Lecture_1_G.ppt

David Gardiner To contact me: email (dmgardin@uci.edu), office hours, before/after lecture My research: limb regeneration/regenerative medicine Why are we here? Acquire knowledge and understanding about embryonic development and regeneration What you can do to facilitate learning: PARTICIPATE Come to lecture/office hours Ask questions Provide feedback in real-time Do the reading What I can do to facilitate learning: Stay organized and focused on the important concepts (summary for each lecture) Encourage participation (sample exam questions study guide/ask you questions) Respond to feedback The reality of exams The lecture slides (The exam will be based on the lectures) Not all the lecture material is in the book The reading is important Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. Before we get started - Developmental/regeneration biology is an emerging and rapidly changing field - The ?facts? are always changing- they can be wrong/inappropriate We do not know/understand everything (we can talk outside of class if you want to know more about the mountain of facts) ? I will focus on the concepts and what we understand about those concepts - Nevertheless, the facts are important in terms of illustrating the concepts (specific examples) and speaking the language Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. Sample exam question #1: Understanding developmental biology and regeneration is NOT fundamentally important for understanding which of the following: 1 - World peace 2 - Treating injury 3 - Correcting genetic defects 4 ? Health care reform 5 ? Preventing/curing cancer 1 2 and 3 1 and 4 2, 3 and 5 None of the above Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. The fundamental concept of development and regeneration Cells are the functional unit of development and regeneration ? they are the output of genetic regulation Where did they come from? How did they get there? What do they do? What do they look like? What do their progeny do? Why do they do it? How do they interact? What do they know? Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. Reading: pg 185 ? 197 Development of nematodes Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. The nematode worm, Caenorhabditis elegans (C. elegans) is an important ?model? system for understanding the relationship between genetics and cell biology - Simple anatomy = Simple development - Rapid development - 15 hours to hatching of larvae, 50 hours for mature adults - Can do Genetics ? easy to breed and hermaphroditic (self-fertilizing) - Transparency of embryos allows easy scoring of mutant phenotypes - Can follow cell lineage - a small number of cells ? 558 cells at hatching and 959 in the adult (after 131 cells have died) - lineage is invariant - 1998 ? first genome of a multicellular organism to be sequenced- it is small (about 3% the size of the human genome), but contains about 19,000 genes (human has about 25,000 genes) Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. The newly hatched larva is similar to the mature adult, but is sexually immature and must undergo 4 additional larval molts to form the adult. C. elegans life cycle Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. Early cleavages and cell fates The first division gives a large anterior AB cell and a small P1 cell P1 (progenitor of the germ cells) produces P2 cell and EMS cell (endoderm/mesoderm). The AB cell divides into anterior ABa (neurons, epidermis plus pharynx mesoderm) and the posterior ABp (neurons, epidermis and specialized cells). Cleavage is invariant and fate map is fixed Therefore development of this embryo is considered to be mosaic and since the body axes depend on asymmetric cell division and inheritance of cytoplasmic factors, development is also determinant This type of development is considered classically to be different than regulative development (controlled by cell-cell interactions) Now understand that cell-cell interactions are required (the ?facts? have changed) Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. Goldstein Lab, UNC http://www.bio.unc.edu/faculty/goldstein/lab/movies.html Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. The pattern of cleavage is invariant The history of every cell can be observed Consequently, the lineage of every cell is known Gastrulation (formation of multiple germ layers) is initiated at the 28 cell stage, when descendants of the E cells invaginate to form the gut (endoderm) which is surrounded by the mesoderm which in turn is surrounded by the ectoderm C. elegans fate map Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. During first three divisions, P1 - P3 cells give rise to a number of somatic lineages but the P4 cell gives rise to germ cells only. E.g. P1 divides into P2 and EMS. EMS divides to make MS (mesodermal pharynx) and E (gut). C. elegans fate map: P cells behave like stem cells (self-renewing) and give rise to the germ cells Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. Asymmetric localization of P granules as a mechanism for mosaic development No polarity exists in the unfertilized egg and P granules are uniformly distributed Point of sperm entry controls the first cleavage and determines the future posterior end The sperm brings in a centriole that become a microtubule organizing center (MTOC) and positions the first cleavage plane, which is displaced toward posterior pole (not everything is controlled by genes) After the first cleavage (cleavage plane is asymmetric), the larger AB cell becomes the anterior and smaller P cell becomes posterior P granules are markers for the P lineage and are involved in the specification of the germ lineage Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. Brangwynne, et al., Science 2009 http://www.bio.unc.edu/faculty/goldstein/lab/movies.html Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. - Developmental/regeneration biology is an emerging and rapidly changing field - The ?facts? are always changing- they can be wrong/inappropriate We do not know/understand everything ? focus on the concepts and what we understand about those concepts Although C. elegans development looks mosaic and determinant (e.g. P lineage and P granules), it is not that simple (it is actually much more interesting and involves local cell-cell interactions) Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. How do you interpret these data? 1- At 2-cell stage: If you isolate P1 ? you get all the P1 derivatives, but nothing more Mosaic 2 - At 2-cell stage: If you isolate AB, do not get all the AB derivatives Not mosaic and must need additional signals (from where?) 3 - At 4-cell stage: If you isolate EMS (derived from P1) it will not form endoderm (E), but if you recombine with P2 cell, it will form endoderm Regulative - Signals from P2-derived cells induce endodermal fate in progeny of EMS cell Local cell-cell interactions regulate the determination of cell fate (other than the P cell-germ cell lineage) If you experimentally switch the positions of ABa and ABp you still get a normal worm. P1 progeny are also altered - the position of the EMS daughter cell with respect to the AB cells is reversed. This results in reversal of D/V axis. The non-P cell lineages are dependent on short range cell-cell interactions, i.e. not mosaic, not determinant, and not diffusible morphogen gradients Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. The cell next to P2 will become ABp ? in the absence of P2 signaling, the cell fate becomes ABa P2 signaling involves the maternal genes glp-1 and apx-1, that encode homologs of Drosophila /vertebrate Notch and Delta ABa and ABp both express Glp-1 protein (receptor) and P2 expresses Apx-1 protein (ligand) on their surface. Apx-1 interacts with Glp-1 in the adjacent cell that is in contact with the P2 cell. This early cell-cell signaling induces the descendants of the ABa and ABp cells to respond differently to signals from the MS cell What are the genes involved in the local cell-cell interactions that establish the A/P axis? Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. What are the genes involved in the local cell-cell interactions that establish the D/V axis? ?Mom? mutants: (More Mesoderm) formed when endoderm induction fails - e.g., Wnt (mom-2; ligand) interacting with Frizzled (mom-5; receptor) ?Pop? mutants: (Posterior pharynx defect) because of the extra gut: MS adopts an E-like fate. Pop is homolog of Lef-1/Tcf which is a repressor of Wnt response - in the absence of POP, both daughters of EMS behave as though they have received Wnt signaling (no longer repressed in MS) By 32 cell stage ? lineages are more or less determined and ablation results in missing lineage Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. Homeobox genes ? conserved function (pattern formation) with different outcome (context dependent) Despite the absence of apparent segmentation, a small cluster of homeobox genes specify cell fate along the A/P axis. Four homeobox genes (lin-39, ceh-13, mab-5 and egl-5), similar to the HOX genes, and a less related homeobox gene (ceh-23), make up the C. elegans Hox cluster. They are expressed in different positions along the A/P axis. Their spatial expression corresponds to order on the chromosome. Not lineage restricted (mosaic) e.g. not all mab-5 expressing cells are from same lineage Mutations result in homeotic transformations Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. Expression of lin-14 mRNA is regulated post-transcriptionally by lin-4 (inhibits translation). Therefore loss-of-function mutations of lin-4 behave like gain-of-function mutations in lin-14 Lin-4 does not code for proteins ? it is a miRNA (microRNA) Code for short (21 nucleotides long) RNA Bind to complementary sequences in the the target mRNA cause degradation RISC ? RNA-induced silencing complex Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. IMPORTANT: - Experimentally - test gene function by inhibition of translation - Clinically ? - disregulation leads to diseases, e.g. cancer - target pathogens by targeted delivery of miRNA designed against microbes or genetic pathways Lecture 1 Concepts to understand/Questions to answer 1- Cells are the functional unit of development and regeneration ? they are the output of genetic regulation 2 - The classic distinction between ?mosaic? and ?regulative? development is more apparent than real 3 - Cell fate can be determined by the inheritance of cytoplasmic ?determinants? (cell history is important) 4 - Cell fate is not always specified/determined by diffusion gradients of morphogens 5 - Short range cell-cell interactions can control developmental fate 6 - The establishment of asymmetry (e.g. at fertilization) is important 7 - Not everything is controlled by regulating gene expression 8 - The intracellular environment is not homogeneous and is dynamically regulated 9 - Genes that control early development and developmental fate are conserved in evolution 10 - Not all genetic regulation is control by proteins (e.g. morphogens or transcription factors) ? e.g. post-transcriptional regulation by miRNA Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved. (1) (0.5 pts) ______T/F_____ Cells of the same developmental lineage can have different fates. (2) (2 pts) What triggers and regulates the redistribution and localization of P granules to the P cell in the C. elegans zygote?   (3) (2 pts) What is the developmental consequence in terms of the fate of the EMS blastomere if Wnt signaling from the P2 blastomere is inhibited during early C. elegans embryogenesis? Study Guide for Lecture #1: Winter 2011, Bio 104, K. Arora, K. Cho and D. Gardiner, UCI - Copyright: All rights reserved.