Gene Regulation during Development

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Gene Regulation during Development

• 200431060022 ???

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Outline

• Part One Three Strategies by which
  Cells Are instructed to express Specific Sets of
  Genes during Development
• Part Two Examples of the Three Strategies
• Part Three The Molecular Biology of Drosophila
  Embryogenesis

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Part One Three Strategies for Specific Genes
Expression

• Some mRNAs Become Localized within Eggs and
  Embryos due to Intrinsic Polarity in the
  Cytoskeleton
• Cell-to-Cell Contact and Secreted Cell Signaling
  Molecules both Elicit Changes in Gene Expression
  in Neighboring Cells
• Gradients of Secreted signaling Molecules Can
  Instruct Cells to Follow Different Pathways of
  Development based on Their Location

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mRNA Localization

• mRNAs serve as a critical regulatory molecule
• They are transported along elements of the
  cytoskeleton which have intrinsic polarity
• The transportation is realized by an adapter
  protein

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Adapter proteins

• Containing two domains
• One recognizing the 3 UTR of the mRNA
• The other associates with the components of the
  cytoskeleton
• Thereby it crawls along the actin filament

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Cell to Cell Contact and Secreted Cell Signaling
Molecules

• Three steps
• The synthesized signaling molecules are deposited
  in the membrane or secreted into the
  extracellular matrix.
• They are recognized by the receptor on the
  surface of recipient cells
• The changes in gene expression in the recipient
  cell is achieved through the signal transduction
  pathways

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The Signal Transduction Pathways

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

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Gradients of Secreted Signaling Molecules
Influences Development through Cells Location

• The influence of location on development is
  called positional information
• Signaling molecules that control position
  information are sometimes called morphogens
• The morphogens are distributed in extracellular
  gradient

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Part Two Examples of the Three Strategies

• The Localized Ash1 Repressor Controls Mating Type
  in Yeast by Silencing the HO gene
• A Localized mRNA Initiates Muscle Differentiation
  in the Sea Squirt Embryo
• Cell-to-Cell Contact Elicits Differential Gene
  Expression in the Sporulating Bacterium, B.
  subtilis
• A Skin-Nerve Regulatory Switch Is Controlled by
  Notch Signaling in the Insect CNS
• A Gradient of the Sonic Hedgehog Morphogen
  Controls the Formation of Different Neurons in
  the Vertebrate Neural Tube

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No.1 The Localized Ash1 Repressor Controls Mating
Type in Yeast by Silencing the HO gene

• After budding to produce a daughter ,a mother
  cell can switch mating type
• The switching is controlled by the product of the
  HO gene
• The HO gene is activated in the mother cell but
  kept silent in the daughter cell

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No.1 The Localized Ash1 Repressor Controls Mating
Type in Yeast by Silencing the HO gene

• Ash1 mRNA is localized during budding
• The mRNAs are transcribed into repressor which
  represses the transcription of HO gene in the
  daughter cell
• Thereby mating type is controlled

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Ash1 mRNA Distribution
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No.2 A Localized mRNA Initiates Muscle
Differentiation in the Sea Squirt Embryo

• Macho-1 mRNA is initially distributed throughout
  the cytoplasm of unfertilized eggs
• 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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Macho-1 regulatory protein

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

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NO. 3 Cell-to-Cell Contact Elicits Differential
Gene Expression in the Sporulating Bacterium, B.
subtilis
Relationship between the two cells

• The septum produces two cells remain attached
  through abutting membranes.
• The smaller cell, called forespore, ultimately
  forms the spore.
• The larger cell ,the mother cell, aids the
  development of the spore.
• The forespore influences the expression of genes
  in the neighboring mother cell.

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NO. 3 Cell-to-Cell Contact Elicits Differential
Gene Expression in the Sporulating Bacterium, B.
subtilis

• Steps for influences
• The active form sF in forespore activates spoIIR
  gene
• The product spoIIR is secreted into the space
  between the mother and the daughters membranes
• SpoIIR triggers the activation of the sE in the
  mother cell through proteolytic process
• Activated sE initiates transcription of the
  target genes

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Asymmetric gene activity in the mother cell and
forespore in the B.subtilis
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No.4 A Skin-Nerve Regulatory Switch Is Controlled
by Notch Signaling in the Insect CNS

• Neurogenic ectoderm is a sheet of cells that will
  develop into nerve cord in insect embryos
• It can be divided into two cell populations
• One group remains on the surface of the embryo
  and forms epidermis
• The other moves inside the embryo to form the
  neurons of the ventral nerve cord
• The developing neurons contain a signaling
  molecule on their surface called Delta
• The Deltas receptor called Notch is on the skin
  cells surface

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No.4 A Skin-Nerve Regulatory Switch Is Controlled
by Notch Signaling in the Insect CNS
Steps for the Skin-Nerve Regulatory Switch

• Notch receptor is activated by Delta
• The intracytoplasmic domain of Notch is released
  to enter nuclei and associate with Su(H)
• Su(H)-NotchIC complex activates genes that encode
  transcriptional repressors which block the
  development of neurons

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No.4 A Skin-Nerve Regulatory Switch Is Controlled
by Notch Signaling in the Insect CNS

• Notch signaling does not cause a simple induction
  of the Su(H)
• In the absence of signaling, Su(H) is bound to
  repressor proteins including Hairless, CtBP, and
  Groucho
• When NotchIC enters nucleus, it displaces these
  repressor proteins
• Su(H) now activates the very same genes that it
  formerly repressed

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No.5 A Gradient of the Sonic Hedgehog Morphogen
Controls the Formation of Different Neurons in
the Vertebrate Neural Tube

• In all vertebrate embryos, there is a stage when
  cells located along the dorsal ectoderm move
  toward internal regions of the embryo and form
  the neural tube.
• Cells located in the ventralmost region of the
  neutral tube form floorplate, where the secreted
  signaling molecule Sonic Hedgehog(Shh) is
  expressed. Shh functions as a gradient
  morphogen.

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No.5 A Gradient of the Sonic Hedgehog Morphogen
Controls the Formation of Different Neurons in
the Vertebrate Neural Tube

• Shh diffuses through the extracellular matrix of
  the neutral tube and forms a gradient.
• The graded distribution of the Shh protein leads
  to the formation of distinct neuronal cell types
  in the ventral half of the neural tube.
• High and intermediate levels lead to the
  development of the V3 neurons and
  motorneurons,respectively.Low and lower levels
  lead to the development of the V2 and V1
  interneurons.

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No.5 A Gradient of the Sonic Hedgehog Morphogen
Controls the Formation of Different Neurons in
the Vertebrate Neural Tube
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No.5 A Gradient of the Sonic Hedgehog Morphogen
Controls the Formation of Different Neurons in
the Vertebrate Neural Tube
Through what pathways the differential activation
of Shh receptors function

• The activation of the Shh receptor allows a
  previously inactive form of Gli transcription
  activator to enter the nucleus in an activated
  form.
• The gradient of Shh leads to a corresponding Gli
  activator gradient
• 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.

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No.5 A Gradient of the Sonic Hedgehog Morphogen
Controls the Formation of Different Neurons in
the Vertebrate Neural Tube

• V1 genes can be activated by low levels of Gli
  because they have high-affinity recognition
  sequences
• In contrast, the V3 target genes might contain
  regulatory DNA with low-affinity Gli recognition
  sequences that can be activated only by peak
  levels of Shh signaling

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Part Three The Molecular Biology of Drosophila
Embryogenesis
Terminologies of Drosophila Embryogenesis

• Cellularization a process that the zygote
  transforms to cellular bastoderm
• Totipotential the ability to give rise to any
  cell type

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Part Three The Molecular Biology of Drosophila
Embryogenesis
Events of Drosophila Embryogenesis

• Insemination
• Zygotic nucleus formation
• Synchronous divisions and formation of
  syncitium-single cell with multiple nuclei
• Nuclei migration to cortex and three more
  divisions
• Cell membranes formation and loss of
  totipotential
• Transformation into cellular blastoderm

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A Morphogen Gradient controls Dorsal-Ventral
Patterning

• The specific morphogen is the Dorsal protein
• Dorsal protein enters nuclei in ventral and
  lateral regions but remains in the cytoplasm in
  dorsal regions
• 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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A Morphogen Gradient controls Dorsal-Ventral
Patterning

• 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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A Morphogen Gradient controls Dorsal-Ventral
Patterning
Three thresholds of gene expression and three
types of regulatory DNAs

• The twist 5 regulatory DNA contains two
  low-affinity Dorsal binding sites
• The rhomboid 5 enhancer contains a cluster of
  dorsal binding sites but only one has high
  affinity
• The sog intronic enhancer contains four
  evenly-spaced optimal Dorsal binding sites

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Three types of regulatory DNAs
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A Morphogen Gradient controls Dorsal-Ventral
Patterning

• 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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A Morphogen Gradient controls Dorsal-Ventral
Patterning

• The binding of Dorsal also depends on
  protein-protein interactions between Dorsal and
  other regulatory proteins bound to the target
  enhancers.
• For example, intermediate levels of Dorsal are
  sufficient to bind due to their interactions with
  another activator protein Twist, they help one
  another bind to adjacent sites within the
  rhomboid enhancer.

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

• Two localized mRNAs in the egg at the time of
  fertilization
• The bicoid mRNA - Located at the anterior pole
• The oskar mRNA Located at the posterior pole.

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The oskar mRNA

• Functions
• Encoding an RNA-binding protein that is
  responsible for the assembly of polar granules
• The polar granules control the development of
  tissues that arise from posterior regions of the
  early embryo

• Movements
• Synthesized within the ovary of mother fly
• First deposited at the anterior end of the
  immature egg by nurse cells
• Transported to posterior region when mature egg
  forms

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Location of maternal mRNAs

• Click here and look into the adapter proteins
  for more details

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Location determination

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

• The Bicoid regulatory protein
• Synthesized prior to the completion of
  cellularization
• Simply diffuses across the syncitium, which
  differs from the case of the Gli and Dorsal
• Produces multiple thresholds of gene expression
• Binds to DNA as a monomer, interacts with each
  other to foster the cooperative occupancy of
  adjacent sites.

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

• High concentrations of Bicoid protein activate
  the expression of the orthodenticle gene
• High and intermediate concentrations of Bicoid
  are sufficient to activate hunchback

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

• This differential regulation of orthodenticle and
  hunchback depends on the binding affinities of
  Biciod recognition sequences.
• The orthodenticle gene - 5 enhancer that
  contains a series of low-affinity Biciod binding
  sites,
• The hunchback gene - 5 enhancer contains
  high-affinity binding sites.

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Hunchback Expression Is also Regulated at the
level of Translation

• Maternal promoter leads to the synthesis of a
  hunchback mRNA that is evenly distributed in the
  unfertilized eggs
• The translation of the maternal transcript in the
  posterior region is blocked by Nanos
• Nanos mRNA is located in posterior regions
  through interactions between its 3 UTR and the
  polar granules
• Nanos binds to NREs, located in the 3 UTR of the
  maternal mRNA, and causes a reduction in the
  hunchback poly-A tail, which inhibits its
  translation

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Hunchback protein gradient and translation
inhibition by Nanos
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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, Krüppel, knirps and
  giant.
• High levels of the Hunchback protein repress the
  transcription of Krüppel, whereas intermediate
  and low levels of the protein repress the
  expression of the knirps and giant, respectively.

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The Gradient of Hunchback Repressor Establishes
Different Limits of Gene Expression

• Not the binding affinities but the number of
  Hunchback repressor sites may be more critical
  for distinct patterns of Krüppel, 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.

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Hunchback and Gap proteins produce Segmentation
Stripes of Gene Expression

• The eve protein coding sequence contains five
  separate enhancers that together produce the
  seven different stripes of eve expression

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Regulation of eve stripe 2

• Eve stripe 2 contains binding sites for four
  different regulatory proteins Bicoid, Hunchback,
  Giant, and Krüppel.

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Regulation of eve stripe 2

• 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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Hunchback and Gap proteins produce Segmentation
Stripes of Gene Expression

• Krüppel mediates transcriptional repression
  through two distinct mechanisms.
• Competition. Two of the three Krüppel binding
  sites directly overlap Boicoid activator
  sites,precludes the activator to bind.
• 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
  repressors Hunchback establishes the anterior
  border, while Knirps specifies the posterior
  border.
• The differential regulation of the the two
  enhancers by the repressor gradient produces
  distinct anterior borders for the eve stripes.

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Gap Repressor Gradients Produce many Stripes of
Gene Expression

• The eve stripe 3 enhancer is repressed by high
  levels of the Hunchback gradient but low levels
  of the Knirps gradient
• 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 Transcriptional Repressors Permit
Different Enhancers to Work Independently

• 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.

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Brief Summary

• There are three major ways to regulate gene
  expression at the level of transcription
  initiation
• The segmentation of the Drosophila embryo depends
  on a combination of localized mRNAs and gradients
  of regulatory factors

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May God be with you!