytial blastoderm formed by
13 very rapid mitotic divisions
without any cell division
Cellular blastoderm formed by
cellularization that begins during
interphase of 14th division
Figure 18.17a
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Drosophila development
after formation of the
cellular blastoderm
Gastrulation begins immediately
after cellularization
Furrows lead to establishment of
three embryonic germ layers:
mesoderm, ectoderm, endoderm
First visible signs of segmentation
appear 40 min after gastrulation
begins
By 10 hrs after fertilization, 14 body
segments are formed
Figure 18.18a - c
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Segment identity is preserved throughout
Drosophila development
Each embryonic segment
defines a specific structure
in the adult
• 3 head segments
• 3 thoracic segments
• 6 abdominal segments
Figure 18.18d
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Genetic screens for mutations affecting early
development of Drosophila embryos
Christiane Nusslein-Vollhard and Eric Wieschaus (both
shared the Nobel Prize with Edward Lewis in 1995)
Two mutagenesis screens to identify genes that control
embryonic development:
1. Screened for abnormal embryos in homozygous mutant
females
• Identified recessive mutations in maternal-effect genes
2. Screened for abnormal homozygous mutant embryos
• Identified recessive mutations in three classes of
zygotic segmentation genes
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Four classes of genes responsible for segment
formation in Drosophila embryos
Function in a hierarchy that progressively subdivides the
embryo into successively smaller units
• Maternal genes – expressed by mother and the mRNAs
deposited in egg, not translated until after fertilization
• Gap genes – expression begins at syncytial blastoderm
stage and controlled by maternal gene products
• Pair-rule genes – seven zones of expression are
controlled by gap gene products
• Segment polarity genes – expression in 14 segments is
controlled by pair-rule gene products
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Two morphogens control anterior and posterior
patterning in the Drosophila embryo
Morphogen – substance that defines different cell fates in a
concentration-dependent manner
Two maternal-effect gene products [bicoid (bcd) and nanos
(nos)] are morphogens
bcd and nos genes are transcribed by the mother and their
mRNAs are localized to opposite poles of the oocyte
bcd and nos mRNAs are not translated in the embryo until
after fertilization
Each protein forms a gradient in the embryos:
• bcd is highest at anterior and lowest at the posterior
• nos is lowest at anterior and highest at posterior
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Localization of bicoid mRNA and protein
bcd mRNA localizes to
the anterior pole of the
oocyte
bcd protein diffuses from
the anterior pole of the
embryo to produce an
anterior-to-posterior
gradient
bcd protein acts as
transcription factor and
translation repressor
Figure 18.19a, b
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Evidence that
bicoid is the
anterior
morphogen
Dosage of maternal bcd
gene determines how
much of the embryo
becomes head structures
Figure 18.19c
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Distribution of mRNAs of the four
maternal-effect genes within oocytes
Bicoid (bcd) mRNA localizes to anterior pole
Nanos (nos) mRNA localizes to the posterior pole
Hunchback (hb) and caudal (cad) mRNAs are uniformly
distributed
Figure 18.20
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Distribution of the protein products of
maternal-effect genes within the early embryo
Bicoid protein represses translation of caudal mRNA
 Causes posterior-to-anterior gradient of caudal protein
Nanos protein represses translation of hunchback mRNA
 Causes anterior-to-posterior gradient of hunchback protein
Figure 18.20
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Segment number is further specified
by zygotic genes
Bicoid, hunchback, and caudal proteins are transcription
factors that control the spatial expression of zygotic genes
Zygotic gene expression begins in the syncytial blastoderm
stage
Three classes of zygotic segmentation genes:
• 9 gap genes
• 8 pair-rule genes
• 17 segment polarity genes
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Zones of gap gene expression
Gap genes (Krüppel, hunchback, knirps, and giant) are the
first zygotic genes expressed
• Binding sites in promoter regions of gap genes have
different affinities for bcd, cad, and hb proteins
• Some gap genes encode transcription factors that
control expression of other gap genes
Gap gene products control division of the body axis into
rough, generalized regions
Figure 18.21a
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Embryos with segmentation defects
caused by mutations in gap genes
normal
hunchback
Krüppel
knirps
Figure 18.21b
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Mutation of a particular gap gene results in loss of
segments corresponding to its zone of expression
Figure 18.21c
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Two classes of pair-rule genes
Three primary pair-rule genes
• Expression is controlled by transcription factors
encoded by maternal genes and zygotic gap genes
• Upstream region of each pair-rule gene has multiple
binding sites for transcription activation/repression
Five secondary pair-rule genes
• Expression is controlled by transcription factors
encoded by other pair-rule genes
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Pair-rule genes are expressed in seven stripes
at the early blastoderm stage
e.g. even-skipped (eve) and
fushi tarazu (ftz)
Expression of pair-rule genes
divides the body axis into
sharply-defined stripes
• Two-segment periodicity:
each stripe has two segments
In stripe 2, eve transcription
is activated by Bcd and Hb,
but repressed by giant (Gt)
and Krüppel (Kr) proteins
Figure 18.22a, b
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Upstream regulatory region of the eve gene
700 bp region contains multiple binding sites for Krüppel
(Kr), giant (Gt), bicoid (Bcd) and hunchback (Hb) proteins
Binding of Kr and Gt proteins represses eve transcription
Binding of Bcd and Hb proteins activates eve transcription
Figure 18.22c
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Segment polarity genes determine patterns
that are repeated in each segment
e.g. Engrailed (en), hedgehog (hh), and wingless (wg)
Activation occurs after cellularization – diffusion of proteins
within the syncytium no longer plays a role in patterning
Intrasegmental patterning is determined primarily by
diffusion of secreted factors (e.g. Hh and Wg) between cells
Transcription factors encoded by pair-rule genes initiate
expression of segment polarity genes in each segment
Interactions between various polarity genes maintains the
periodicity
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Distribution of engrailed protein in 14 stripes
Segment polarity genes are expressed in stripes that are
repeated with single segment periodicity (one stripe per
segment)
Figure 18.23a
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En, Hh, and Wg are responsible for many
aspects of segmental patterning
En protein is a transcription factor
Hh and Wg proteins are morphogens that bind to specific
receptors on cells in adjacent segments
• Activate signal transduction pathways that contain
proteins encoded by other segment polarity genes
En activates transcription of the hh gene in the posterior
compartment
Hh protein initiates a signal that results in activation of wg
transcription in the adjacent anterior compartment
Wg protein initiates a signal that results in activation of en
and hh transcription in the adjacent posterior compartment
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Segment polarity genes establish
compartment borders
Signal transduction pathways initiated by Hh and Wg result
in a reciprocal loop that stabilizes cell fates at the borders
of adjacent segments
Figure 18.23b
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The genetic
hierarchy leading to
segmentation in
Drosophila
In successive levels of
the hierarchy, genes are
expressed in narrower
bands
Figure 18.24a
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Mutations in
segmentation genes
cause segment loss
These mutant embryos have
lost part of each segment
Often, the remaining part of
the segment will be a mirrorimage duplication
Figure 18.24b
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Segment identity is established by
homeotic genes
Transcription of homeotic genes is controlled by gap, pairrule, and segmentation genes
At the cellular blastoderm stage, each homeotic gene is
expressed within a subset of body segments
Homeotic genes are master regulators that control
transcription of many genes responsible for development of
segment-specific structures
Homeotic mutations cause particular segments to develop
as if they were located elsewhere in the body
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Bithorax (bx) and postbithorax (pbx) mutants
have homeotic transformations in the third
thoracic segment (T3)
Wild type
bx mutant
pbx mutant
Figure 18.25
bx pbx double mutant
bx mutant: anterior T3 transformed into
anterior T2
pbx mutant: posterior T3 transformed
into posterior T2
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Two clusters of homeotic selector genes
control most aspects of segment identity
Both clusters of genes are
on chromosome 3
Genes in Antennapedia
complex (ANT-C) control
segments in the head and
anterior thorax
Genes in bithorax complex
(BX-C) control segments in
the abdomen and posterior
thorax
Figure 18.26
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The bithorax complex of Drosophila
Three BX-C genes:
Ultrabithorax (Ubx)
abdominal-A (abd-A)
Abdominal-B (Abd-B)
Figure 18.27
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The BX-C has multiple regulatory regions that
control expression of three genes
Edward Lewis (shared Nobel Prize in 1995 with NussleinVollhard and Wieschaus)
Identified infra-abdominal (iab) mutations – mutations in the
BX-C that affect each of the abdominal segments
Many of the bx, pbx, and iab mutations affect large cisregulatory regions that control spatial and temporal
expression of Ubx, abdA, and AbdB
Order of regulatory regions corresponds to the anterior-toposterior order of segments
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The Antennapedia complex of Drosophila
ANT-C complex controls segment identities in the head and
anterior thorax
Five genes:
• labial (lab) – expressed in intercalary regions
• proboscipedia (pb) – expressed in maxillary and labial
segments
• Deformed (Dfd) – expressed in the mandibular and maxillary
segments
• Sex combs reduced (Sxr) – expressed in labial and T1
segments
• Antennapedia (Antp) – expressed mainly in T2
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The homeodomain in development
and evolution
Homeobox – a 180 bp region of closely-related sequence
found in homeotic genes (at ANT-C and BX-C), as well as
some non-homeotic genes (bicoid and eyeless)
• Encodes a 60 amino acid homeodomain, which is a
DNA binding domain
Hox gene clusters found in all animal genomes
• More Hox genes are found in animals with a more
complex body plan
 Humans and other mammals have 38 Hox genes that are in
four clusters (see Fig 18.28)
• In all organisms, linear order of genes in each cluster
reflect their anterior-to-posterior expression
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The mammalian Hox genes are organized
into four clusters
Figure 18.28
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Synpolydactyly caused by mutations in
the human HoxD13 gene
Figure 18.29
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Development requires sequential changes
in gene expression
Different cell types express characteristic subsets of genes
Cell fate is progressively refined
• Once a developmental fate is determined, the cell and its
descendants follow a differentiation path that excludes an
alternative fate
Transcriptional regulation plays a key role
Posttranscriptional gene regulation also is important
• e.g. splicing, transport to nucleus, mRNA stability,
translational regulation, protein stability
Earliest stages of development require both maternal and
zygotic gene products
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Development requires
precise control of
the expression of
many genes
Wing imaginal discs of
Drosophila
Each disk was stained with a
fluorescent antibody against
a different protein
Figure 18.31
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Hartwell et al., 4th edition, Chapter 18
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Development exploits asymmetries
Cells must be exposed to different environmental signals or
they must be intrinsically distinct at the biochemical level
In some species, the egg is inherently asymmetrical
• Drosophila egg has an anterior-to-posterior polarity
because of connections to nurse cells at anterior end
In other species, asymmetries occur after fertilization
• C. elegans – site at which sperm enters the egg
• Mammals – asymmetry doesn't occur until after four
rounds of mitosis (16 cell embryo)
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A Drosophila egg chamber
Somatic nurse cells connect to anterior part of oocyte
Bicoid mRNA is transcribed in nurse cells, transported into
oocyte, and associates with microtubules in oocyte
Figure 18.32
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Cell-to-cell communication is essential
for proper development
Cells must obtain information about their positions relative
to other cells
Ligand expressed by one cell binds to a receptor expressed
on the surface of another cell
• Juxtracrine signaling – ligand is membrane-bound and
interacts with a receptor on an adjacent cell
• Paracrine signaling – ligand is secreted and mediates
long- range (e.g. hormones) or short-range signals (e.g.
Wingless and hedgehog)
Different ligand/receptor combinations active different
signal transduction pathways
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