MON -This is not quite ready for 2003 as u can see. Will be I hope by Wed. Keep watching. Rather than wait and load it all at once, I'm giving you parts ASAP.
Topics for Final Exam - Wolpert text - emphasis CHPTS 4,11,9 (pay attention to figures mentioned) As you may infer from the text figures that I suggested in class and below, you will find it fruitful to review Chpt 3 Topics for Exam Questions If you haven't already downloaded out last lecture - Link for Differentiation - Chpt 9 Induction [review from Chpt 3] and new I. Classical experiments provide conceptual information [mostly amphibian] Tissues can direct/influence the further development of another cell population Spemann - optic vesicle transplanted under belly ectoderm induces lens ectoderm is competent to respond Early steps in establishing dorsoventral axis Sperm penetration triggers cortical rotation and localization of the critical signaling center, termed the Nieuwkoop center (NC)F3.4 Induction is progressive Nieukoop center sets up relationships necessary for action of organizer Fig 3.26 The region of the chordamesoderm will set up early axial relationships [organizer concept] Spemann and Mangold - transplantation of dorsal lip of blastopore induces 2nd embryonic axis from host [Fig 1.10 ] Regional specificity of induction Regions of "organizer" induces head, trunk or tail structures Inducer molecules may be diffusible agents - transfilter experiments or act as membrane associated signal molecules Inducer molecules may exist spatially along overlapping gradients Animal cap exps showing different effects of inducer concentrations [F 3.22, 27] Temporal considerations - Inducer signal in criitcal period for response; competent cells must have receptors present to interact; response may be significantly later [F 3.24] II. Molecular events provide mechanisms for induction A. Early events Counterparts for highly conserved Drosophila genes found in vertebrates Evidence from Xenopus and other amphibian models transplants or injection exps animal cap experiments Sperm penetration stimulates cortical rotation and distribution of morphogens [summary box pg 75] Signals in early dev. [sum. box pg 89] Criteria for assessing role of protein in induction Protein must be present in the embryo at the appropriate concentration critical time period "proper" place There must be an appropriate response from responding cells If "inhibited or mutated" the response does not occur Pathways, genes and proteins highly conserved Maternal products - early vegetal signals Mesoderm induction vg-1 and activin in animal cap exps both, at high conc., induce dorsal meso (notoch), muscle Ventralization maintained by BMP and Xwnt B. Setting up the "organizer" Spemann's Organizer Characteristics - ability to form dorsal mesoderm, head mesenchyme and notochord, which in turn induces regional paraxial mesoderm dorsalize lateral mesoderm initiate gastrulation (brachyury - required for gastrulation - morphogenetic movements) dorsalize ectoderm into neural ectoderm induce neurulation Gastrulation positions prechordal plate and chordamesoderm in roof of archenteron Xbra= brachyury- required for gastrulation and morphogenetic movements F3.35,36 Mechanisms Protect itself - must inactivate BMP uses chordin and noggin - proteins which bind BMP [inhibit] Wnt signal protein initiates signal transduction pathway b-catenin + coprotein serve as ts factor to activate siamois siamois gene product is a ts factor, activates goosecoid gene goosecoid protein acts as transcription factor multiple effects proposed activates mechanisms for gastrulation [morphogenetic movements] as prospective chordamesoderm cells migrate goosecoid is still expressed continued dorsalizing and anterior effects activates genes for chordin and other "dorsal" mesoderm genes vg-1 also activates noggin gene chordin, noggin, et al. block effect of BMP and other ventralizing signals *remember dorsal meso gives rise to notochord, head mesenchyme and paraxial/epimeric meso C. Post-gastrulation effects of the "organizer" region [continued/progressive effects] Xnot (Xenopus gene encoding ts factor) needed for diff'n of notochord organizer (= notochord) [producing chordin, noggin et al.] responsible for induction of neural tube [sum pg 94] in anterior region activates cerberus which induces ant. brain structures, eyes and cement gland in posterior - FGF, possibly working in gradient fashion with Xbra, determines tail III. Establishing the Body Plan Gastrulation positions prechordal plate and chordamesoderm in roof of archenteron [ie.the organizer] What is the outcome? Induction Gene regulation - differential gene expression - signaling - signal transduction - cell activity - basic body plan and/or differentiation [phenotype] Often redundancy - more than one control mechanism Multiple control options at each step "and yet it works" A. Patterning in somitic mesoderm : Chick model - easily visualized Notochord - dorsal midline Cells that produce epimeric meso ingress at Hensen's node and form pop. of somitic stem cells which produce progeny that form somitic cords lateral to notochord in U-shaped patttern Stem cells regress with HN [F 2.24, 15] Somitic cords gradually segment into blocks (paired somites) in ant-post progression Sequential pattern determined early F4.2 Excise and invert section of scord before segmented - inverted section forms somites in order of original position Temporal order to molecular pattern - internal "clock" Periodic cycle of gene expression Candidate genes - c-hairy 1: expression sweeps thru cords every 90 min (approx time it takes to form visible somite pairs) Lunatic fringe: protein also cycles and activates Notch signaling pathway [in mutant when pathway impaired - formation of somites not ordered or fail] Somites give rise to specific structures depending on their A-P position Portions of skull - cervical vertebrae - thoracic with ribs-etc Positional identity along AP axis is specified before scord segments grafting exp F4.3 PI controlled by Hox gene expression [Box 4A] Homeotic [homeobox] genes are involved in patterning along AP axis Unique identity of segments first identified in Drosophila - now know segmentation and other "positional" features inherited in similar fashion [F 4.3] Gene activity may provide positional "values" Fs What accounts for this? Homeotic selector genes - master regulatory genes specify positional identity direct future development of segments by controlling other sets of genes required throughout development to maintain specificity Experiments indicate that the genes work in combination Order of genes in complex is the same as the spatial and temporal order of their expression along the antero-posterior axis [F 4.7, 9,10] Somite consists of 3 subpopulations [F 4.4] Subpops have different gene expression Dermamyotome - expresses Pax3 gene Dermatome - dermis Myotome - muscle - expresses ts factor MyoD and muscle prots Sclerotome - vertebrae and ribs - expresses Pax1 gene Specification of these "3" fates requires signals from surrounding tissues Experimental manipulation Excise notoch and nt - apoptosis of somite Implant extra notoch (before segmentation) - somite develops as sclerotome [F 4.5] Multiple signals [F 4.6,7] Dorsalizing signal from epidermal ecto and dorsal nt-BMP-4 Ventralizing signal from notoch and floor plate of nt- Sonic hedgehog Lateralizing signal from lateral plate meso B. Establishing the Nervous System The neural tube forms with a single layer of neuroepithelium - Review neurulation: what events lead to the formaton of a mature brain and spinal cord? In what ways do the regions of the CNS differ from one another? [F 2.7; 8.28-31; 11.1, 13] Patterning the Nervous System Organizer and Henson's node induce axis development and neurulation [F 4.12, 13] Regional differences in kind and amounts of gene products continue to influence development BMP-4 induces ectoderm to form epidermis Ectoderm in region where chordin and noggin are released become neural ectoderm [F 4.17] noggin also impt in inducing neural ectoderm Other regional inducers goosecoid - most active in anterior portion of embryo cerberus - promotes formation of mouth parts, olfactory placodes and eyes, induces cardiac mesoderm and liver many other examples in the sci lit +++++++++++++++++++==below this point - still editing SPINAL CORD DEVELOPMENT Dorso-ventral patterning is established by opposing signals Ventral signal - action of sonic hedgehog in notochord and floor plate receptor- patched positional (ventral)signal for neural tube Dorsal signal - inductive signals from epid. ecto during tube closure; Dorsalin-1 also indicated to send signal in contact-mediated fashion Notochord plays a role in spinal cord organization When notochord segment grafted to lateral site of forming neural tube, a 2nd floor plate is induced as well as an outgrowth of motor neurons Notoch grafts in dorsal area repress Dorsalin-1; excision of notochord - area of Dorsalin-1 expression expands ventrally Specification of cell type Neuroepithelium - proliferation, migration and determination, differentiation Progenitor cells produce cell lines - neuroblasts vs glioblasts Gene interaction effect in progenitor cells -results in multiple cell types [F 11.1] Hypothesized that progenitor cells respond to different conc. of Shh Class I genes repressed Class II genes activated *will try to add links to fig here and elsewhere - check back Neurons arise from asymmetric division [F 11.17] Notch localizes on neuroblast; numb retained on stem cell Identity of neurons specified as cells migrate Notch-bearing cells in the neuroepithelium are specified as neuronal precursors Notch signalling = lateral inhibition F 11.6 Cells initially express: [F 11.10] Neurogenin gene - codes for ts factor which may reg - Delta - codes for a ligand Notch - codes for a receptor Delta-Notch interaction represses Neurogenin Neighboring cell producing more Delta inhibits 2nd cell so it no longer produces Notch Neurogenin then triggers NeuroD, another tsf required for neuronal diff'n Expression of Pax genes becomes restricted to regions of sp cord and helps direct fate of developing neuroblasts Motor columns - regional differences - for ex. specific groups develop in assoc with limbs F 9.28 Motor neurons exhibit changing pattern of gene expression - LIM family of homeobox genes [F 11.14] All motor neurons express LIM proteins lsl-1 and lsl-2 As axonal outgrowth proceeds, different combinations af the LIM group are seen If spinal cord from limb area transplanted to thoracic region, LIM genes appropriate to new site are expressed Regulation by mesoderm implied ------- What roles do glial cells perform in the developing nervous system? Fig 11.15 May result in distinct layering of neurons F 11.1 -------- CNS Patterning Exemplified in region of rhombomeres and activity of neural crest cells F4.25-28 Clonal cells (product of progenitor cell proliferation) in rhombencephalon observed to migrate in region over moderately large distances If progenitor cells labeled later in dev, progeny are restriced in movement by neuromere "boundaries" Also neural crests show pattern of emergence and migration that is associated with specific rhombomeres [Rev fates of NC F 9.27 ] Mechanism for establishing boundary and restricting migration - "cell-lineage restriction" Bidirectional signalling of ephrins and their receptors on adjacent cells modify migration and adhesion ------ How do the axons of neurons grow? What cues the control of the directionality of their growth? After neuroblast reaches its designated location it elaborates processes Axonal guidance best studied Activly growing (extending) tip is termed growth cone can "sample" substrate Mechanisms contact guidance on ECM, can involve CAMs chemoattraction vs repulsion F11.21; diffusible agents F11.29 contact attractrion vs repulsion utilization of guidepost cells F11.22 generally assumed to involve cell signaling and modification of cytoskeleton Example [new] commissural neurons potentially have 3 receptors for 3 prots ( netrin, slit and comm) expressed in midline area interaction causes down regulation of slit receptor and axon can cross midline later reexpresses receptor, can't cross back Non-commissural neurons are repelled by slit and don't cross midline Link for this figure In chick, spinal cord commissural neurons exhibit chemotatic response to netrin F 29, 30 ------ Neuronal targeting F 11.18 refinement - some cells die (PCD) Muscle as target may involve reciprocal interaction Formation of neuromuscular junction F32 - 37 Neurotropins required for out growth of axon and maintenance after connection made Once axon arrives at muscle it releases agrin which stimulates aggregation of ACh receptors on muscle Axon then releases neuregulin - stim musc to make more ACh receptors locally Refinement - extra synapses in vicinity eliminated F 11.38 D. Other neural patterning D/V axis due to BMP from epid ecto (dorsal) and sonic hedgehog and noggin from notochord transplantation of notochord to lateral position induces 2nd floor plate (in neural tube)and motor neurons lateral to plate [F15.24] Dorsalin inhibits motor neuron dev but stimulate neural crest migration Hox genes influence "segmental origin" of neural crest and segmentation of somites[] This next short section is a supplement to the Diff'n link. Differentiation AFTER proliferation and AFTER the organ/tissue primordium has been formed [determined] progressive selective gene activation will establish a pattern of gene expression that is specific for that tissue; most other genes will be turned off The cell type is differentiated when, under the direction of these specifc genes, it produces a specific set of unique proteins that allow it to carry out its unique function Proerythroblasts will synthesize hemoglobin; muscle cells will syn contractile prots,; unique membrane proteins will be inserted into the appropriate cell membrane; etc. Organelles required for the sustained specific function of the cell type will be prominent The cell type is mature and, under most circumstances, this state of differentiation is irreversible; few adult cells have cell division as a function *In cancer, the normal block to cell division has been circumvented Although we examined several examples of this progression of events, one of the best studied is the origin and differentiation of the various blood cells [ F 9.38] Notice that the stem cell via proliferation gives rise to progenitor cells. Under the influence of microenvironments and growth factors, genes are selectively activated or inhibited and the progenitor lines become more restricted [determined]. Finally with the activity of specific function-related genes, cells in each cell lineage becomes differentiated. OK - what else? Review summary boxes for portions of chapters covered. Know major features of Chpt 1 Have a working vocabulary for cell structure, signal transduction and gene expression. Good Luck!