Lecture 13 - Plant Growth & Development PDF

**Lecture 13** Plant growth - Tissues formed by coordinated divisions/expansions - Cells have cell wall - Immobile - Cell fate determined by position - Ontogenetically - Tremendous genetic plasticity Arabadopsis thaliana - Mustard family - 5-10000 seeds in 6 weeks...

**Lecture 13** Plant growth - Tissues formed by coordinated divisions/expansions - Cells have cell wall - Immobile - Cell fate determined by position - Ontogenetically - Tremendous genetic plasticity Arabadopsis thaliana - Mustard family - 5-10000 seeds in 6 weeks - Genome has over 25k proteins - Phases - 1n haploid -- gametophytic - 2n diploid -- sporophytic - Life cycle - Fertilization - Embryogenesis - Germination - Growth + maturation - Gametogenesis Seed morphogenesis - 0-20 days postanthesis - After opening of flower (?) - Embryo body plan established - Root + shoot apical meristems formed by 'heart' stage Seed maturation - Cells expand (rather than divide) - Accumulate starch, lipids, proteins Plant primary tissue systems 1\. protoderm 2\. ground tissue 3\. vascular tissue Plant body plan construction - During embryogenesis a\) zygote asymmetric division b\) radially symmetric 8-cell embryo proper c\) protoderm established d\) dermatogen globular embryo with procambium + quiescent center e\) "heart stage" fully differentiated tissue systems f\) seedling A diagram of the structure of the tooth Description automatically generated Plant pattern formation issues (FASS) - FASS gene mutation = disordered division patterns - Fail to produce microtubules pre-prophase - Direct plane of division - However all cell types are still present Cell fate determination - Governed by - Cell lineage - Position of cell within embryo - Independent of division patterns - New tissues develop thru regulatory signals from neighbors Cell polarity - Controlled by Auxin hormone - Accumulates early in embryo proper - PIN1 gene encodes auxin efflux carrier - Loss of PIN1 results in a PIN-like bolt and no lateral organs - PIN 2,3,4,7 regulate development Genetic defects - Hormone traffic/perception defects produce altered body parts - GNOM (encodes ADP -\> GTP factor ARF-GEF) - Regulates membrane vesicle traffic - Mutants lose apical basal polarity - GNOM required for PIN1 localization Meristem indeterminate growth - Shoot meristems regulate organ formation - Balance maintenance of undifferentiated cells and commitment of cells and their eventual differentiation Arabadopsis' primordia - 3 regions - Central zone - Contains stem cell core - Peripheral zone - Site of lateral organ primordia - Rib zone - Gives rise to undifferentiated meristem cells - Picture a dome with a smaller dome in middle - Inner/middle/smaller dome is rib zone - Divide arc above it by 3 - Top one is central zone - L/R top ones are peripheral zones Shoot apical meristem (primordia) layers - L1 - Outermost - Undergoes anticlinal divisions to create epidermis - L2 - Gives rise to ground tissue - L3 - Forms body of new tissues including vasculature Homeodomain proteins - Promote shoot apical meristem activity - Maintenance of undifferentiated cells in meristem STM gene - Prevents premature recruitment of cells into undifferentiated pathways - Mutants will lack apical meristem at end of embryogenesis - Due to role in maintaining stem cell population - Overexpression increases meristematic activity - Increased SAM size, flowers, seed yields Wus gene - Maintains pool of stem cells - Mutants also lack SAM - Fails to establish - Repeated meristem termination results in failure to specify central stem cells needed to repopulate peripheral meristem - Required for stem cell identity CLAVATA mutants - Possess enlarged SAMs relative to wildtype - CLAVATA limits meristem size - CLV1 -\> SAM receptor kinase protein - CLV2 -\> similar receptor-like protein - CLV3 -\> peptide ligand for CLV1/2 complex - CLVs promote organ initiation CLV-Wus feedback loop - SAM maintenance depends on organ initiation & stem cell self-renewal - \(1) CLVs (2) Wus - Wus maintains renewal of stem cells that will be used in CLV processes which make organs **Lecture 14** The fruit fly - DROSOPHILA MY BELOVED - Small, easy to maintain, observe - High fecundity (500 eggs/life) - 13600 genes - Developmental processes conserved in other species Drosophila development overview 1. Fertilization a. Gives organism new genome 2. Cleavage b. Division into blastomeres 3. Gastrulation c. Slowing mitotic divisions, cell rearrangement, blastomere movement 4. Organogenesis d. Chemical signal exchange between germ layers e. Organ production as a result of \^ 5. Formation of body axes f. Anterior posterior (head to tail), dorsal lateral (back to belly), lateral medial (left to right) Drosophila fertilization 1. Sperm enters ALREADY activated egg a. Inflow of Ca2+, many mitosis, translation of mRNAs 2. Micropyle allows sperm to pass b. One at a time, polyspermy prevention 3. Sperm + egg DON'T fuse, but nuclei do Drosophila cleavage - Superficial cleavage - Karyokinesis (nuclear division) occurs without cytokinesis (cell division) to create syncytium (cell containing several nuclei) - 9^th^ cycle - \~5 nuclei reach surface of posterior pole - Nuclei become enclosed by cell membranes -\> pole cells - Pole cells produce gametes - 10^th^ cycle other nuclei move to periphery - Each nuclei contained within own territory of cytoskeletal proteins - Energids are the nuclei & associated islands of cytoskeletal proteins - 13^th^ cycle - Egg cell membrane folds inward between nuclei, partitions off each energid into single cell - Process creates cellular blastoderm in which all cells are arranged in almost a jacket shape around egg-yolky core - Basically: - Superficial forms syncytium - 9^th^ forms pole cells - 10^th^ forms energids - 13^th^ forms blastoderm Mid blastula transition - Slowdown of division, cellularization, new RNA transcription - Maternal effect genes = Genes belonging to the maternal genome used to make mRNAs - Maternal messages -- mRNAs in oocyte - Maternal -\> zygotic transmission - Begins \~ cycle 11, greatly enhanced at 14 cycle - Control of gene expression shifts from being derived from mRNAs within oocyte to being controlled by new transcription from zygotic genome Drosophila gastrulation - Begins shortly after mid-blastula transition - First gastrulation movements - Segregate presumptive mesoderm, endoderm and ectoderm - Mesoderm folds inward to produce ventral furrow - Anterior and posterior midgut invaginations - Presumptive endoderm invaginates to form two pockets - Presumptive ectoderm bends to form cephalic furrow - Ventral furrow pinches off from surface to become ventral tube - Internalization of pole cells - Germ band extension Germ band extension - Ectodermal cells on surface and mesoderm undergo convergence & extension - Migrate toward ventral midline - Forms GERM BAND - Includes all cells that will form trunk of embryo - At end: - All 3 germ layers now formed - Beginning of organogenesis & segmentation - Dividing ectoderm and mesoderm - Ma, Mx, Lb correspond to mandibular (lower jaw), maxillary (upper jaw) , and labial (lips) Germ band retraction - Upon appearance of body segments - Dorsal closure - After germ band retracts, at dorsal surface - 2 sides of epidermis brought together **Lecture 15** Axis-formation importance - Crucial in establishing body plan - In drosophila, consistent in embryo, larva & adult stages - Repeating segmental units present between head and tail ends - 3 segments compose thorax - 8 form abdomen - Each segment presents unique identity Anterior-posterior pattern formation - Bicoid mRNA and nanos mRNA at anterior and posterior ends respectively - Both diffuse toward middle and form opposing gradients - Bicoid anterior -\> posterior gradient - Nanos posterior -\> anterior gradient - Morphogen gradients establish embryo's anterior posterior polarity and create differences in regions - Bicoid mainly responsible for acron, head, thorax - Nanos mainly responsible for abdomen Other maternal mRNAs - Hunchback & caudal - Transported from nurse cells into oocyte - Distributed ubiquitously throughout oocyte - Hunchback anterior -\> posterior gradient - Caudal posterior -\> anterior gradient - All 4 MATERNAL protein gradients necessary for activation of zygotic genes for segmentation to begin Segmentation genes 1. Gap genes a. Encode transcription factors i. Initiate transc. of pair-rule genes b. define broad -- connected -- domains (ex. A1-A5) ii. Mutation would remove that segment but leave rest normal 2. Pair-rule genes c. Divide embryo into periodic units iii. Precursors of segmental body plan d. Zebra stripe pattern iv. Due to genes such as eve (EVEN-SKIPPED) v. Activator binding sites (above) and repressor binding sites (below) interact competitively, located close together e. Activate segment-polarity genes 3. Segment-polarity genes f. Protein products divide embryo into 14 segment-wide units g. Ex. engrailed (en), wingless (wg), hedgehog (hh) - All 3 segmentation gene types regulate homeotic selector genes - Determine developmental fate of each segment Homeotic genes in drosophila - Give identity to segments - They are HOX GENES in this case - Hox genes are a subtype of homeotic gene that determine order of expression - Antennapedia complex - Head and thorax (until t2) components - Bithorax complex - Thorax (t3 onward) and abdomen components - Both ANT and BIT are on chromosome 3 - Mutations of homeotic genes can lead to development of segments that have abnormal identities (will assume identity of a different segment) - Ex. mutation or deletion of Ubx gene causes T3 to become another T2, no halteres, 4 wings - 'most posterior' gene usually exerts dominance - Represses activity of previously activated anterior gene to determine new identity Thoracic segments 1. Segment exclusively has legs 2. Has legs and wings 3. Contains legs and Halteres (flight balancing organs, not wings) **Lecture 16** Xenopus Laevis (phrog) - Large, manipulable, culturable embryo - Can be obtained in large numbers - Rapid development to tadpole - External development Phrog fertilization and cortical rotation - Unfertilized egg has polarity - Looks like voltorb - Dense, vegetal yolk (lower) - Less, animal yolk (upper) - Sperm can enter anywhere in animal hemisphere - Entry point will become ventral side, 180º opposite will be dorsal Phrog cleavage - 1^st^ cleavage bisects exposed gray crescent - Due to cortical cytoplasm rotating relative to internal cytoplasm - 2^nd^ is at right angles to the first - Meridional - Vegetal yolk impedes cleavage, 2^nd^ division begins before first has completed - 3^rd^ is equatorial - However its displaced asymmetrically toward animal pole (doesn't have to be right between 2 yolks) - As cleavage progresses vegetal hemisphere contains larger macromeres and fewer blastomeres, animal hemisphere maintains micromeres Amphibian blastocoel - Prevents cells beneath it from prematurely interacting with cells above it - Basically a big hollow dome - Roof of blastocoel = animal cap - Placing cells from animal cap next to vegetal cells forming a blastocoel base, the cap cells differentiated into mesoderm instead of ectoderm Phrog gastrulation 1. Blastocoel roof cells spread to cover entire embryo (at least the outer layer) 2. Floor of blastocoel moves upward toward animal cap (vegetal rotation) 3. Bottle cells (epithelium) invaginate and form blastopore dorsal lip 4. Mesodermal precursors involute under animal cap 5. Archenteron (new big hollow hole) forms and displaces blastocoel 6. Cells migrate from lateral and ventral blastopore lips into the embryo 7. Convergence & extension of mesodermal & ectodermal cells 8. Blastocoel is obliterated (L) 9. Ectoderm surrounds embryo 10. Endoderm internalized 11. Mesodermal cells positioned between other 2 germ layers Specification of germ layers - Late blastula stage - Ectoderm & endoderm specified by maternal factors - Animal cap cells differentiate into ectoderm, vegetal cells differentiate into endoderm - Mesoderm fate induced by interactions of animal and vegetal cells - Vegetal cells induce cells immediately above them to become mesoderm Differentiation of vegetal cells into endoderm vs. mesoderm - mRNA for TF VegT allocated to vegetal cells during cleavage - endoderm - VegT activates set of genes including Sox17 befORE mid-blastula transition actually - But sox17 plays critical role in specifying fate of vegetal cells as endoderm - Mesoderm - vegT activates paracrine factor Nodal that signals cells above to accumulate phosphorylated Smad2 - Phosphor. Smad2 upregulates eomesodermin for specification of mesoderm - Eomes & Smad2 create positive feedback loop for mesoderm maintenance Amphibian axis formation - Spemann and mangold transplanted dorsal lip of gastrula to opposite region (fated to become ventral) - As a result, dorsal structures were made in ventral part (ventral tissues gave rise to dorsal structures) - Dorsal lip cells were able to induce host tissues to form complete neural tube - Can induce a second main body axis, and create twinned embryo The "organizer" - Dorsal blastopore lip and its descendants - Formation: - Dorsal most vegetal cells in Nieuwkoop center are capable of inducing organizer - B-catenin is major factor that specifies Nieuwkoop center - Synergy with vegetal signals Nieuwkoop center (diagram slide 21 L16) - Dorsal-side vegetal cells induce dorsal mesoderm components, somites, notochord, and act as organizer - As opposed to ventral vegetal cells, which induce ventral mesoderm, mesenchyme, blood - B-catenin synthesized from maternal mRNA accumulates in dorsal side, covering Nieuwkoop center and organizer region - Accumulation due to translocation of disheveled, GSK3-binding GBP, Wnt11 mRNA from vegetal pole - GSK3 stimulates degradation of B-catenin so -\> Dsh & GBP inactivate GSK3 and -\> Wnt11 stabilize Dsh and GBP then -\> B-catenin enters nuclei & binds Tcf3 to activate Siamois and Twin - Siamois and Twin bind to Noggin, Chordin, Goosecoid which involved in organizer function Synergy with vegetal signals - Siamois and twin alone can't activate organizer - VegT produces Nodal which induces mesoderm - Recall via pSMAD2 - High B-catenin also helps Nodal-related expression - So Vegetal VegT/Nodal/pSMAD2 path + Dorsal B-catenin/Siamois&Twin path = synergy that activates organizer genes **Lecture 17** Recall mammalian fertilization 1. Translocation of sperm and egg 2. Acrosome reaction 3. Digestion of egg membranes 4. Binding/recognition 5. Membrane fusion 6. Sperm entry 7. Genetic fusion 8. Egg metabolism - Begin embryogenesis woo hoo - Now initiation of first cleavage Mammalian cleavage - 1^st^ occurs during oviduct -\> uterus journey - 2^nd^ = rotational cleavage - One blastomere divides meridionally - One blastomere divides equatorially - Mammalian blastomeres don't all divide at same time -- asynchrony - Zygotic genes activate at \~8 cell stage Compaction - In mice, blastomeres arranged loosely up to 8-cell stage - After 3^rd^ cleavage, they cluster & form tight ball (compaction) - Outer cells form tight junctions - Primarily become trophoblast cells (trophectoderm) - Inner cells form gap junctions for signal exchange - Give rise to ICM inner cell mass (embryo) Cavitation - Morula initially lacks internal cavity - Cavitation = trophoblast cells within morula secrete fluid to create cavity and pump in Na+ - Accumulation of sodium draws in water osmotically which enlarges & creates blastocoel - As blastocoel expands, ICM positions on 1 distinct side of trophoblast ring as blastocyst Hatching - Blastocyst enters uterus & fluid in blastocoel creates pressure from trophoblast against zona pellucida - Uterine glands also have zona digesting proteases - Eventually lytic enzymes & proteases cause zona to rupture & release blastocyst Recall blastocyst pathways - Trophoblast -\> cytotrophoblast -\> syncytiotrophoblast - Icm -\> hypoblast and epiblast Implantation steps 1. Apposition - Orient blastocyst w/ icm facing uterus - L-selectin & ligands mediate recognition of trophoblast by uterine cells 2. Adhesion - LIF (leukemia inhibiting factor) secreted epithelial cells attracts embryo to strongest adhesive region - Integrins mediate adhesion of trophoblast and uterine cells 3. Progression (invasion) - Trophoblast metalloproteinases digest ECM of uterine cells - Embryo breaks thru & embeds into uterine cells - Formation of syncytiotrophoblast (secretes hormones for maintenance) 4. Decidualization - Stromal cells form spongy core (chemical support for embryo) - Uterine cells suppress immune response to protect embryo ICM segregation - Hypoblast - Primitive endoderm - Positions site of gastrulation - Regulate cell movements - Maturation of blood cells - In contact with blastocoel (so it's the lower layer) - Epiblast - Upper layer (closer to uterine cells) - Forms embryo \~gastrulation starts around here\~ Bilaminar germ disc - Epiblast + hypoblast - Separates amniotic cavity and yolk sac - Upon completion the Amniotic cavity fills with amniotic fluid for shock absorption - Epiblast cells replace hypoblast cells - Undergo EMT epithelial-mesenchymal-transition - Gastrulation begins at posterior end of embryo where Primitive Streak arises Recall EMT - Epithelial cells - Adhere to one another and can form sheets and tubes - Polarized - Stationary - Mesenchymal cells - Migrate individually or collectively - Mobile - Invading Result of epiblast cell EMT - Ingressing cells fan out to form mesodermal layer - Migrate thru primitive streak to form a node (thick bulb at anterior end) Mammal anterior-posterior axis formation - Primitive streak (mammalian version of blastopore) establishes antero-posterior axis - 2 signaling centers: node, AVE (anterior visceral endoderm) - DVE cells -\> Cerebus -\> block Nodal & Wnt -\> DVE cells migrate to anterior to become AVE - AVE produces Dickkopf (Wnt antagonist) - Wnt3 and Nodal have no effect on anterior side of embryo - BMP4 from trophoblast instructs epiblast cells to produce Nodal and Wnt3 at posterior side leading to mesoderm generation and node formation - Node secretes chordin (ok?) HOX code hypothesis - Similarity of Hom-C cluster (drosophila) and Hox gene cluster (mouse) = homeotic gene evolutionary conservation - Equivalent genes hoxa4,b4,c4,d4 = paralogues - Similar in sequence due to ancestral events - Sequential activation of Hox genes begins during gastrulation, ends as primitive streak appears - All known mammals contain 4 copies of Hox complex per haploid genome on 4 different chromosomes (hox genes labelled 1-13) - As cells migrate anteriorly, anterior cells = 1, posterior = 13 - Regional identity suggests posterior dominance Evidence for Hox code - Comparative anatomy - Mouse & chick have similar number of vertebrae, but distributed differently - Hox gene code presents along A-P axis which determines type of vertebrae formed (both mice and birds) - Homeotic vertebrae transformations - Knockout of hox10 paralogues converts lumbar vertebrae into ribbed thoracic vertebrae - Knockout of hox11 converts sacral to lumbar - See slide 30 and 31 L17 for overview and flow chart **Lecture 18** Developmental disorders - Congenital malformations = any defects an individual is born with - 3 major pathways lead to disorders - Genetic alterations (mutations/chromosomal) - Environmental mechanisms (chemicals altering development signals, phenotypic changes) - Random events (back luck) Genetic alterations: mutation - Gene mutations/chromosome \# changes are caused by intrinsic genetic events - Pleiotropy = same gene producing different effects in different parts of body - Ex. kit gene - Expressed in blood stem cells, pigment stem cells, germ stem cells (promotes proliferation) - Mutation: syndrome -\> anemia (no rbcs), albinism (no pigment), sterility (no germ cells) - Relational pleiotropy = defective gene in one part of embryo causes defect in another part, even if gene not expressed in 2^nd^ tissue - MITF mutation in retina causing small eyes (lack of gene causes small retina which can't contain normal amount of vitreous humor so eye fails to enlarge) Genetic alterations: chromosome \# changes - High proportion (50-70%) of cleavage-stage embryos have wrong \# - Aneuploidy is a leading cause of miscarriages and infertility - Sex chromosome aneuploidies, autosomal aneuploidy - Often due to alterations in oocyte meiosis - Nondisjunction (failure to separate homologous chromosomes or sister chromatids) Sex chromosome aneuploidies - Turner syndrome 45 X0 - 1/3000 females - Webbed neck, short stature, delayed puberty - Nonfunctional ovaries, infertility - Congenital heart defect - Klinefelter syndrome 47 XXY - 1/500 males - Tall stature, small balls - Infertility, cognitive impairment - XYY counts as normal male Autosomal aneuploidy - Only 3 can produce live births - Trisomy 13, 18, 21 - Trisomy 21 (down syndrome) - Facial muscle changes - Heart & gut anomalies - Cognitive problems Genetic screening - Many abnormalities can be diagnosed before birth - Ultrasound scan (fetal growth patterns) - Maternal serum test (placental proteins, hormones) - AFP test - Cell-free fetal DNA test - Amniocentesis (amniotic fluid sample) - Chorionic villus sample - Percutaneous umbilical blood sampling Environmental disruption - Agents (usually chemicals) can cause deleterious phenotypic changes - Most that cause congenital anomalies = teratogens - Drugs, chemicals, viruses, heavy metals, maternal conditions, alcohol, endocrine disruptors - Teratology - Max. fetal susceptibility to teratogens = weeks 3-8 - Organogenesis Fetal alcohol spectrum disorder - Small brain - Avg iq 68 (intellectual disability - Behavioral abnormalities - A single occurrence of having a couple beers or glasses of wine can be enough - 1/1000 canadian children has it FASD mechanisms - Ethanol - Easily diffuses thru cell membranes - Binds to receptors - Alters neurotransmitter function - Acetaldehyde - Toxic byproduct of ethanol - Carcinogen - Short-lived - Contributed to physical and mental effects of alcohol Endocrine disruptors - Exogenous (come from outside body) chemicals that interfere with hormone functions - Mimic effect of natural hormone - Act as antagonist (inhibit hormone binding receptor) - Affect synthesis, elimination or transportation of a hormone - Prime organism to become more hormone sensitive Infertility - Usually - Man failure to produce or produce enough sperm - Woman failure to ovulate a mature oocyte - Also - Physical blockage of either male or female ducts - Immunological incompatibilities between sperm & egg or reproductive tract BPA - Chemical used to make hard, clear plastic - Water bottles, food containers, receipts, dental fillings - Exposure causes testis anatomy abnormalities - Disrupts spermatogenesis - Decreases sperm motility - Causes meiotic defects, chromosome abnormalities, aberrant follicle formation - Can be transgenerational - Behavioral changes up to 4 generations in mice - Dna methylation differences several too Chance alterations - Bad luck is always a factor - Dynamic developmental processes, specifications are prone to fluctuations by small chance in amounts of paracrine or transcription factors, receptors etc - Identical twins raised in same environment can still have different phenotype **Lecture 19** \~Leopard phrog organogenesis (L19-21)\~ Nervous system development - CNS = brain + spinal cord - PNS = everything else (nerves/neurons) - 86 billion neurons The ectoderm - Forms the nervous system - 1 part becomes the neural plate (neural tissue induced by prechordal plate & notochord during gastrulation) - Later forms CNS - Another part becomes epidermis (skin) - Between the 2 parts is the neural crest - Forms PNS and pigment cells - Neurulation = processes by which 3 ectodermal regions made distinct from each other - Right after gastrulation Neurulation principal models 4. Primary neurulation - Neural plate cells proliferate, invaginate into body, separate from surface ectoderm to form hollow tube 5. Secondary neurulation - Neural tube arises from aggregation of mesenchyme cells into solid chord that forms cavities to become hollow tube Transition zone - Between primary & secondary neurulation - They're divided spatially in many embryo species - Primary neurulation forms anterior - Secondary forms posterior portion of neural tube - Tube complete when 2 separate tubes join together by transition zone - Junctional neurulation Notochord - Mesodermal cells compose - Extends to base of head, supports embryo development - Only present in embryo in fish, mammal, reptile, bird - Vertebrae replace it during later stages Primary neurulation 1. Elongation a. Neural plate (dorsal ectoderm) will eventually become neural ectoderm b. Neural plate cells are elongated i. Cell divisions within neural plate are in anterior -\> posterior direction 2. Folding c. Medial Hinge Point MHP cells anchor plates to notochord d. Simultaneously the epidermal cells move toward dorsal midline ii. Both slightly arching 3. Elevation of neural folds e. Neural folds elevate as epidermis continues to approach midline f. Creation of furrow (Neural Groove) at midline iii. MHP cells changing shape 4. Convergence g. 2 dorsolateral hinge points DLHPs induced & direct rotation of cells around them h. Continued convergence pushes toward midline & causes folds (peaks; see diagram if necessary) to converge i. Important for invagination; folding inward, not outward 5. Closure j. Neural tube closes at paired folds at dorsal midline k. During fusion, cells at apex of folds delaminate and become neural crest cells iv. Open end at top of tube (above meeting folds) = anterior neuropore v. Open end at bottom of tube = posterior neuropore 6. Fusion/Separation l. Tube eventually forms closed cylinder that separates from ectoderm vi. Mediated by adhesion molecules (N- & E-cadherin) Epithelial cell bending - Rectangular box-like epithelial cells can't bend, need to adopt wedge shape at hinge location - Apical Constriction = contraction of actinomyosin complexes at apical border reducing size of apical half of cell compared to basal part - Nucleus moves to base - Cells packed tightly - Similar to drosophila midgut invagination Morphogen regulation of hinge points - Notochord induces neural plate cells to form MHP by secreting sonic hedgehog Shh - Noggin expressed in dorsal neural folds inhibits BMP causing DSHP formation in folds - BMP inhibits MHP and DLHP formation - Apical constriction occurs only in cells w/ low BMP and low Shh Defects of neural tube closure - Failure to close anterior neuropore -\> anencephaly-lethal - Forebrain remains in contact with amniotic fluid -\> degeneration - Failure to close posterior neuropore -\> spina bifida - Failure to close entire tube = craniorachischisis Secondary neurulation - Takes place in caudal/most posterior region of embryo - Mesenchymal cells from prospective mesoderm & ectoderm condense to form medullary chord beneath ectoderm - Central portion of medullary chord undergoes cavitation to form hollow spaces (lumens) - Lumens coalesce into single cavity Neural tube polarity - Dorsal -- ventral - Specification of axis is initiated by 2 major paracrine factors - Shh (originating from notochord & floor plate cells) - Differentiates into motor neurons & interneurons - TGF-B proteins (from dorsal ectoderm) - Specify dorsal cells - BMP4, BMP7, Dorsalin, Activin **Lecture 20** Human brain vesicles - 3 primary vesicles (eventually subdivide) - Forebrain (leads to olfactory lobes, hippocampus, cerebrum, optic vesicle, hormone thalamuses) - Midbrain (leads to midbrain -- temp., motor control, motivation, emotions) - Hindbrain (cerebellum, pons, medulla i.e. movement stuff) - Anterior -\> posterior patterning of hindbrain controlled by series of genes that include Hox complexes Neural crest cell derivation - Neural crest cells are multipotent - PNS -\> neurons, ganglia (sympathetic, parasympathetic, sensory), schwann cells, glial cells - Endocrine derivatives -\> adrenal medulla, calcitonin cells, carotid type 1 cells - Pigment cells -\> epidermal pigment - Facial cartilage and bones -\> (just that) - Connective tissue -\> corneal endothelium/stroma, tooth papillae, smooth muscle, dermis, adipose tissue, gland connective tissue, artery smooth muscle Neural crest regions 1. Cranial/cephalic a. Bones b. Cartilage c. Glia d. Pigment cells e. Connective tissues of face 2. Cardiac f. Muscular connective tissue i. Large arteries g. Septum (between aorta/pulmonary artery) 3. Trunk h. Sympathetic ganglia i. Pigment cells j. Adrenal medulla 4. Vagal/sacral k. Parasympathetic nerves, ganglia Neural crest cell lineage - Ex. mice - Single cell labelling - Cre-mediated recombination -- "confetti" (multicolor) - Progeny of each labelled cell showed multiple lineages - Original multipotent cell divides and progressively refines potentials - Precursors of glia, neurons, cartilage, bone, melanocytes Neural crest cell migration: delamination - Neural tube pushes crest cells out of dorsal region - Mediated by cadherins - E-cadherin (surface ectoderm, from BMP/Wnt) - N-cadherin (neural tube, from sox2/snail2) - Cadherin-6B (pre-migratory neural crest, from snail2) - Only in apical half of pre-migratory NC cells Neural crest cell migration: contact inhibition - Facilitates migration/dispersal by preventing backward migration over other cells - 2 migrating NC cells make contact -\> cytoskeleton change halt protrusive activity along surfaces -\> new protrusive extensions form away from contact point Collective migration - Kinda the opposite - Self-propelled cells exerting coordinated forces upon each other to migrate together - Co-attraction - Cells on leading edge produce protrusions to guide and drive the cluster's movement - External factors (ex. chemotaxis) NOT required Migration of Trunk NCCs - Ventral pathway - Early migrating cells - Cells differentiate into sensory (dorsal root) and autonomic neurons, adrenomedullary cells, schawnns, glials - Negative correlation between regions of Ephrin and NCC presence, so NCCs bind to no-ephrin regions - Involves sclerotome (somites that give rise to spine cartilage) - Dorsolateral pathway - late-migrating cells - Cells become melanocytes, melanin-pigment cells - Skin pathway (Eph signalling) - NC-derived melanoblasts upregulate Ephrin & endothelin receptor - NCC in melanocyte lineage migrates on ECM containing ephrin and endothelin - Same molecules that repelled glial lineage cells Migration of Cranial NCCs - Placode cells set up chemoattractant 'SDF1' gradient - Cranial NCCs have sdf1 receptor (cxcr4) and migrate toward placode - Once NCCs and placode meet, contact inhibition causes placode cells to migrate away - "chase and run" Neural connections - All neuronal development is biologically intentional - Sets up proper numbers of stuff for coordination of functional connections with targets - Connectivity permitted by neuronal body processes (axons and dendrites) - Driven by growth cone (locomotory apparatus at front of axon) Growth cone - Migrates, senses environment - Moves via elongation & contraction of filopodia microspikes - Fan out in front of cone, each spike 'samples' environment & returns info - Actin polymerization within spike -- treadmilling -\> \*poly.\* -\> retrograde flow - Axon navigation depends on guidance molecules in environment Guidance cues - 4 protein families - Ephrins, semaphorins, netrins, slits - Attractiveness or repulsiveness depends on - Type of cell receiving signal - Time when a cell receives the signal Axonal connection specificity - Pathway selection -\> axons travel along to particular region of embryo - Target selection -\> in area, axons bind to set of cells with which they can stably connect - Targets secrete chemotactic proteins (endothelins, neurotrophins) - Address selection -\> initial patterns refined such that axon binds to small subset of possible targets - Based on axon competition - When one motor neuron is active it suppresses other neurons' synapses Synapse formation - Where axon contacts target cell - Growth cone contacts myotube -\> neural agrin induces aCh receptors to cluster -\> synaptic basal lamina forms -\> eventually B2 laminin binds to act as 'stop' of signal (stop accumulation of neurotransmitters) - No specializations in either membrane Important summary: NCCs vs. Neural Plate & Neural Tube derivatives - NCCs - PNS - Adrenal medulla - Melanocytes - Facial cartilage - Dentine - NP/NT - CNS - Brain - Spinal cord - Motor neurons - Retina - Neural pituitary **Lecture 21** Limb formation (general) - Organogenesis with mesoderm - As opposed to ectoderm for nervous system stuff - Tetrapods -- vertebrates with 4 limbs - Limb axes - Proximal (close) vs. distal (far) ends - Anterior -- posterior (thumb to pinkie) - Dorsal -- ventral (knuckle to palm) Skeletal regions - Stylopod -- adjacent to body wall - Zeugopod (middle part) - Autopod -- distal part - Ex. humerus -\> radius/ulna -\> hand - Hox genes involved in limb region identity Limb field specification - Limb field = specific positions along body axis where limb forms - At-location mesoderm promotes formation, flank mesoderm actively represses - Position is constant with respect to level of hox gene expression along A-P axis - Forelimb buds found at most anterior part of hoxc6 region - Level with first thoracic vertebrae - Axial level hox genes regulate fgf8 & retinoic acid in mesoderm Forelimb or hindlimb? - Tbx4 -\> hindlimb - Tbx5 -\> forelimb - Determined by RA-FGF8 antagonism Limb buds - Bilateral bulges at limb field - Under-ectodermal accumulation of mesenchyme cells from Lateral Plate Mesoderm and somites - 3 domains - Progress zone - Highly proliferative mesenchyme - Zone of polarizing activity - Posterior region of progress zone - Apical ectodermal ridge - Thickening ectoderm at apex of developing bud (distal, outer part) Progress zone - FGF10 (downstream of tbx5 and 4 transcription factors) - Primary inducer - Drives cell proliferation Apical ectodermal ridge - If removed at any time during development, **distal** portion of limb development ceases (proximal fine) - FGF8 is major growth factor - Can be substituted for AER to re-induce limb growth PZ -- AER feedback loops - FGF8 in AER establishes loops between fgf10, fgf8, wnt3a for bud growth - Proximal-distal axis established by opposing RA gradients (from proximal/more internal flanks) and FGF/Wnt gradients (from distal AER) Anterior-posterior limb axes - Sonic hedgehog Shh expression - Based on amount of time shh expressed and concentration of shh protein the cells receive - Digit 1 -- shh independent - Digit 2 -- low shh - 3 -- brief shh expression, high shh concentration - 4 -- moderate expression, high concentration - 5 extended expression, high concentration - Mutation/improper location of shh = polydactyly (most was cat w/ 28 digits) Limb dorsal-ventral axis specification - Dorsal cell fates induced by Wnt7a (thru Lmx1b) - Ventral limb patterning regulated via BMP (thru engrailed 1 "En1") Termination of limb development - Shh -\> grem1 -\> fgf feedback loop drives bud outgrowth - Increased fgf concentrations eventually (go back and) inhibit grem1 and result in grem1 distancing from signaling centers (terminates outgrowth) **Lecture 22** Regeneration - Reactivation of developmental mechanisms in postembryonic life - Restoration of missing/damaged tissue - Varying degree of what can be regenerated between species - 4 modes (stem-cell mediated, epimorphosis, morphallaxis, compensatory) Regeneration steps 1\. prior to injury, cells/tissues need to have an idea of their own identity \- morphological memory map 2\. after injury, cells/tissues recognize change and that replacement needs to be made 3\. rapid wound-closing response 4\. commence regenerative response \- same mechanisms used in embryonic development \- cell proliferation, tissue growth, repatterning of cells 5\. regeneration ends once proper proportions met Stem-cell mediated regeneration - New cells routinely produced to replace ones that die - Ex. hair follicular stem cells, bone marrow hematopoietic stem cells Epimorphosis regeneration - Adult structures undergo dedifferentiation to form undifferentiated mass of cells - Blastema - Then redifferentiate into new structure - Ex. amphibian limb Morphallaxis regeneration - Re-patterning of existing tissue w/ little to no growth - Rather cell death and change in cell type - Transdifferentiation (into different cell fate) - Results in rescaling of whole organism (as well as regenerating of missing part) - i.e. it gets smaller - ex. hydra Compensatory regeneration - differentiated cells divide but maintain differentiated functions - ex. mammalian liver regeneration vs. development - similarities - utilize mechanisms of embryogenesis - require context-specific adaptations - differences - immune response - induced reprogramming - system integration - size recognition & termination whole body animal regeneration - hydra - constantly undergoing mitosis & being displaced due to extremities of column - routine replacement by 3 types of stem cells - endodermal and ectodermal (unipotent) - hydra have no mesoderm - interstitial (multipotent) - within ectodermal layer (generates neurons, secretory cells, gametes, nematocytes) - if cells separated and reaggregated, a new hydra will still form routine cell replacement organization - head activation gradient (highest at hypostome) - hypostome = organizer - when transplanted, it induces host tissue to form 2^nd^ body axis - produces activation signal - only self-differentiating region of hydra - produces head inhibition signal that suppresses forming of new organizing centers - major head inducer is set of Wnt proteins acting w/ B-catenin pathway - foot activation gradient (highest at basal disc) morphallaxis in hydra - in event of decapitation, if cut is made below hypostome - wnt3 upregulated in epithelial cells near cut - remodeling of existing cells to form head - no proliferation, just transdifferentiation epimorphic regen. in Hydra - if cut at midsection - interstitial stem cell derivatives undergo apoptosis immediately below cut site - produce burst of Wnt3 before dying which activates B-catenin in interstitial stem cells below - surge of B-catenin causes wave of proliferation in interstitial stem cells + remodeling in epithelial cells salamander limb regeneration - blood and immune cells flood amputation & form clot - activation of stem/progenitor cell proliferation - epidermal cell migration to form wound epidermis - epidermis thickens into apical epidermal cap - AEC signals progenitor cells to develop the regeneration blastema - From differentiated cells that are dedifferentiated - Continued proliferation of blastema -\> limb outgrowth Regeneration blastema - Genes expressed in differentiated tissues are downregulated while genes associated w/ proliferating Progress Zone mesenchyme are expressed - Dedifferentiated blastema cells paired with activated stem cells continue to proliferate and eventually redifferentiate to form new limb - AEC acts similarly to AER in normal limb development - Blastema cells retain specification even when they dedifferentiate - Blastema = assortment of restricted progenitor cells Blastema growth - Depends on nerves and AEC - AEC - Stimulates growth by secreting FGF8 - Nerves - Both sensory and motor axons innervate blastema - Sensory axons contact AEC - Motor axons terminate in blastema mesenchyme - if limb is denervated before amputation, no regeneration occurs Liver compensatory regeneration - proliferation & enlargement of existing tissue - no dedifferentiation - division of differentiated cells to recover structure & function - mature cell rejoins cell cycle and proliferates - hepatocytes, duct cells, fat cells, endothelial cells, hepatic macrophages) - quiescent hepatic progenitor cells activate **Lecture 23** Development and the environment - metamorphosis - developmental changes where immature organism given new (usually sexually mature) form - hormone triggered - adaptation to new environment/lifestyle - phenotypic plasticity - ability of organism to alter phenotype/behavior in response to environmental inputs - when difference occurs in embryonic/larval stages, ability to change phenotype = developmental plasticity - 2 types (reaction norm and polyphenism) Reaction norm - Continuous range of potential phenotypes - Genome-environment interaction determines most adaptive phenotype - Ex. plants and light/water/nutrient levels Polyphenism - Discontinuous (either/or) phenotypes - Still induced by environment - Ex. bees - Diet induced - Royalactin (protein-lipid mix) fed to larvae that will become queens induces ovaries - Royal jelly - Activation of yolk proteins for egg production - Ex. ant - Also larval feeding differences - Ex. caterpillar - Eat young oak leaves -\> develop cuticle that resembles oak flowers -\> spring caterpillar - Eat mature oak leaf -\> develop 'young-twig' like cuticle -\> summer caterpillar Phenotype interactions - Anatomical phenotype differences can induce behavioral changes - Ex. horned vs. hornless male dung beetles - Horned males find mate, have egg, guard egg and mate - Hornless male will burrow around guarding horned male to get to mate Dna methylation and diet - Agouti gene gives mice yellow fur - Affects lipid metabolism -- mice become fatter - If agouti promoter methylated, no transcription - Dark fur, metabolism normal - Offspring to mothers given folate = sleek and dark Predator induced polyphenism - Red eyed tree frog - Embryos detect vibrations of specific frequency & interval and hatch prematurely - Ex. snake slithering - Hatched embryos fall into pond and transform into free-swimming larvae - Will be worse off development-wise but at least they're alive Temperature and development - Le moth - 'Distal-less protein' transcription factor determines size of eyespot - 20E hormone sustains & expands distal-less expression - 20E production elevated by higher temperature Stress and development - Spadefoot tadpole - Live in desert ponds which may dry up at any time or may last ages depending on rain - If pond dries, wider mouth and jaw = carnivore - If pond stays, less powerful jaw and smaller = omnivore Symbioses - Symbiosis = any close association between organisms of different species - Host and symbiont - Parasitism - Mutualism - Symbionts can be critical in organism development - Humans have indigenous microbiota all over and in bodies, everywhere except blood - Holobiont = combination of host and persistent symbionts Developmental symbiosis - Vertical transmission - Transfer of symbionts from one generation to next - Maternally (ex. via eggs) - Ex. Wolbachia bacteria in drosophila oocyte via maternal microtubules - Wolbachia-infected flies make 4x as many eggs - Provide virus resistance - Horizontal transmission - Host is born free of symbionts but becomes infected - Environment or other species member - Infection with Wolbachia in male pill bugs transforms them into egg-producing females - Ex.2: Squid + vibrio fischeri bacterium = building of light organ for bioluminescence Obligate developmental mutualism - Neither species could survive individually - Ex. wasp - Remove Wolbachia from female wasps and ovaries only produce 36 oocytes - As opposed to 228 - Ovaries undergo apoptosis and cease egg production Bacteria and gut development - Zebrafish - Proliferation of intestinal stem cells - Mice - Important in nutrient absorption - Helps blood vessel formation - Fortifies intestine ECM Gut microbes and capillary + pancreas development - Zebrafish - Aeromonas bacteria make BefA protein which increases insulin-secreting beta cells in embryo pancreas - Mice - Gut bacteria inoculation increases capillary network - Further increased by bacteroides thetaiotaomicron Gut microbes and pregnant mice - Send essential metabolites to fetus - Circulate thru mother's blood, enter fetus via placenta - Short chain fatty acids bind to fetal intestine, pancreas, SNS, and activate genes for metabolism - TMAV & hippuric acid promote maturation of fetal brain neurons in auditory region Symbiotic bacteria and postnatal brain development - Mammalian brain - In situ hybridization of Egr1 mRNA in frontal cortex - Egr1 expression usually depends on symbiotic microbes - Correlates with behavioral differences Gut microbiome during pregnancy - Changes dramatically - Associated with weight gain, insulin insensitivity - Bacteria from early pregnant women transplanted into mice - Normal mice phenotype - Bacteria from late pregnancy - Induced pregnancy-like metabolism - Symptoms like weight gain, insulin resistance **Lecture 24** Evolutionary Developmental biology - Views evolution as result of changes in development - Studies mechanisms - Produces model of evolution that integrates developmental + population genetics to explain biodiversity - Analyzes environmental change effects Genetic modularity - Preconditions for evolution - 1\. Multiple enhancers for each gene - 2\. Each enhancer region can have multiple TF binding sites - Ex. pelvic spines in sticklebacks - Modularity = recruitment of modules into new phenotype - Elytra = hardened forewings in beetles - Apterous protein activates exoskeleton gene in forewing & suppresses in hindwing - New wing emerges from recruitment of genetic module for exoskeleton development (into dorsal forewing development) Molecular parsimony - Development within all lineages uses same types of molecules (despite enormous lineage to lineage differences) - BMPs = dorsal-ventral axis in animal kingdom - Wnt + Hox genes = anterior-posterior axis in all bilaterians - Pax6 = light sensing organs in all seeing-animals Gene families - Created via duplication of original gene, subsequent mutation of original duplicates - Hox genes, globin genes, collagen genes, paracrine factor families (Wnt genes) Expansion of human cerebral cortex - SRGAP2 in mammalian brain decreases length & density of dendritic processes - SRGAP2 duplicated 3 times in humans - Eventually truncated version SRGAP2C inhibits activity of normal SRGAP2 made from complete genes - As a result, dendrites are larger and have more connections in human brain than non-human mammals Chimpanzee-human similarity? - Share 98-99% of dna - Protein encoding nearly identical - Coding genes in all mammals similar - Mice & humans share \~25000 genes - Similar genes fulfil similar functions in different organisms - Important differences are when, where, how much genes are activated Evolutionary mechanisms at gene expression level - Heterotopy - Change in location - Heterochrony - Change in time - Heterometry - Change in amount - Heterotypy - Change in type Heterotopy - Ex. autopods of chick vs. duck - Chick has webbing in embryo but ends up with (almost) toes - Duck foot expresses BMP4 inhibitory protein Gremlin in webbing and retains webbed feet - Bmp4 induces apoptosis - Turtle shell - Rib cells migrate laterally into dermis instead of forming ribcage - Attracted by fgf10 - Ribs of other vertebrates do not enter dermis because fgf10 signals blocked in dermis Heterochrony - Birds arose thru heterochronic development of dinosaur skulls - Dolphins - Evolved from terrestrial animals - Longer duration of fgf signalling from forelimb AER - Longer fingers - Blocking of BMP activity - Webbing (can't see said fingers) - Hindlimb ZPA ceased early in development - AER can't be sustained without ZPA, hindlimb doesn't develop Heterometry - Differences in beak morphology - Changes in Bmp4 expression during beak development - Heterochronic and heterometric - Expressed earlier and at higher levels in ground finches - Broader, deeper beaks to crack open seeds Heterotypy - Affects coding sequence - Insects have 6 legs as opposed to other arthropods w/ many more - Legs only found in 3 thoracic segments, but not abdominal - Relationship between Ubx protein and Distal-less gene polyalanine region represses distal-less transcription in abdominal segments Evolution & developmental symbiosis - Selectable variation - ex. pea aphid (numerous symbionts) - Buchnera aphidicola can provide higher fecundity and greater heat tolerance - Rickettsiella alleles can alter aphid's color - Hamiltonella defensa can provide proteins that defend host aphid against wasps - Reproductive isolation - Biological or geographical barriers preventing members of same species making offspring - Results in necessary formation of new species - Often cytoplasmic incompatibility (wasp egg bacteria don't match if contain incompatible symbionts) - Symbiogenesis = new organisms form by acquiring symbionts Symbiogenesis - Single celled organism choanoflagellates were likely common ancestor of all animals - Proliferates asexually in filtered seawater - Form rosettes (cells connect by ecm & cytoplasm) if cultured w/ bacterium algoriphagus machipongonensis - Beginning of multicellularity that led to animals Symbiogenesis and the evolution of placental mammals - Retroviruses critical in uterus & placenta formation (allow development to occur inside mother) - RNA based viruses that integrate into host genome, heritable - \~8% of human genome is retroviral inserts - SYNCYTIN-1 retrovirally derived gene encoded a protein that allowed virus to enter animal cells - Has since been repurposed to fuse trophoblast cells & form syncytiotrophoblast - Placenta layer that separates maternal/fetal blood vessels Developmental constraints - Physical - Blood can't circulate to a rotating organ, thus vertebrate with wheeled appendages can't exist - Morphogenetic - Middle digit never shorter than surrounding digits - Zeugopod never more proximal than stylopod - Due to rates of morphogen diffusion, inhibitors - Pleiotropy - Recall "one gene having different functions in different cells" - Ex. Hox genes specify vertebrae type, stem cell proliferation - However hox gene changes might facilitate skeleton vertebrae type/\# changes (can be lethal) or mis-regulate stem cells and lead to cancer **Nobel Prize Winners** - 2024 - Ambros, ruvkun -- microRNA and post-transcriptional gene regulation - 2022 - Svante pääbo -- Genomes of extinct hominins and human evolution - 2012 - Gurdon, Yamanaka -- mature cells can be reprogrammed to become pluripotent - 2010 - Robert g edwards -- development of in-vitro fertilization - 2007 - Capecchi, evans, smithies -- principles for introducing specific gene modifications in mice by use of embryonic stem cells - 2002 - Brenner, Horvitz, Sulston -- genetic regulation of organ development and apoptosis - 2000 - Carlsson, greengard, kandel -- signal transduction in nervous system - 1995 - Lewis, nüsslein-volhard, wieschaus -- genetic control of early embryonic development - 1935 - Hans spermann -- organizer effect in embryonic development