Lecture 1: Gastrulation and Neural Induction PDF

Module 3: Neurodevelopment Dr. Orkun Akin Asst. Professor, Dept. Neurobiology, DGSOM, UCLA Office Hours: Fridays, 3:30-4:30 pm (Zoom link in Bruinlearn) [email protected] Di...

Module 3: Neurodevelopment Dr. Orkun Akin Asst. Professor, Dept. Neurobiology, DGSOM, UCLA Office Hours: Fridays, 3:30-4:30 pm (Zoom link in Bruinlearn) [email protected] Discussion Assignments: Posted early in the week, Due Fridays at 8 am. Textbooks: None assigned Lectures and discussions are comprehensive Development of the Nervous System, Sanes et al. (any edition)  Module 3, Lecture 1: Gastrulation and Neural Induction Outline Model organisms in the study of development Gastrulation and Germ Layers: Comparative view across phyla Neural Induction: How is the ectoderm turned into nervous tissue? The Spemann-Mangold Experiment Deep Dive: Identification of Neural Inducers Modern Synthesis: The Default Model of Neural Induction The Nervous System: The Family Tree Phylum Members Porifera Sponges Cnidarians Jelly fish, Sea anemone Nematodes Round worms Platyhelminthes Flatworms Molluscs Snails, Slugs, Octopus Protostomes Bilateria Annelids Earthworms Arthropods Crabs, Spiders, Insects Echinoderms Starfish, Sea Deuterostomes cucumbers Chordates Sea squirts, Pigs Tunicates Vertebrates The Nervous System: Deep Time No neurons, but some synaptic genes Neurons, Diffuse Nerve Nets 750-620 MYa Organized Nerve Nets, Centralized Nervous Systems, and Brains 700-600 MYa The Nervous System: Shared Heritage Hodgkin and Huxley Eric Kandel studied the established the ionic molecular basis of learning and mechanisms of action potential memory in the propagation in the sea slug Aplysia. Squid giant axon. The Nervous System: Why Development? Practical: If understand how we are built, better chance of fixing ourselves when we break. Implicit: Our bodies are not put together from ready-made parts, they are grown— developed—following a program. Intellectual: Development has been and remains a grand scientific challenge. The Nervous System: Why Development? https://www.hhmi.org/news/unveiling-the-biggest-and-most-detailed-map-of-the-fly-brain-yet This is the fruit fly brain. It has ~150,000 neurons. The mouse has ~70 million. Humans have ~86 billion. The Nervous System: Why Development? Practical: If understand how we are built, better chance of fixing ourselves when we break. Implicit: Our bodies are not put together from ready-made parts, they are grown— developed—following a program. Intellectual: Development has been and remains a grand scientific challenge. 1850s: Gregor Mendel's genes and rules of heredity (peas!) 1910s: Thomas Hunt Morgan showed genes are on chromosomes (flies) 1953: Watson & Crick (working with Rosalind Franklin's data) gave us the double helix. 2003: Human Genome Project completed. So, how does it work? How do we take the information in the genome and build an animal (preferably one with a brain)? The Nervous System: Model Organisms What makes a good model organism? Accessible: Not dangerous, rare, or endangered. Convenient: Can be maintained in the lab, develop and reproduce rapidly, and are cheap to maintain. Experimental advantages: Genetics, robust to manipulation, external development, transparent, inbred stocks, etc. Momentum: The more people use it, the more powerful they become. The Nervous System: Model Organisms Nematode worm (Caenorhabditis elegans) Fruit fly (Drosophila melanogester) Purple sea urchin (Strongylocentrotus purpuratus) Echinoderms have bilaterally symetric larvae. Zebrafish (Danio rerio) African clawed frog (Xenopus laevis) Chicken (Gallus gallus domesticus) House mouse (Mus musculus) The Nervous System: Model Organisms Nematode worm (Caenorhabditis elegans) Lives on petri dishes, eats bacteria. Transparent: Great for microscopy. Females are hermaphrodites: Self-fertilization makes maintenance and genetics easy. Lives fast: Reproductive cycle is 3 days. Invariant development: 959 somatic cells in the hermaphrodite; 1031 in the adult male. All cell lineages mapped. Great for studying cell fate determination. Connectome: Connectivity of all ~300 neurons known. The Nervous System: Model Organisms Nematode worm (Caenorhabditis elegans) Fruit fly (Drosophila melanogester) Cheap to maintain by the thousands. Backed by 100+ years of research momentum: established workhorse of classical genetics, modern bleeding edge of cellular and systems neuroscience. Still fast: 10-day reproductive cycle. Unparalleled genetic and molecular tools. Genome is 60% homologous to humans; 75% known human disease genes have homologs in flies. Connectome: Many parts of the adult brain already available at high resolution; complete connectome on the way. The Nervous System: Model Organisms Nematode worm (Caenorhabditis elegans) Fruit fly (Drosophila melanogester) Purple sea urchin (Strongylocentrotus purpuratus) Echinoderms have bilaterally symetric larvae. Easy to maintain in the lab and get large numbers of synchronized embryos. Transparent embyro and larval stage. Model for cell fate decisions of early embryogenesis (i.e. gene regulatory networks.) The Nervous System: Model Organisms Nematode worm (Caenorhabditis elegans) Fruit fly (Drosophila melanogester) Purple sea urchin (Strongylocentrotus purpuratus) Echinoderms have bilaterally symetric larvae. Zebrafish (Danio rerio) A single female can spawn hundreds of eggs every 2-3 days. Transparent embryo, external development: Microscopy! Reproductive cycle is 90 days. (Not bad for a vertebrate.) Advanced tools and techniques for genetic screens. First forward genetic screen in a vertebrate system carried out in Zebrafish. (A forward genetic screen is when you randomly mutate a lot of individual animals with the goal of finding genes involved in a biological process you are interested in.) The Nervous System: Model Organisms Nematode worm (Caenorhabditis elegans) Stalwarts of classic embryology. Fruit fly (Drosophila Robust, externally developing embryos melanogester) (can put a 'window' in a chicken egg.) Embyros readily available in quantity. Not genetic animals; but, canPurple cut andsea graft,urchin inject material to (Strongylocentrotus mark/alter parts of develeoping animals. Critical, purpuratus) unique Echinoderms have features. bilaterally symetric larvae. Large Xenopus eggs provide material for protein biochemistry. Zebrafish Modern molecular techniques being incorporated. (Danio rerio) African clawed frog (Xenopus laevis) Chicken (Gallus gallus domesticus) The Nervous System: Model Organisms Nematode worm (Caenorhabditis elegans) Fruit fly (Drosophila melanogester) Purple sea urchin (Strongylocentrotus Common model organism purpuratus) most closely related to humans (last common ancestor lived ~75 million Echinoderms years ago) have bilaterally symetric larvae. Female can produce 8 litters of 8 pups per year. 60-day reproductive cycle—faster than Zebrafish! Zebrafish In utero development; specialized (and costly) rearing facilities required. (Danio rerio) 'Designer' mice possible with advanced embyronic stem cell and in vitro fertilization techniques; now even more powerful African clawed frog with CRISPR. (Xenopus laevis) Emerging reverse genetic powerhouse. (Reverse genetics is when you target a specific gene for disruption Chicken to study its role in a biological process.) (Gallus gallus domesticus) House mouse (Mus musculus) The Nervous System: Model Organisms Nematode worm (Caenorhabditis elegans) Fruit fly (Drosophila melanogester) Purple sea urchin (Strongylocentrotus purpuratus) Echinoderms have bilaterally symetric larvae. Zebrafish (Danio rerio) African clawed frog (Xenopus laevis) Chicken (Gallus gallus domesticus) House mouse (Mus musculus)  Gastrulation: Tubes, layers, and making bellies. (Almost all) Bilaterians have a tube-within-a-tube body plan. Fundamentally, we are feeding tubes with two openings and some tissue around it. This geometry separates our tissues ito three layers: 1. Outer layer: Ectoderm 2. Inner Layer: Endoderm 3: Middle Layer: Mesoderm  Gastrulation: Tubes, layers, and making bellies. Early cell divisions after fertilization generates a hollow ball: Blastula. The ball invaginates at the blastopore creating a small pocket: Archenteron. This is the future gut. The embyro made a small belly; it Gastrulated. The three germ layers are forming. All outer layer (skin, hair, tooth enamel) Muscle, bone, cartilage, connective Digestive system, Central nervous system tissue, fat, circulatory and lymphatic liver, pancreas, Peripheral nervous system systems, etc. bladder, lungs, etc.  Gastrulation: A closer look with an echinoderm (Sea urchin). Blastocoel: Cavity inside blastula/gastrula. ('blasto-seal') Mesenchyme: Loosely organized mesodermal cells Animal / Vegetal poles: Before gastrulation, blastula already polarized. Animal (non-yolky) / Vegetal (yolky) poles are set up during egg maturation.  Gastrulation: A closer look with an echinoderm (Sand dollar). Richard A. Cloney, University of Washington NATIONAL SCIENCE FOUNDATION GRANT DB10 COPYRIGHT 1968 Educational Development Center, Inc. All rights reserved. Distributed by the University of Massachusetts Amherst under license agreement with the Educational Development Center, Inc.  Gastrulation: Priorities Protostome: Mouth forms from the blastopore. ('Mouth first') Deuterostome: Anus forms from the blastopore. ('Mouth second') Phylum Members Porifera Sponges Cnidarians Jelly fish, Sea anemone Nematodes Round worms Platyhelminthes Flatworms Molluscs Snails, Slugs, Bilateria Octopus Protostomes Annelids Earthworms Arthropods Crabs, Spiders, Insects Coelom: Body cavity between Echinoderms Starfish, Sea Deuterostomes digestive tract and body wall cucumbers ('seal-um'). e.g. in humans: thoracic, Chordates Sea squirts, Pigs abdominal, and pelvic cavities. Tunicates Vertebrates  Remember: Memento Mori. You are a deuterostome. (Remember: You are mortal)  Gastrulation: Vertebrates—Modified geometries, Same principle Amniote: Amnion membrane surrounds embyro; exchanges gases and waste. Allowed animals to develop on land. No larval stage. e.g., Reptiles, birds, mammals. Anamniote: Eggs laid in water which facilitates gas and waste exchange. e.g., Fish, amphibians. Gastrulation: Fish (anamniote) Gastrulation: Fish Gastrulation: Fish Phillip J. Keller Lab, HHMI Janelia Research Campus Gastrulation: Fish Royer, L. A. et al. Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms. Nat Biotechnol 34, 1267–1278 (2016). Gastrulation: Amphibians (anamniote) Dorsal Lip of the Blastopore Gastrulation: Amphibians Gastrulation: Avians (amniote)  Gastrulation: Avians (amniote) Blastula -> Blastodisc Blastopore -> Primitive streak/groove Dorsal lip -> (Hensen's) node Gastrulation involves cells moving in through the primitive streak/groove to form the three germ layers.  Gastrulation: Mammals The details of in utero development are complex; the zygote builds both embryonic and extra-embryonic tissues. But, gastrulation follows the reptilian / avian archetype with a node and a streak and involuting cells to build the germ layers. Uterine lining Gastrulation: Mammals Mouse embyro developing in culture Expressing Histone-GFP in all cells 2 days of imaging McDole, K. et al. In Toto Imaging and Reconstruction of Post-Implantation Mouse Development at the Single-Cell Level. Cell 175, 859-876.e33 (2018). Gastrulation: 3 Germ Layers  Gastrulation: 3 Germ Layers Endoderm – gut, pharynx, liver, lungs, bladder, urethra Mesoderm – skeleton, muscles, heart, spleen, connective tissue, circulatory system, and notochord (transiently) Ectoderm – develops into skin and nervous system Even before first cleavage, the fertilized egg has two perpenticular axes: 1. Animal-Vegetal Axis 2. Dorsal-Ventral Axis (Gray Crescent) Normal development requires contributions from both. Dorsal blastopore lip is derived from the Gray Crescent Hans Spemann (1869-1941) Hilde Mangold (1898-1924) The Spemann-Mangold Experiment Second dorsal lip produced a second body and nervous system. Most cells of the second nervous system were derived from the host embryo. The dorsal lip of the blastopore is now known as the Spemann-Mangold organizer. Equivalent structures in other vertebrates, including mammals, are called (embryonic) organizers. (Recall: the Node.) Molecular Biology of the Cell, 5th Edition Alberts, Johnson, Lewis, Raff, Roberts, & Walter ISBN: 978-0-8153-4105-5 Watch closely for a glimpse of UCLA's own Eddy DeRobertis!  The Neural Induction Model A question of ectodermal fate: skin or nervous tissue? Animal cap Isolated isolated from the animal cap Animal cap pre-gastrula isolated from embryo develops the gastrula into epidermis. develops into neural tissue. Hypothesis: Organizer induces ectoderm to adopt neural fate. Induce means to influence. Implies inter-cellular communication. It took about 70 years to find the molecules involved in this communication. Deep Dive: Identification of Noggin as a neural inducer (1/5) Hyper Dorsalized Ventralized Embryo Embryo UV Inference: Neural inducer (molecules) are Microtubule Cytoskeleton absent in the UV-treated case and over- abundant in the Lithium-treated case. Gray Crescent Organizer Approach: Recover material from Li-treated and put it into UV-treated. See if you can rescue Neural Induction neural induction. (Richard Harland and colleagues) Deep Dive: Identification of Noggin as a neural inducer (2a/5) Deep Dive: Identification of Noggin as a neural inducer (2b/5) complementary DNA (cDNA) library: Deep Dive: Identification of Noggin as a neural inducer (3/5) cDNA library Bacterial Colonies Pooled DNA Pooled mRNA UV treated Screening Assay: Neural Induction Rescue Deep Dive: Identification of Noggin as a neural inducer (4/5) + Noggin UV treated mRNA Result: Noggin mRNA alone can rescue neural induction None (negative in a dose dependent manner. control) Control Low Medium High Deep Dive: Identification of Noggin as a neural inducer (5/5) Noggin protein Conclusion: Neural Noggin is a Genes soluble protein Result: Soluble Noggin protein acts secreted by the dorsal lip like the organizer. that induces overlying ectoderm to adopt a neural fate. Result: Noggin mRNA localizes to the organizer.  Neural Induction Neural Inducers: Noggin (1993, Harland and colleagues) Follistatin (1994, Melton and colleagues) Chordin (1994, DeRobertis and colleagues – UCLA!) All three genes encode for secreted proteins. mRNA for all at the organizer during gastrulation. (these are cut in half, we're seeing cross section of the embryo.) Triple Knock Control Down Knocking down all three leads to severe loss of neural structures. This is called a loss-of-function (LOF) experiment. (single and double knock downs have milder defects.) Localization of neural gene Neural Induction (The Third Act Twist!) Hypothesis: Organizer induces I S E ectoderm V fate. R E neural to adopt Dissociated pre-gastrula animal cells grow into neurons. Dissociated pre-gastrula animal cells cultured with BMP grow into skin. Intact pre-gastrula animal caps cultured with BMP inhibitors grow into neurons. + BMP inhibitors Conclusion: BMP made by the animal cap cells inhibits neural fate.  Neural Induction Bone morphogenic proteins, or BMPs, are members of a very large group of secreted proteins: TGF-β super family (named after the first member, Transforming Growth Factor beta) BMP cell signaling cascade: 1. BMP binds to its receptor from the outside of the cell. 2. This binding leads to activation of the intracellular part of the receptor. 3. Activated receptor phosporylates an intracellular protein called SMAD. 4. Pi-SMAD binds co-SMAD and they move to the nucleus. 5. In the nucleus, they regulate gene expression (in a specific fashion).  Neural Induction Noggin, Chordin, and Follistatin prevent BMP from binding its receptor, and the signalling cascade that selects epidermal fate over neural is not switched on. Neural Induction Noggin Chordin BMP Neural Fate (from ectoderm) (in ectoderm) Follistatin (from organizer) Revised Hypothesis: aka "The Default Model" The default fate of ectoderm is neural; this fate is repressed by neighboring cells through BMP signalling. The organizer de-represses neural fate by inhibiting BMP signaling.  Neural Induction No Noggin or Wildtype No Noggin Chordin In the mouse, loss of Noggin leads to mild defects. The head is nearly absent with loss of both Noggin and Chordin