1. Gene Regulation during Development
陈国荣

2. 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

3. 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

4. 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”

5. “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

6. 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

7. 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.

8. 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

9. 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

10. 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

11. 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

12. Ash1 mRNA Distribution

13. 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

14. 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.

15. 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.

16. NO. 3 Cell-to-Cell Contact Elicits Differential Gene Expression in the Sporulating Bacterium, B. subtilis
Steps for influences:
The active form σF in forespore activates spoIIR gene
The product spoIIR is secreted into the space between the mother and the daughter’s membranes
SpoIIR triggers the activation of the σE in the mother cell through proteolytic process
Activated σE initiates transcription of the target genes

17. Asymmetric gene activity in the mother cell and forespore in the B
Asymmetric gene activity in the mother cell and forespore in the B.subtilis

18. 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 Delta’s receptor called Notch is on the skin cells’ surface

19. 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

20. 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

21. 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.

22. 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.

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

24. 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.

25. 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

26. 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

27. 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

28. 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

29. 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.

30. 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

31. Three types of regulatory DNAs:

32. 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.

33. 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.

34. 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.

35. The oskar mRNA Movements: Functions:
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
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

36. Location of maternal mRNAs
Click here and look into the “adapter” proteins for more details

37. Location determination
The localization of the bicoid mRNA in anterior regions also depends on sequences contained within its 3’ UTR. Therefore, the 3’UTR is important in determine where each mRNA becomes localized.
If the 3’UTR from the oskar mRNA is replaced with that from biciod, the hybrid oskar mRNA is located to anterior regions (just as biciod normally is).

38. 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.

39. 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

40. 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.

41. 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

42. Hunchback protein gradient and translation inhibition by Nanos

43. 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.

44. 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.

45. 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.

46. 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

47. Regulation of eve stripe 2
Eve stripe 2 contains binding sites for four different regulatory proteins: Bicoid, Hunchback, Giant, and Krüppel.

48. 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.

49. 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.

50. 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.

51. 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.

52. 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.

53. 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.

54. 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

55. May God be with you!