Gene expression in development is controlled at several levels,

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Molecular and Cellular Mechanisms
Gene expression in development is controlled at
several levels,
and it is controlled combinatorially. e.g. by
multiple transcription factors acting positively
and negatively with each other and with DNA
regulatory elements.
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• True or false? The fairly common observation
  that the same transcription factor can activate
  transcription of a gene in one cell type and
  repress transcription of the same gene in another
  cell type can be explained by
• 1) the presence of different DNA regulatory
  (response)
• elements for that gene in the two
  different cell types.
• 2) different combinations of other
  transcription
• factors present in the two cell
  types.
• a) Both these statements are true.
• b) 1 is true 2 is false.
• c) 1 is false 2 is true.
• d) Both are false.

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Molecular and Cellular Mechanisms
Signaling pathways (a small number) allow cells
during development to communicate with each other,
and they act combinatorially to control gene
expression, cell behavior e.g. by multiplexed
signals from growth factors and ECM that can
affect activity of transcription factors and
cytoskeletal components.
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• Which of the following can determine
  how a cell
• responds to a ligand?
• a) The receptors and downstream signaling
  proteins it has synthesized previously.
• b) The transcription factors, splicing
  factors, and other mRNA binding proteins it has
  synthesized previously.
• c) The presence of other signaling
  pathways in
• the cell and ligands in the cells vicinity.
• d) All of the above.
• e) All but one of (a), (b), and (c).

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Using genetics to understand development
Forward genetics with certain organisms is a
powerful approach to finding the genes that
control developmental processes. e.g. screening
for mutants, mapping, gene identification,
protein identification
Reverse genetics is a powerful approach to
defining functions of cloned genes, in addition
to experiments using mutants. e.g. Targeted
knockouts, RNAi (and morpholinos), reporter
constructs, mosaic and chimera experiments.
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Dorsal
Anterior
Posterior
Bicoid mRNA
Ventral
21-55 (see G 9.36)
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Choose from the following list in answering the
next four questions choose the most useful
approach in each case a) biochemical analysis
of gene product activities. b) epistasis
testing. c) forward genetics. d) reverse
genetics. e) mosaic analysis. The components
of the signaling pathway that determines which
side of a Drosophila embryo will be dorsal were
identified by application of -- ?
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Choose the most useful approach from the
following list a) biochemical analysis of
gene product activities. b) epistasis
testing. c) forward genetics. d) reverse
genetics. e) mosaic analysis.
The site of action (e.g. in follicle cells, in
the oocyte) of the various gene products was
determined by application of ?
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Choose the most useful approach from the
following list a) biochemical analysis of
gene product activities. b) epistasis
testing. c) forward genetics. d) reverse
genetics. e) mosaic analysis. The order of
interactions among these gene products in the
pathway was determined by -- ?
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You know the following information about mutants
in the pathway of genes that help to specify the
vulva Mutant gene Phenotype
(lf mutations)
let-60 Vul all Pnp cells make epidermis (3o
fate) lin-1 Muv all Pnp
cells 1o or 2o vulval fate let-60 lin-1 Muv
all Pnp cells 1o or 2o vulval fate Of the
pathways shown below, which best describes the
above data? (Pathways show a genes normal
function.) a) lin-1 -- let-60 ? vulval
fate b) lin-1 ? let-60 -- vulval fate c)
let-60 -- lin-1-- vulval fate d) let-60 ?
lin-1 -- vulval fate
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Add slidessee adding section
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Understanding embryonic development
Development requires establishment of asymmetries
first in the early embryo, later in other cells
and tissues "Give me one asymmetry, and I can
explain development." Sydney Brenner, 1960s
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Which of the following statements about what
happens during cleavage is FALSE? a) The three
orthogonal axes of the embryo and their
polarities are established. b) The zygote
contents are partitioned into separate cells as
the embryo grows in size. c) Some cell
identities are determined and certain cells are
separated from each other. d) A hollow ball or
disc of cells is formed called the blastula. e)
Control of development shifts from maternally
derived to embryonically produced gene
products.
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A
P
The first cleavage in the C. elegans embryo is
unequal because a) factors in the cortex that
specify the position of the future cleavage
furrow are positioned posterior to the
middle of the embryo. b) the spindle is
pulled toward the posterior end of the embryo
prior to cytokinesis. c) the spindle is pulled
toward the anterior end of the embryo prior to
cytokinesis.
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The polarity of the animal-vegetal axis in a
Xenopus embryo is first decided a) before
fertilization. b) by the process of
fertilization. c) during cleavage. d) during
gastrulation. e) after gastrulation.
10-5
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The polarity of the dorsal-ventral axis in a
Xenopus embryo is first established a) before
fertilization. b) by the process of
fertilization. c) during cleavage. d) during
gastrulation. e) after gastrulation.
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Mesoderm arises near the equator (in the marginal
zone) of the Xenopus blastula because a) animal
hemisphere cells in this region are autonomously
determined to become mesodermal precursors. b)
vegetal hemisphere cells in this region are
autonomously determined to become mesodermal
precursors. c) animal hemisphere cells in this
region receive signals from underlying vegetal
hemisphere cells and become mesodermal
precursors. d) vegetal hemisphere cells in this
region receive signals from overlying animal
hemisphere cells and become mesodermal
precursors.
10-7 1 point
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The dorsalizing factors that induce formation of
the organizer in Xenopus embryos a) arise at
the point of sperm entry and move across the
animal pole toward the organizer region. b)
arise at the vegetal pole and move dorsally
toward the organizer region. c) arise at
the animal pole and move dorsally toward the
organizer region. d) are present in ventral
vegetal cells before the organizer forms. e)
are present in dorsal animal cap cells before
the organizer forms.

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Which of these statements is true in the Xenopus
embryo? a) The organizer induces formation of
neural ectoderm by locally inhibiting an
inhibitor of neural cell fates in the animal
cap. b) The organizer induces formation of
neural ectoderm by locally inducing animal cap
cells that would otherwise adopt epidermal
fates. c) The organizer moves during
gastrulation toward the future anterior of the
body axis and then retracts again toward the
ventral side, leaving notochord tissue behind
it. d) The organizer cells contain activated
?-catenin as a master transcriptional
regulator. e) The organizer secretes factors
that activate formation of the Nieuwkoop
center.

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Add two slides here (See adding section)
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The initial positional information that
ultimately specifies different segmental
identities along the A-P axis of the Drosophila
embryo is provided by a. A global A-P gradient
of small-molecular weight morphogen(s). b.
Overlapping gradients of diffusible transcription
factors acting as morphogens. c. Signals between
cells in the cellular blastoderm.
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• Which one of the following classes of Drosophila
  genes does not play a role in the process of
  segmentation along the A-P axis?
• egg polarity genes
• gap genes
• pair-rule genes
• segment polarity genes
• Hox genes
• What establishes the anterior boundary of each
  Hox gene?
• gap gene combinations
• pair rule gene combinations
• A and B
• segment polarity genes
• the boundary of expression of other Hox genes

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• In-class work
• During the course, I and my group have been
  sitting
• a) in the front two rows
• b) in the back two rows
• c) in the middle rows

THANK YOU!!!!
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Review Session for final cumulative exam Friday,
230 - 400 PM, Porter B121 Final Cumulative
Exam Saturday, 1030-100, Hum 250 (this room)
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Start here
Therapeutic cloning to restore ability of
Rag2-defective B lymphocytes to make antibodies
21.25
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Understanding embryonic development
Development requires establishment of asymmetries
first in the early embryo, later in other cells
and tissues "Give me one asymmetry, and I can
explain development." Sydney Brenner, 1960s
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Understanding embryonic development
Cells in an embryo become "determined" for
different developmental fates by two major
mechanisms asymmetric segregation of
cytoplasmic factors during cell division, and in
response to signals from other cells.
But now that we understand the mechanisms, both
are just gene expression and signaling pathways
all over again.
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Understanding embryonic development
In general, cells become more restricted in their
developmental potential as embryonic development
proceeds.
But we now know something about how these
restrictions can be reversed.
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Understanding embryonic development
Sheets of cells (epithelia) can rearrange and
move,
and cells can leave an epithelium to become
mesenchymal or as mesenchymal cells, rejoin an
epithelium. e.g. during gastrulation.
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Understanding embryonic development
Single cells can migrate through tissues,
and receive instructions on where to go and what
genes to express from chemical signals along the
way. e.g. neuronal pathfinding, neural crest
cells, other stem cell migrations. (Note just
signaling pathways again.)
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Understanding embryonic development
Patterns of cells with different fates and
behaviors can arise in response to gradients of
diffusible factors (morphogens),
and then maintained by local cell interactions.
Morphogens can be intracellular (e.g.
transcription factors in the Drosophila syncytial
blastoderm embryo), but are usually
extracellular. Again, signaling pathways, for
both pattern determination and maintenance.
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Understanding embryonic development
Overall patterning of animal body plan involves
segmentation (establisment of repeating units of
pattern) and then control of segment identity by
Hox genes and chromatin remodeling
using mechanisms that are still not well
understood. Hox proteins are master
transcription factors
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Understanding embryonic development
Animals have evolved a variety of chromosomal and
gene control mechanisms for determining sex and
maintaining an optimal sex ratio,
and for dosage compensation of sex-chromosome-link
ed genes in heterogametic species with
chromosomally determined sex.
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Add the following slides These next three slides
go after let-60 epistasis slide
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Using mosaic analysis to understand the
functions of genes Different techniques used in
different species Ultimate outcome Some
wild type cells and some homozgyous mutant cells
are next to each other. Depending on
phenotype of genotypically mutant cells, the
general function of the gene can be
determined gene product acts
cell-autonomously (as a receptor or transcription
factor) or gene product acts cell
non-autonomously (ligand)
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• A mosaic fly has a patch of mutant cells (red
  cells) next to wild type (heterozygous) cells
  (white). Assume juxtacrine signaling.
• Which of the following results would indicate
  that the gene of interest
• acts cell autonomously?
• Cells in Columns 1 and 2 have a wild type
  phenotype. Cells in
• Columns 3,4,5 have a mutant phenotype.
• Cells in Columns 1, 2, and 3 have a wild type
  phenotype. Cells in
• Columns 4 and 5 have a mutant phenotype.
• c. Only cells in Column 1 have a wild type
  phenotype

What if the gene acts non-autonomously?
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Tissue specific knockouts or overexpressions

• Drosophila GAL4 UAS system
• Mouse Cre-Lox system

What are the common principles? both use tissue
specific promoter driving one non-native protein
(Gal4, Cre) both use another marker to target
specific gene (UAS, Lox)
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Gastrulation
Which mesodermal precursor cells enter the
blastopore first in Xenopus gastrulation? a.
presumptive head (anterior, dorsal) mesoderm b.
presumptive tail (posterior, ventral) mesoderm.
The bottle cells that initiate involution at the
blastopore are a. presumptive ectodermal
cells. b. presumptive mesodermal cells. c.
presumptive endodermal cells.
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The principle of Lateral Inhibition

• 1. In a normal developing Drosophila embryo,
  which of the two cells above will become a
  neuroblast?
• a. A
• b. B
• c. Neither
• d. Both
• 2. In a Dl loss of function Drosophila embryo,
  which cell will become a neuroblast?
• a. A
• b. B
• c. Neither
• d. Both
• 3. If B is mutant for Dl but A is not (ie, a
  mosaic), which cell will become a neuroblast?
• a. A
• b. B
• c. Neither
• d. Both

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Delete the following slides
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What will be the phenotype of cells in Columns 4,
5 and 6 if the gene acts non-cell autonomously
and the arrangement of cells is as shown below?
Assume only juxtacrine signaling again.
1 2 3 4 5
6

• a. All cells in Columns 1, 2 and 3 will have a
  mutant phenotype
• All white cells will have a wild type phenotype
  all red cells will have a mutant phenotype
• All cells contacting the white cells will have a
  wild type phenotype
• All cells contacting the white cells will have a
  mutant phenotype
• d. none of the above is correct

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For a loss-of-function bcd mutation, the
phenotype of a heterozygous bcd-/bcd embryo
produced by mating a wild-type male to a
homozygous bcd-/bcd- mother will be a) inviable,
lacking anterior structures. b) inviable, lacking
posterior structures. c) inviable, lacking
anterior and posterior structures. d) you cant
do the experiment because the bcd-/bcd- mother is
dead e) wild-type, because the embryo is
heterozygous
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• You transfect ES cells from a black mouse
  (dominant marker) with this construct
• Following transfection, the culture will include
  
• cells that have lost the transgene without
  inserting it
• cells that have inserted the transgene by
  homologous
• recombination at the YFG locus
• cells that have inserted the transgene at a
  random site by end-joining to a nicked strand
  followed by DNA repair.
• 3) In non-selective growth medium (no neomycin or
  gancyclovir) the most frequent class of cells
  will be__

4) In medium containing gancyclovir alone, the
most frequent class of cells will be ___.
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• cells that have lost the transgene without
  inserting it
• cells that have inserted the transgene by
  homologous
• recombination at the YFG locus
• cells that have inserted the transgene at another
  locus by end-joining to a nicked strand followed
  by DNA repair.

5) In medium containing neomycin alone, the most
frequent class of cells will be___ .
6) In medium containing neomycin and gancyclovir,
the most frequent class will be ___.
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DELETE

• How are segment polarity (SP) genes regulated by
  pair-rule genes?
• A unique combination of pair-rule gene products
  activates each SP gene in each parasegment.
• A unique combination of pair-rule gene products
  activates all the SP genes in each parasegment.
• c. The same combination of pair-rule gene
  products activates a given SP gene in every
  parasegment.
• d. One of two possible combinations of pair-rule
  gene products activates a given SP gene in each
  parasegment.

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• Using the figures above, decide why the early
  isolated animal caps did not make neural cells.
• Chordin and noggin must be present in the early
  stage animal cap
• Chordin and noggin are not present in the early
  stage animal cap but BMPs are
• The animal cap cells are initially fated to be
  epidermis that fate is changed to neural
  ectoderm because of inductive events during
  gastrulation
• The animal cap cells can no longer be inhibited
  by BMPs after gastrulation

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DELETE
What kind of mesoderm are somites derived
from? a. Ventral b. Intermediate c. Paraxial d.
Axial

• MyoD is
• Is a signaling molecule that activates division
  of myoblasts
• Is a signaling molecule that inhibits division of
  myoblasts
• Is a transcription factor that activates
  muscle-specific proteins for differentiation
• Is a transcription factor that inhibits
  muscle-specific proteins
• A molecule with both activating and inhibiting
  functions

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DELETE

• Of the following, which experiment best tests the
  hypothesis that cells in the limb bud have a
  pre-specified fate?
• Remove AER, see what cells types are made
• Mark distal cells in an early limb and see what
  they become
• Remove factors required for proliferation see
  what cell types are made
• Transplant AER from an older limb bud to tip of
  younger limb bud

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• Normal formation of organs often relies upon
• Signaling between epithelium and mesenchymal
  parts of the organ
• Signaling between ectodermal and endodermal parts
  of the organ
• Signals from nearby developing structures
• A and B
• A and C

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• If Shh did not act as a morphogen but still
  affected limb development, what result would you
  have expected from a bead of Shh implanted on the
  anterior side?
• No change, still would be mirror image
  duplication of digits
• An extra digit 4 would be made
• An extra digit 2 would be made
• The duplication would occur but would not be a
  mirror image

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• If you a produce a chick with cardia bifida by
  separating the two heart primordia, what
  experiment would convince you that each of the
  two heart were complete, functioning hearts
  rather than two half hearts?
• If they were both beating
• If they both looped
• If they both expressed left and right-specific
  heart molecules
• If they both expressed markers for ventricular
  and atrial myosin

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• What does the data below indicate about the role
  of Dax-1 in gonad development?
• Dax-1 is inactive in XY individuals
• Dax-1 only specifies ovary development when
  present in two copies
• One copy of SRY represses one copy of Dax-1
• Relative levels of Dax and Sry determine ovarian
  or testes development