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Chapter 18 Gene Regulation during Development

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• 200431470062

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• Outline
• Three strategies by which cells
• are instructed to express specific sets
• of genes during development
• Examples of the three strategies
• for establishing differential gene
• expression
• The molecular biology of Drosophila
• embryogenesis

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Three strategies by which cells are instructed to
express specific sets of genes during development
? mRNA localization ?Cell-to-cell
contact ?Signaling through the diffusion of
secreted signaling molecules
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Some mRNAs Become Localized within Eggs and
Embryos due to an Intrinsic Polarity in the
Cytoskeleton
The asymmetrically distributed mRNA is
transported along element of the cytoskeleton
from to the growing end. Adaptor protein
binds to 3untranslated trailer (3UTR) region of
the mRNA, has two domains one recognizes the 3
UTR,the other associates with myosin.
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Cell-to-Cell Contact and Secreted Cell Signaling
Molecules both Elicit changes in Gene Expression
in Neighboring Cells

• Cell-to-Cell Contact signaling molecules remain
  on the surface control gene expression only in
  cells which are directly, physically contact with
  the signaling cell.
• ? A given signal is recognized by a specific
  receptor on the surface of recipient cells,
  triggers changes in gene expression in them.
• ? Then signal transduction pathways is involved
  in the communication from the cell surface
  receptor to the nucleus.

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Signal Transduction Pathways

1. Ligand-receptor interaction induces kinase
   cascade that modifies regulatory proteins present
   in nucleus.
2. Activated receptor cause the release of
   DNA-binding protein so it can enter the nucleus,
   regulate gene transcription.
3. The intracytoplasmic domain of the activated
   receptor is cleaved to enter the nucleus and
   interact with DNA-binding protein.

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Gradient of Secreted Signaling Molecules

• Two concepts
• Positional information
• a cells development is influenced by its
  location within the developing embryo.
• Morphogens
• signaling molecules that control position
  information

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• Cells located near the source of morphogen
  receive high concentration of the signaling
  molecule, experience peak activation of
  receptors, determine most regulatory protein
  enter the nucleus while the situation of cells
  locate far from the source is just the opposite.
• The different levels of the regulatory factor
  lead to the expression of different sets of gene.

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Topic 2Examples of the Three Strategies for
Establishing Differential Gene Expression
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mRNA Localization Controls Mating Type in Yeast

• A haploid yeast cell budding to produce a mother
  cell and a smaller daughter cell.
• The daughter cell cant switch due to localized
  Ash1 repressor,it cant express HO which initiate
  switching.
• The mother cell can switch it lacks Ash1,and is
  able to express HO.

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• The ash1 gene is transcribed in the mother cell
  during budding.The encoded mRNA localized within
  the daughter cell by sliding along polarized
  actin filaments, depends on She2 and Sh3 adapter
  proteins that bind the 3UTR and myosin.
  

• Once localized within daughter cell, ash1 mRNA is
  translated into a repressor protein that binds to
  and inhibits the transcription of HO gene.

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• General principle broadly distributed activators
  and localized repressors interplay to establish
  precise patterns of gene expression within
  individual cell.

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A Localized mRNA initiates Muscle Differentiation
in the Sea Squirt Embryo

• Macho-1 regulatory protein is a major determinant
  to form muscle .
• The Macho-1 mRNA encodes a zinc finger
  DNA-binding protein that id believed to activate
  the transcription of muscle-specific genes,such
  as actin and myosin.
• These genes are expressed only in muscles because
  Macho-1 is made only in those cells.

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• Macho-1 mRNA is initially distributed throughout
  the cytoplasm of unfertilized eggs but becomes
  localized to the vegetal(bottom)region shortly
  after fertilization, ultimately inherited by two
  cells of the eight-cell embryos,thus the two
  cells go on to form the tail muscles.

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Cell-to-Cell Contact Elicit Differential Gene
Expression in the Sporulating Bacterium,
B.subtilis

• B.subtilis spore formationa septum form at an
  asymmetric location within the sporangium,
  produce two cells remain attached through
  abutting membranes.The smaller cell is forespore,
  it ultimately forms the spore.The larger cell is
  the mother cell,it aids the development of the
  spore.
• The forespore influences the expression of genes
  in the neighboring mother cell.

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• sF factor in forespore activated the spoIIR gene.
• The encoded SpoIIR protein is secreted and
  associate with the septum where it triggers the
  proteolytic processing of an inactive form of
  sE(pro-sE)in the mother cell.The pro-sE protein
  contains an N-terminal inhibitory domain that
  blocks sE activity and tethers the protein to the
  membrane of the mother cell.
• After the cleavage of the N-terminal peptide, the
  activated sE protein leads to the transcription
  of target genes.

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Asymmetric gene activity in the mother cell
and forespore in the B.subtilis
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• SpoIIR functions as a signaling molecule that
  acts at the interface between the forespore and
  the mother cell,elicits differential gene
  expressions.
• Induction requires cell-to-cell contact because
  the forespore produces small quantities of SpoIIR
  which are insufficient to elicit the processing
  of sE in the other cells except the abutting
  mother cell.

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Delta-Notch Signaling control skin-nerve
regulatory in the Insect CNS

• In insect embryo, Neurons of the ventral nerve
  cord arises from neurogenic ectoderm, the other
  cell population is ventral skin.
• Signaling between the two populations decide
  which to become skin or neuron.

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• The developing neurons contain a signaling
  molecule Delta on their surface,which binds to
  and activates the Notch receptor on the skin
  cells.
• Activation causes the intracytoplasmic domain of
  Notch (NotchIC) to be released from the cell
  membrane and enter the nuclei,then it associates
  with the DNA-binding protein Su(H).

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• NotchIC displaces the repressor proteins in
  complex with Su(H),turning Su(H) into a activator
  instead.
• The Su(H)-NotchIC complex activates genes that
  encodes transcriptional repressors which block
  the development of neurons.

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A Gradient of the Shh Morphogen Controls the
Formation of Different Neurons in the Vertebrate
Neural Tube
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• The activation of the Shh receptor allows a
  previously inactive form of Gli transcription
  activator to enter the nucleus in an activated
  form.
• Once in the nucleus,Gli activates gene expression
  in a concentration-dependent fashion.
• The different binding affinity of Gli recognition
  sequences within the regulatory DNAs of the
  various target genes is important in the
  differential regulations of Shh-Gli target genes.
• Thus,V1 genes have high-affinity
  recognition sequences for the activator in the
  nearby regulatory DNA so they can be activated by
  low levels of Gli.

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Topic3The Molecular Biology of Drosophila
Embryogenesis
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• Localized determinants and cell signaling
  pathways are both used to establish positional
  information that result in gradients of
  regulatory proteins that pattern the
  anterior-posterior (head-tail) and dorsal-ventral
  (back-belly) body axes.
• A recurring theme is the use of complex
  regulatory DNAs to bring transcriptional
  activators and repressors to genes whose product
  define different regions of the embryo.

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An Overview of Drosophila Embrogenesis

• A single sperm
  cell enter a mature egg
• Form diploid zygotic nucleus
• Series of synchronous divisions syncitium(a
  single cell with multiple nuclei)
• nuclei migrate to cortex
• formation of monolayer
• 1-hour period cell membranes form between
  adjacent nuclei
• Just after cellularization, nuclei become
  irreversibly determined to differentiate into
  specific tissues.

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A Morphogen Gradient Controls Dorsal-Ventral
Patterning of the Drosophila Embryo

• The dorsal-ventral patterning of the early
  Drosophila embryo is controlled by a regulatory
  protein called Dorsal.
• Regulated nuclear transport of the Dorsal protein
  is controlled by the cell signaling molecule
  Spätzle,which is distributed in a
  ventral-to-dorsal gradient within the
  extracellular matrix.

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• After fertilization, Spätzle
• binds to the cell surface Toll
  receptor.Depending on the concentration of
  Spätzle,Toll is activated to greater or lesser
  extent.Peak activation of Toll is in ventral
  regions,where the Spätzle concentration is
  highest.
• Toll signaling causes the degration of a
  cytoplasmic inhibitor Cactus,and the release of
  Dorsal from the cytoplasm into nuclei.

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• The Dorsal gradient specifies three major
  thresholds of gene expression across the
  dorsal-ventral axis of embryos undergoing
  cellularization.
• The highest levels of the Dorsal gradient
  activate the expression of the twist gene in the
  ventralmost 18cells that forms the mesoderm. The
  twist 5 regulatory DNA contains two low-affinity
  Dorsal binding sites, so peak levels of the
  Dorsal gradient are required for the efficient
  occupancy of these sites. The rhomboid gene is
  activated by intermediate levels of the Dorsal
  protein in the ventral neurogenic ectoderm.The
  enhancer in it has a cluster of mostly
  low-affinity Dorsal binding sites but one
  high-affinity site. Thus, the rhomboid enhancer
  can be activated by both the high and the
  intermediate levels of Dorsal protein.
• The lowest levels of the Dorsal protein
  are sufficient to activate the sog
• gene in both the ventral and the dorsal
  neurogenic ectoderm. The enhancer
• of the gene contains four high-affinity
  Dorsal binding sites.

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• Both rhomboid and sog gene are kept off by the
  transcriptional repressor Snail in the mesoderm
  for they have binding sites for it .Thus the
  Snail repressor and the affinities of the Dorsal
  binding sites together determine specific gene
  expression.

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Segmentation Is Initiated by Localized RNAs at
the Anterior and Posterior Poles of the
Unfertilized Egg

• In fertilization,Drosophila egg contains two
  localized mRNAs.One,the bicoid mRNA,is located at
  the anterior pole, while the other,the oskar mRNA
  at the posterior pole.
• The oskar mRNA is first deposited at the
  anterior end of the oocyte,then transported from
  anterior to posterior regions.

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• Like ash1 mRNA in the yeast, the oskar mRNA
  interacts with adapter proteins which associate
  with the growing ends of the microtubules
  depends on specific sequences within the 3UTR
  region, thereby transported into the posterior
  plasm.
• After fertilization the cells that inherit the
  localized oskar mRNA form the pole cells.

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• The ash1 gene is transcribed in the mother cell
  during budding.The encoded mRNA localized within
  the daughter cell by sliding along polarized
  actin filaments, depends on She2 and Sh3 adapter
  proteins that bind the 3UTR and myosin.
  

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• The localization of the bicoid mRNA in anterior
  regions also depends on sequences contained
  within its 3 UTR. Therefore, the 3UTR is
  important in determine where each mRNA becomes
  localized.

• If the 3UTR from the oskar mRNA is replaced with
  that from biciod, the hybrid oskar mRNA is
  located to anterior regions (just as biciod
  normally is).

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• Localized bicoid mRNA initiates anterior regions

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The Bicoid Gradient Regulates the Expression of
Segmentation Genes in a Concentration-Dependent
Fashion

• The Bicoid regulatory protein diffuses away from
  its source of synthesis at the anterior pole and
  simply distributed across the syncitial embryo.
• There are peak levels of the Bicoid protein in
  anterior regions,intermediate levels in the
  central regions and low levels in posterior
  regions.

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• Only high concentrations of Bicoid activate the
  expression of orthodenticle,while both high and
  intermediate concentrations are sufficient to
  activate hunchback.
• This differential regulation of orthodenticle and
  hunchback depends on the binding affinities of
  Biciod recognition sequences.The orthodenticle
  gene is regulated by a 5enhancer that contains a
  series of low-affinity Biciod binding sites,while
  hunchback gene is regulated by a 5enhancer
  contains high-affinity binding sites.
• The Bicoid protein binds to DNA as a monomer,
  Bicoid monomers interact with each other to
  foster the cooperative occupancy of adjacent
  sites.

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Hunchback Expression Is also Regulated at the
level of Translation

• The hunchback gene is actually transcribed from
  two promotersone activated by the Bicoid
  gradient,the other is maternal promoter.
• Nanos is an RNA-binding protein which block the
  translation of the maternal transcript in
  posterior regions.

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• This dual regulation of hunchback expression
  produces a steep Hunchback protein gradient with
  the highest concentrations located in the
  anterior half of the embryo and sharply
  diminishing levels in the posterior half.

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The Gradient of Hunchback Repressor Establishes
Different Limits of Gene Expression

• Hunchback functions as a transcriptional
  repressor to establish different limits of
  expression of the gap genes, Kr üppel, knirps and
  giant.
• High levels of the Hunchback protein repress the
  transcription of Kr üppel, whereas intermediate
  and low levels of the protein repress the
  expression of the knirps and giant, respectively.

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• Not the binding affinities but the number of
  Hunchback repressor sites may be more critical
  for distinct patterns of Kr üppel, knirps and
  giant expression.

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Hunchback and Gap proteins produce Segmentation
Stripes of Gene Expression

• The eve gene is expressed in a series of seven
  alternating or pair-rule stripes that extend
  along the length of the embryo.
• The eve protein coding sequence is less than 2kb
  in length while the 12kb of the regulatory DNA
  contains five separate enhancers that together
  produce the seven different stripes of eve
  expression.
• Eve stripe 2 contains binding sites for four
  different regulatory proteins Bicoid, Hunchback,
  Giant, and Krüppel.

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We will consider the expression of eve stripe 2
for example.
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• In principle, Bicoid and Hunchback can activate
  the stripe 2 enhancer in the entire anterior half
  of the embryo where they both present, Giant and
  Krüppel function as repressors that form the
  anterior and posterior borders, respectively.

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Regulation of eve stripe2
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• Krüppel mediates transcriptional repression
  through two distinct mechanisms.
• One is competition. Two of the three Krüppel
  binding sites directly overlap Boicoid activator
  sites,precludes the activator to bind.

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• The other is quenching. The third Krüppel is able
  to inhibit the action of the Bicoid activator
  bound nearby.
• It depends on the recruitment of the
  transcriptional repressor CtBP, which contains a
  enzymatic activity that impairs the function of
  neighboring activators.

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Gap Repressor Gradients Produce many Stripes of
Gene Expression

• The same basic mechanism of how eve stripe 2 is
  formed applies to the regulation of the other eve
  enhancers as well. The stripe borders are defined
  by localized gap repressorsHunchback establishes
  the anterior border, while Knirps specifies the
  posterior border.
• However,the differential regulation of the the
  two enhancers by the repressor gradient produces
  distinct anterior borders for the eve stripes.

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• The eve stripe 3 enhancer is repressed by
  high levels of the Hunchback gradient but low
  levels of the Knirps gradient, while the stripe 4
  enhancer is just the opposite, this differences
  are due to the number of repressor binding sites.

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Short-range Transcriptional Repressors Permit
Different Enhancers to Work Independently

• There are additional enhancers that control eve
  expression,this type of complex regulation is
  common.
• The mechanism that repressors bound to one
  enhancer do not interfere with activators in the
  neighboring enhancers is short-range
  transcriptional repression,which ensures enhancer
  autonomy.

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Short-range repression and enhancer autonomy

• Stripe 3 activator is not repressed by the
  Krüppel repressors bound to the stripe 2 enhancer
  because it lacks the specific DNA sequences that
  are recognizes by the Krüppel protein and they
  map too far away.