Chapter 18 Gene Regulation during Development 张慧琼 200331060115

Outline Topic 1 Three Strategies by which Cells Are Instructed to Express Specific Sets of Genes during Development Topic 2 Examples of the Three Strategies for Establishing Differential Gene Expression Topic 3 The Molecular Biology of Drosophila Embryogenesis

Topic 1: Three Strategies by which Cells Are Instructed to Express Specific Sets of Genes during Development

Three Strategies: mRNA localization Cell-to-cell contact Signaling through the diffusion of secreted signaling molecules

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 3’untranslated trailer (3’UTR) region of the mRNA, has two domains: one recognizes the 3’ UTR,the other associates with myosin.

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.

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.

Gradient of Secreted Signaling Molecules Two concepts: Positional information: a cell’s development is influenced by its location within the developing embryo. Morphogens: signaling molecules that control position information

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.

Topic 2:Examples of the Three Strategies for Establishing Differential Gene Expression

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 can’t switch due to localized Ash1 repressor,it can’t express HO which initiate switching. The mother cell can switch: it lacks Ash1,and is able to express HO.

The ash1 gene is transcribed in the mother cell during budding 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 3’UTR 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. General principle: broadly distributed activators and localized repressors interplay to establish precise patterns of gene expression within individual cell. In yeast,the SWI5 protein is responsible for activating expression of HO gene, but Ash1 keeps the HO gene off in the daughter cell despite the presence of SWI5.

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.

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.

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

σF 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 σE(pro-σE)in the mother cell.The pro-σE protein contains an N-terminal inhibitory domain that blocks σE activity and tethers the protein to the membrane of the mother cell. After the cleavage of the N-terminal peptide, the activated σE protein leads to the transcription of target genes.

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

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 σE in the other cells except the abutting mother cell.

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.

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).

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.

A Gradient of the Shh 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.

Shh is secreted from the floorplate and diffuses through the extracellular matrix of the neutral tube. Cells located near the floorplate receive the highest concentrations of Shh,have a high number of Shh receptors activated on their surface;while those located far away are just the opposite. 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.

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.

Topic3:The Molecular Biology of Drosophila Embryogenesis

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.

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.

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.

After fertilization, Spätzle binds to the cell surface Toll receptor 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.

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.

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.

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.

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.

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 3’UTR region, thereby transported into the posterior plasm. ash1 mRNA transportation After fertilization the cells that inherit the localized oskar mRNA form the pole cells.

The ash1 gene is transcribed in the mother cell during budding 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 3’UTR and myosin.

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).

Localized bicoid mRNA initiates anterior regions

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.

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 5’enhancer that contains a series of low-affinity Biciod binding sites,while hunchback gene is regulated by a 5’enhancer 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.

Hunchback Expression Is also Regulated at the level of Translation The hunchback gene is actually transcribed from two promoters:one 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.

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.

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.

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.

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.

We will consider the expression of eve stripe 2 for example.

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

Regulation of eve stripe2

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.

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.

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. However,the differential regulation of the the two enhancers by the repressor gradient produces distinct anterior borders for the eve stripes.

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.

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.

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.

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