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Development of the nervous system - I Raghav Rajan Bio 334 – Neurobiology I August 8th 2013 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I 1 Early embryonic development ● ● 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I Development of the nervous system begins after gastrulation Nervous system and skin develop from ectoderm 2 Stages of neural development ● Neural induction ● Birth and differentiation of neurons ● Polarity, segmentation of nervous system ● Migration of neurons to their final position ● ● Growth of axons and axon guidance to their post-synaptic targets Changes in synapses 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I 3 Model systems for studying development C-elegans Zebra fish Drosophila Chicken Mouse Xenopus 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I 4 Hydra (diploblast)– neurons develop from a precursor in the epidermis – interstitial cell ● 08th August 2013 Molecular mechanisms poorly understood Bio 334 - Neurobiology I - Development of nervous syste ms I 5 C-elegans – development of nervous system 08th August 2013 ● ~1000 cells ● 302 neurons and 56 glial cells Bio 334 - Neurobiology I - Development of nervous syste ms I 6 C-elegans ● ● ● ● 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I Endoderm and mesoderm precursors migrate inward – end of gastrulation Proliferation phase Ventral indentation – start of morphogenesis – 4 molts Neurons form and migrate inward 7 Drosophila – neurons generated from ventrolateral part of embryo (ectoderm) ● ● ● ● 08th August 2013 Cellularisation results in cellular blastoderm Ventral furrow marks start of gastrulation Mesoderm invagination Ventral midline – future site of neurogenesis Bio 334 - Neurobiology I - Development of nervous syste ms I 8 Drosophila ● Continuation of neurogenic region into anterior region gives rise to brain ● Neuroblasts delaminate ● Divide into GMC and Nb ● ● ● GMC gives rise to neurons or glia Larval nervous system Morphogenesis – additional neurogenesis 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I 9 Frog ● ● ● ● ● 08th August 2013 Blastopore – small invagination – point of initiation of gastrulation First cells to invaginate occur at the dorsal side of blastopore – opposite to sperm entry point Spemann's organiser Involuting marginal zone – first cells form head, last form tail (mesoderm) Neural plate Bio 334 - Neurobiology I - Development of nervous syste ms I 10 Frog ● ● ● ● 08th August 2013 Neural plate folds over to form neural tube Gives rise to neurons and glia of CNS Neural crest – junction between neural tube and ectoderm Unique to vertebrates – gives rise to PNS Bio 334 - Neurobiology I - Development of nervous syste ms I 11 Zebra fish – small differences in gastrulation due to differences in amount of yolk ● ● ● ● 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I Epiboly 50% epiboly marks start of gastrulation at future dorsal margin – shield Mesoderm delaminates, moves inside Neural plate forms 12 Chick – “yolky” egg ● ● ● ● 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I Blastodisc floating on yolk No epiboly Mesoderm invagination in this disc through blastopore-like primitive streak Hensen's node – posterior end of primitive streak – like dorsal lip of blastopore 13 Human – no yolk ● ● ● ● 08th August 2013 Inner cell mass of blastula gives rise to embryo Primitive streak – line of cells migrating into blastocoel to form mesoderm Overlying ectoderm induced to form neural tube Tube rolls up and forms brain and spinal cord Bio 334 - Neurobiology I - Development of nervous syste ms I 14 Involuting mesoderm “induces” overlying ectoderm to form neural tissue ● 08th August 2013 Culture each part of embryo separately to determine time of fate determination ● Frog ● Hans Spemann Bio 334 - Neurobiology I - Development of nervous syste ms I 15 Spemann and Mangold identify Spemann organizer ● ● ● ● 08th August 2013 Dorsal lip of blastopore can induce formation of second body axis including second brain and spinal cord Host and donor cells contribute to new axis Dorsal lip – Spemann organizer – not only neural inducer but also organizes body axis Who organizes Spemann organizer? Bio 334 - Neurobiology I - Development of nervous syste ms I 16 Spemann organizer organized by Nieuwkoop center ● ● ● ● 08th August 2013 Nieuwkoop found that mesoderm is induced at the junction of animal cap (ectoderm) and vegetal cap (endoderm) Dorsal-most part of vegetal pole can induce Spemann organizer Possibly β-catenin But, again, how is this setup – sperm entry, cortical rotations, gray crescent, etc... Bio 334 - Neurobiology I - Development of nervous syste ms I 17 Induction of neural tissue – studying it with the isolated animal cap assay ● ● 08th August 2013 Do candidate factors induce neural tissue directly or indirectly? Assay looked for increased expression of neural genes without increased expression of mesoderm genes Bio 334 - Neurobiology I - Development of nervous syste ms I 18 Noggin is a secreted molecule that can induce neural tissue ● ● ● ● 08th August 2013 Ventralized and hyperdorsalized embryos cDNA from dorsal lip (Spemann organiser) was divided into smaller and smaller collections to rescue UV treated embryos Noggin mRNA expressed in gastrulating embryos by cells of dorsal lip of the blastopore Richard Hartland's group Bio 334 - Neurobiology I - Development of nervous syste ms I 19 Chordin identified as another molecule secreted by organizer ● ● ● ● 08th August 2013 DeRobertis' group Identify genes that were expressed in the organizer region Chordin a secreted molecule expressed when neural induction occurs Overexpressin results in formation of second axis just like noggin Bio 334 - Neurobiology I - Development of nervous syste ms I 20 Neural fate is the default for endodermal cells ● ● ● ● ● Melton's lab Identified follistatin as a potential neural inducer Known to inhibit activin, a TGF-B family member Was actually studying the role of follistatin in mesoderm induction ● Truncated activin ● No mesoderm ● 08th August 2013 Noggin, chordin – not clear what they were doing Also lots of neural tissue despite the absence of mesoderm!! Bio 334 - Neurobiology I - Development of nervous syste ms I 21 More experiments to show that neural fate is default for animal cap cells ● ● 08th August 2013 Dissociated animal cap cells form neural tissue Follistatin also expressed in the organizer region at the time of neural induction Bio 334 - Neurobiology I - Development of nervous syste ms I 22 Vertebrate embryos are inverted insect embryos! ● ● ● ● 08th August 2013 sog (like chordin) inhibits dpp (like BMP4) in Drosophila to induce neural tissue – only on the ventral side Different neural inducers antagonize BMP signaling BMP4 inhibits neural tissue formation in animal caps (dissociated animal cells too) – even with noggin, chordin, follistatin Antisense BMP4 RNA – neural tissue forms without addition of inducers Bio 334 - Neurobiology I - Development of nervous syste ms I 23 Wnt and BMP signaling pathways – two that are important for development 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I 24 All three neural inducers block BMP signalling in a similar fashion ● ● 08th August 2013 Noggin and chordin bind to BMP4 directly Follistatin binds to related BMP7 and activin Bio 334 - Neurobiology I - Development of nervous syste ms I 25 Inhibiting BMP signaling – not enough in mammals ● ● Deletion of noggin and chordin affect head formation and cerebral hemisphere formation Some neural tissue does form 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I 26 Current understanding of neural induction : Old problem, new findings, yet more questions Stern CD Development (2008): Old problem, new findings, yet more questions 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I 27 Next step – formation of neuroblasts ● ● ● 08th August 2013 Drosophila, proneural clusters achaete-scute complex genes expressed by clusters of proneural cells before delamination One neuroblast develops from each cluster Bio 334 - Neurobiology I - Development of nervous syste ms I 28 Achaete-scute complex genes are required for formation of the one neuroblast ● ● ● 08th August 2013 Only one of the proneural cells becomes a neuroblast in a normal fly Flies mutant for proneural genes like achaete scute – neuroblasts don't form Flies mutant for notch or delta – multiple neuroblasts form Bio 334 - Neurobiology I - Development of nervous syste ms I 29 These genes are basic-helix-loop-helix transcription factors 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I 30 Delaminating neuroblast inhibits neighbouring cells from becoming a neuroblast ● ● 08th August 2013 Ablation of a delaminating neuroblast makes the neighbouring ectodermal cell become a neuroblast Neuroblast inhibits neighbouring cells from becoming neuroblasts Bio 334 - Neurobiology I - Development of nervous syste ms I 31 Notch signaling pathway is involved in lateral inhibition Fortini ME Developmental Cell (2009) The Notch pathway: Core signaling and its posttranslational regulation 08th August 2013 Bio 334 - Neurobiology I - Development of nervous syste ms I 32