DB-Lec 11

DB-Lec 11 - Biology 442 Biology Developmental Biology...

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Unformatted text preview: Biology 442 Biology Developmental Biology Developmental Lecture 11 Axis Formation and Mesoderm Induction During cleavage, blastula and early gastrulation progressive differentiation of cells results in the formation of the 3 germ layers germ 1. Ectoderm – skin and nervous system 2. Endoderm – linings of the digestive tube, bladder, pancreas liver etc. 3. Mesoderm – muscle, bone, cartilage, etc. During cleavage we saw how cytoplasmic localization of cytoplasmic determinants in the oocyte (egg) gave rise to germ cells and we saw localization of determinants that will later play a role in predisposing cells to form the germ layers. Now we are going to look: Now 1) iinteractions between cells that induce the formation of 1) nteractions mesodermal structures and set up the body axes. mesodermal 2) the nature of Inductive Interactions 3) the molecular biology of the “inducers” 3) 4) the receptors and downstream signal transduction 4) systems through which competent cells respond to inducers. inducers. In our discussion of Axis Formation and Mesoderm Induction we are going to focus on Amphibians (Frogs) because much Amphibians of the most detailed information has come from studies on amphibians. 3 different axes have to form: 1. The anteroposterior axis develops during gastrulation anteroposterior from the animal-vegetal axis which originates during animal-vegetal oogenesis. 2. The dorsoventral axis is formed after fertilization and is dorsoventral fixed before the first cleavage. 3. The left-right asymmetry is not obvious until the heart left-right and other internal organs become asymmetrically located during later embryogenesis BUT the molecular cues to left-right asymmetry are evident during blastula and early gastrula. and The concept of Regulative Development. The An isolated blastomere has a potency greater than its normal embryonic fate. (That potency might have been restricted as a result of cytoplasmically localized determinants). A cell’s fate is further determined by interactions further between neighboring cells – Inductions or Inductive Interactions. Interactions We will first look at the role of the cytoplasmically localized determinants that set up the animal –vegetal pole and then look at how these confer a “reduced” potency on cells and then how inductive interactions between cells result in the later steps in axis formation. A Review of the Body Axes Axes Body Distal Body Proximal 1) The Animal-Vegetal Axis originates by oriented transport of materials during oogenesis As a result of the cytoplasmic localization events that we discussed last time two key molecules are localized at the vegetal pole. 1. VegT – a transcription factor 2. Vg1 – The transforming growth factor –beta ligand which is a paracrine factor VegT Fate Map of Xenopus Embryo Fate The surface of the animal half of the egg will give rise to the ectodermal cells. The cells that form from surface of the vegetal half of the egg will form the endoderm. The cells that form from the internal cytoplasm of the egg will become mesoderm. VegT and Vg1 initially VegT impose this fate map on the embryo on If you use antisense oligos to reduce VegT, the embryos do not make normal fate maps. Endoderm is missing and mesoderm is reduced. Embryos that lack Vg1 also are missing endodermal cells and show reduced mesoderm. The correct allocation of cells to the three germ layers The depends on prelocalized cytoplasmic determinants laid down in the egg during oogenesis! down Isolation Experiments give a clearer picture of how this works. In Isolation – make ectoderm ectoderm In Embryo – make mesoderm mesoderm In isolation & embryo -- endoderm In Isolation –make mesoderm mesoderm Between the 32 and 128 cell stages the descendents of the B/C blastomeres become determined to form mesoderm. Commitment of Marginal Zone blastomers to form Mesoderm results from an interaction between the vegetal blastomeres and the Animal Cap cells. Nieuwkoop demonstrated that if you Nieuwkoop mixed animal cap cells and vegetal blastomeres in culture Mesodermal tissues developed. Fluorescent labeling demonstrated that all mesodermal tissues arose from animal cap cells NOT vegetal cells. The Principle of Induction The The interaction between 2 Non-equivalent cell types – the vegetal blastomeres that would form endoderm and the animal cap cells that would form ectoderm to cause one cell type – the animal cells to form a different cell type – the mesoderm cells, is an INDUCTIVE Interaction or Induction. INDUCTIVE Induction Cells undergoing a change in their determined state are called responding cells. responding Cells that cause the change are called inducing cells inducing As a result of early inductive interactions, inducing and responding cells often form adjacent precursor cells or rudiments of a tissue. Inductive interactions increase the complexity of the cell types. 2) Formation of the Dorsal—Ventral Axis The events that will further position materials for specifying the dorsal-ventral axis start with the cortical rotation that the occurs at fertilization. The side where the sperm enters marks the future belly or ventral side. ventral When the cortical cytoplasm shifts, the internal cytoplasm is also reorganized and the grey crescent region becomes the area where gastrulation will initiate – future dorsal side. dorsal Internal cytoplasm also shifts as does material in the sheer zone between the cortex (cortical cytoplasm) and the endoplasm. This shifting localizes key factors to the future dorsal side and grey crescent. Spemann had also demonstrated that the grey crescent region was important for formation of dorsal tissues. If he divided the fertilized egg so that each half got grey crescent material then both formed normal tadpoles. If he cut it so that only one side got the grey crescent, the side that did not get it developed into ventral tissue and died. The cells that arise from the grey crescent become the dorsal lip of the blastopore which is the site where gastrulation initiates. In 1924 Mangold and Spemann showed that the dorsal lip of the blastopore could induce neighboring cells to form a complete secondary axis. Spemann called the dorsal lip cells and their derivatives (notochord, prechordal mesoderm) the Organizer. Organizer The organizer initiates a series of sequential inductions that 1) induce the host ventral tissue to form neural tube and dorsal mesoderm and 2) organize host and donor tissue into a new secondary embryo. The key inductions carried out by the organizer are called the “primary primary embryonic induction” to distinguish them embryonic from all of the secondary induction events. But how does the organizer arise ? We know that the cortical rotation plays a key role but what does it do ? 1984 –Gimlich and Gerhart used transplantation experiments on 64 cell stage embryos to show that the 3 dorsal most vegetal blastomeres that were just under the dorsal grey crescent area were able to induce overlying cells to form the dorsal lip of the blastopore and a complete dorsal dorsal axis on UV irradiated embryos or a second axis on normal embryos. embryos. So that says that a dorsalizing factor must be located in the vegetal region of the embryo underneath the grey crescent area. The Nieuwkoop experiment showed that vegetal blastomeres could induce animal cells to make mesoderm and now we see the 3 dorsal most vegetal blastomeres dorsal inducing overlying animal cells to make the dorsal lip of the blastopore (dorsal mesoderm). This suggested that their might be regional specificity in the inductive ability of the vegetal blastomeres. 1987 Dale and Slack combined single vegetal blastomeres (32 cell stage embryos) with animal caps and showed regional specificity of induction. specificity DBL Putting together: 1) the role of the vegetal blastomers in inducing animal cells to make mesoderm, 2) the 3 Dorsal vegetal blastomers to induce overlying grey crescent cells to form the organizer 3) the role of the organizer in inducing dorsal structures etc. We arrive at a model for the organization of anterior posterior patterning and the start of dorsal ventral patterning. Progressive patterning of the anteriorof posterior and dorsalventral axes by ventral cytoplasmically localized determinants and sequential inductions. inductions. What are the actual molecules that carry out the inductions and how do VegT and Vg1 initiate this sequence ? Molecular Biology of Induction Or How are Signals Between Inducer and Responder Transmitted? Between Two major types of inducing molecules: Juxtacrine Factors Juxtacrine and Paracrine Factors. Paracrine – Secreted or matrix bound Inducer binds to transmembrane receptor on responding cell. Juxtracrine - Inducer and responder receptor are both transmembrane proteins 4 General Classes of Paracrine Factors General Fibroblast Growth Factor Family – work through tyrosine kinase receptors. kinase Activated responding proteins can activate a variety of different pathways; PI3Kinase, MAPkinase, Phospholipase C, Jak-Stat, etc. Stem Cell Factor JAKSTAT is a subset of tyrosine kinase motifs Hedgehog Family – Often function to create boundaries Hedgehog between tissues and to induce some specific cell types. It often works together with members of the other paracrine family of inducing molecules. Mediates formation of Left – right axis. right In the absence of Hedgehog – C1 protein is anchored to microtubules where it can be phosphorylated by PKA and cleaved by Slimb to make a transcriptional repressor that repressor blocks transcription of certain genes. protease Hedgehog binding to patched receptor allows smothened to inhibit PKA and Slimb and lets C1 act as a transcriptional activator. activator. The TGF-b (beta) Superfamily. Over 30 Superfamily Over members that regulate some of the most important interactions in development. The proteins of this family are processed so that the COOH terminus contains the mature peptide. These peptides dimerize to form homo or hetero dimers which are secreted from the cell. cell. The TGF-Beta superfamily factors work by binding as either homo or heterodimers to Serine-Theronine kinase Receptors and activate the Smad transcription factors. and Amino acids at the N terminus of the TGF-beta ligands determine how far they can diffuse from the secreting cell. Some N termini bind to proteoglycans in ECM of surrounding cells, restricting their diffusion. The Wnt family – Are cysteine rich glycoproteins. Many The Wnt proteins are vital in establishing the polarity of structures like wings and limbs. structures Wnt binding to frizzled receptor activates Disheveled which inhibits GSK3. When GSK3 is active Beta Catenin is bound to APC which causes it to be degraded. When GSK3 is inactive Beta Catenin is released from APC and enters the nucleus and function as a transcription factor. Dual functional components -- GSK3 also functions in glycogen metabolism, Beta Catenin is part of cell adhesion complexes and APC is a tumor suppressor Molecular Biology of the Nieuwkoop Center: Beta-Catenin in the Wnt pathway is a key player. Beta Catenin is initially synthesized from maternal mRNAs throughout the embryo but it’s degradation is catalyzed by GSK3 phosphorylation. Disheveled which inhibits GSK3 is found in the Vegetal Cortex of the unfertilized egg. Upon fertilization cortical rotation causes Disheveled to move along the microtubule complex to the future dorsal side of the fertilized egg. On the dorsal side Disheveled inhibits GSK3 allowing Beta Catenin to go to the nucleus of dorsal cells and act as a transcription factor. Beta catenin activates the transcription of another transcription factor, Siamois transcription Siamois activates transcription of “organizer” specific genes. A major one is the Goosecoid Transcription factor which is responsible for transcribing most of the organizer genes. most Transcription of Goosecoid also requires factors expressed through the action of the TGF-beta pathway. of Vg1, Nodal and Nodal related TGF beta ligands Vg1 and several other TGF-beta paracrine factors (Nodal related proteins Xnr 1-4) are cytoplasmically localized in the vegetal blastomeres. Dorsally located Beta-Catenin plus vegetally located, Vg1 interact to form a dorsal ventral gradient of Nodal related proteins (Xnr proteins, all TGF-b family members) proteins The Nodal related proteins that specify mesoderm cause expression of Dorsal or ventral mesoderm genes. Goosecoid transcription factor in the organizer and BMP & Xwnt 8 paracrine factors in ventral mesoderm. Siamois Low Nodal related proteins High goosecoid BMP4 & Xwnt8 TGF ligand Ability of goosecoid mRNA to Induce a New Axis goosecoid Goosecoid is a major transcription factor in the organizer. When goosecoid mRNA is injected into ventral blastomeres it can induce a second developmental axis. It transcribes many organizer genes. A complex chorography of the secreted paracrine factors acting through various signaling pathways together with transcription factors will further restrict the potential of mesoderm, ectoderm and endodermal cells and pattern the subsequent tissues and organs. and ...
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