Sept 8 (DOC)

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					Sept 8: Preneurological development Morula: 12-16 Blastomeres formed from cleavage Blastula: blastocoel cavity on animal hemisphere Gastrulation: ecto-, endo-, meso- derm form. Nervous tissue forms from ecto. Mesoderm forms most of body. Endoderm forms respiratory epithelial lining, digestive organs. Neurulation Neural Tube – becomes cells of CNS. becomes ventricular system of brain, central canal of spinal cord Neural Tube layers: ependymal layer (innermost layer, the ventricular zone) Mantle layer (intermediate, becomes neurons/macroglia of CNS) Marginal layer (white matter, axons of mantle layer cells) Neural Crest – becomes PNS (sensory cells, Schwann, paraganglia, adrenal medulla, mealnocytes (non-neural), enteroendocrine (non-neural)) In mammals, embryonic disk (between amniotic cavity and tolk sac) involutes along primitive streak. Goes from border to center. Then streak regresses caudally. Ventral midline mesoderm becomes notochordal plate. Notochord formation begins at end of primitive streak and extends rostrally to prechordal plate (location of oral cavity). Hensen’s node (mammals) analogous to the Speeman organizer. Both can induce neural tissue in any host (amphibian or mammal). Notochord causes induction of neural plate from ectoderm. Neural plate midline is neural groove. Neural plate edges are neural folds. Neural folds curl up to form the neural tube. BMP is highest laterally and lowest medially. Forms gradient for neural tube folding. Notochord forms from mesoderm. Somites are distinct bone, muscle, tissue segments; the bone segments become vertebrae. Located around the midline of the embryo. Tube closure begins at somites 4-6. Rostral of somite 4 = brain. Caudal of somite 4 = spinal cord. Spina bifida, failure of caudal closure. Anencephaly, failure of closure at rostral end (lethal). Neuroblasts migrate along radial glial cells to mantle layer to become neurons. Sulcus Limitans groove divides spinal cord into dorsal/ventral

Dorsal region/alar plate, afferent functions (to CNS) Ventral region/basal plate, efferent functions (to periphery) Sept 13: Gastrulation occurs on dorsal side. Bottle cells form and invaginate to form the blastopore. Epithelial cells become bottle cells via cytoskeleton changes and actin. Actin tightens one end and there is elongation along direction of microtubules. Bottle cell movement because of loss of E-cadherin. Speeman organizer, piece of dorsal blastopore lip that organizes ingressing tissue. Can induce a new embryonic axis/neural tissues if transplanted to ventral side. Dorsal (but not ventral) animal cap required for proper brain formation. Heterochronic transplants: They work in early gastrula stage (cells can become other types of tissue), but late gastrula cells are committed to fate. Ectoderm destined to become neural tissue, is normally hindered by inhibitory molecules. Inductors inhibit the inhibition. Transforming growth factor beta (TGF betas – polypeptide growth factors): Activin: mesoderm induction, reproductive regulation BMPs: induction of chondrogenesis Inhibin: reproductive regulation Vg-1 mesoderm induction BMPs 4 and 7 are the inhibitory signal for neuralization. TGF receptors are transmembrane receptors, ligands/kinases in close proximity phospohorylate each other. Dimerization – intracellular domains must have a partner to phosphorylate. A mutant TFG receptor blocks mesoderm formation. Speeman organizer blocks BMP effects or antagonizes TGF beta receptors. Peptides expressed by Speeman organizer at gastrula stage that block BMP signaling: noggin, follistatin, and chordin. They have some functional redundancy but double

knockout inhibits brain development. They induce primitive neural tissue rather than mature neurons. Each has different affinity. Follistatin binds activin with high affinity. BMP forms gradient for ectoderm tissue: low = neural plate, medium = neural crest, high = epidermis. Formation of anterior-posterior axis: Early tissues going ingressing through blastopore become forebrain structures; later tissues ingressing become more posterior structures. The signaling for establishing polarity is radial (Einsteck Experiment – transplanting dorsal mesoderm from gastrula to blasocoel, anterior regions induce brain, posterior regions induce spinal cord, Holtfreter Experiment – exogastrulation causes no neural induction) Neural inducers create anterior markers (NCAM, Otx2, Bf1) initially. Transplantation: Early organizer induces head structures, late organizer induces late structures Posteriorization accomplished through increased contact with a constant signal or timing of posteriorization signal with involuting mesoderm Posteriorization signals include Retinoic acid, Wnts, and FGFs. Retinoic acid binding its receptor causes posteriorization. FGF 4 – produces posterior markers like otx2 (midbrain) Combined with noggin in produces anterior and posterior markers (Bf1, Hoxb3) WNT binds frizzled receptor to produce posteriorization. Organizer expresses soluble form of frizzled receptor that competes for WNTs. Sept 15 Anteriorization signals: Antivin- inhibits activin (TGF-beta), which can cause conversion of forebrain to posterior region in large doses Cerberus – binds BMP and WNTs, can cause multiple heads DKK – inhibits WNT receptor, can induce full head structure

Frzb – soluble form of WNT receptor (so inhibitory), expressed in prechordal plate mesoderm Factors that promote neutralization via inhibition of BMP: noggin, follistatin, chordin, and Cerberus Factors that inhibit WNT receptor to cause anteriorization: Cerberus, dkk. Frzb Anterior Visceral Endoderm in mice induces anterior structures, expresses the factors Otx2 and Lim1. Lim 1 needed for a mouse head to develop. Otx2 for both anterior competence/induction as well as later maintenance (forebrain formation and proper development). May be regulated by planar signaling from Row 1 or ANR cells. Neuromeres – subdivision of the neural tube. Bf-1: factor in telencephalon needed for proper development of this region Planar signaling is used by centers at rostral end of neural plate to regionalize the telencephalon. Row 1 cells – ectodermal cell which produces a planar signal necessary for telecenphalic marker expression. (zebrafish, chicks) Anterior Nerual Ridge – produces FGF 8 which enhances telecenphalic marker expression (Bf-1). ANR transplant can induce telencelphalon. Both row 1/ANR cells express FGF-8, but it is only necessary in ANR animals for proper development. Neuralation stages: 1) 3 vesicle = prosencephalon, mesencephalon, rhomencephalon 2) 5 vesicle = telencephalon , mesencephalon, metencephalon Diencephalon mylencephalon 3) Cephalic flexure 4) Rotation of cerebral hemispheres – anterior regions become ventral, anterior tip of neural plate becomes hypothalamus. Dorsal Telencephalon rotates, portions become ventral and form basal ganglia.

Developmental control genes become more specified for each region. Telencephalon – Cortex and Striatum (connects cortex to thalamus) Dorsal is the cortex, genes are Emx1, Emx 2, Pax 6. Gradient of emx2 and pax 6 responsible for patterning. Emx 1 and Emx 2 are controlled via Gli3 expression. Ventral is the striatum, genes are Dlx and Nkx Gradients of otx 2 and otx 1 and emx 1 and emx 2 may control regionalization of telencephalon, diencephalon, and mesencephalon. Otx2 and emx2 specify head segments and neuromeres. Emx2 expression limited to ventricular zone. Ventral zone of telencephalon – 2 regions Dorsolateral portion (lateral ganglionic eminence) – striatum (caudate + putamen) Ventromedial portion (medial ganglionic eminence) – globus pallidus (Nkx 2.1) expressed in hur Without Nkx 2.1 you’ll see increased striatum at the cost of palladial tissue. Boundary between dorsal and ventral telencephalon is established by adhesion molecules (dorsal cells express R-cadherin, Lewis-x). Expression of these adhesion molecules controlled by Pax 6. Community effect for signaling, a single transplanted neuron (dorsal to ventral) will be assimilated but a tissue transplant will not be converted. Migration takes place because of Dlx1 and Dlx2, allow temporary movements, these neurons become cortical inhibitory neurons Sept 20: Diencephalon regionalization (contains the thalamus): Anterior forms parencehphalon (divided by zli, which expresses SHH) -Ventral thalamus (anterior) expresses Dlx1 and Dlx2 -Dorsal thalamus (posterior) expresses Gbx2 and Wnt3 Posterior forms synencephalon (divided by posterior commissure) -Future anterior synencephalon (anterior) -Pretectum (posterior) Here Diencephalic Pax 6 represses mesencephalic engrailed and Pax 2 to form barrier between diencephalons/mesencephalon Mesencephalon regionalization

Dorsal is the tectum. Includes superior and inferior colliculi (visual and audio input, respectively). Ventral is the substantia nigra and the ventral tegmental area, also called tegmentum (dopamingeric neurons for both SN and VTA) Tegmentum contains SN, reticular formation, periaqueductal gray, red nucleus Anterior Optic tectum gets input from temporal retina Posterior Optic tectum gets input from nasal retina Temporal retina axons have Eph A3 receptors. Tectum gradient of Eph A2 Ligands: high in the posterior, low in the anterior. Temporal axons stopped by low concentrations in the anterior because they have the receptors in high concetrations. Nasal axons don’t have as high of concentration so go farther back. Isthumus connects midbrain vesicle to hindbrain, does signaling for posterior-anterior orientation of midbrain/rostral hindbrain. Development of isthmus is controlled by Pax 2, Pax5, and Pax 8. Anterior of Isthumus otx2 is expressed. Posterior of isthumus gbx2 is expressed. Overexpression of one shifts isthmus expression the other way. Isthmus also expresses En1 and en2 (engrailed genes) with strongest expression at isthumus, spreading out rostrally to mesencephalon and caudally to first rhombomere (hindbrain). En1 expression increased before En2 expression and downregulated prior to en2 downregulation. En1 needed for regionalization of entire region of expression, en2 just for cerebellum. En expression control the polarity of the tectum (and thus where retinal axons go). Isthmus controls en expression, and transplanted isthmus tissue can induce optic tectum. Pax 2 binding sites exist on the En2 enhancer region, a lack of Pax 2 causes a lack of En2 and a lack of isthmic development. WNT 1 (at isthmus) necessary for maintenance of EN expression but not induction. FGF 8 expression is posterior to WNT 1 expression, increases expression of itself, WNT 1, and En2. Decreases expression of Otx2. If otx2 is expressed at the hindbrain, it represses Gbx2, the new boundary forms an isthmus more caudally. Otx 2 also increases Pax 2, which triggers En2 and FGF 8; FGF 8 induces a tectum in the cerebellar region. Response to signal depends on tissue competence. An isthmus transplanted to the diencephalons will produce a tectum, while an isthmus in the hindbrain will produce a cerebellum.

Dorsal ventral patterning of midbrain depends on SHH. Alar (dorsal) plate forms tectum. Basal (ventral) plate forms tegmentum. Increased SHH furthers the tegmentum at expense of tectum. SHH decreases isthmic genes (En1 and En2, , fgf 8, pax2, pax5) while increasing ventral genes Isl1, Lim1, and Lim2.

Sept 22: Rhombencephalon becomes metencephalon (pons/cerebellum) as well as mylencephalon Exhibits both dosal/ventral as well as anterior/posterior patterning. Dorsal has sensory interneurons and relay neurons Ventral has motor neurons Anterior/posterior patterning – development of rhombomere segments, cerebellum is rhombomere 1. Each has distinct neurons. Rhombomeres have 2 segment repeat pattern. R2, R4, R6 – branchial motor neurons. Boundaries established by differential cell adhesion and eph receptor ligand system. R3 and R5 express the receptors. R2, R4, and R6 express the ligand. Drosophila HOM-C genes as well as vertebrate Hox genes control rhombomere development. Exhibit collinearity (Collinearity – position of genes on chromosome mirrors expression on the anterior-posterior axis). Rhombomere 4 expresses Hoxb1 exclusively, failure to express it causes r4 to look like r2. Control of hox genes for the rhombomeres Retinoic Acid – RA can bind upstream of Hoxb1 genes at an enhancer site; the binding site is called RARE. If too much RA binds (which can happen if there’s too much vitamin A), it can cause too much Hoxb1 to be expressed, causing too much r4 to form. Krox 20 controls Eph A4 receptor in r3 and r5. No krox 20 = no r3 and r5.

FGF8 expression by isthmus inhibits expression of hox genes, so that r1 doesn’t express these genes and forms the cerebellum instead. Each rhombomere past r1 expressed a unique combination of hox genes to specify it. Dosal/ventral Anterior/Posterior coordination: CVAN neuron example, r4 basal (ventral) neurons are destined to become CVAN neurons. If an r4 dorsal neuron (alar plate) is transplanted to the ventral area of r2, it will become a ventral neuron. This is because it has 1) the r4 cell fate already determined and 2) the signal from SHH by being transplanted to a ventral region. Notochord induces floor plate (in adjacent cells) and motor neurons (at a distance) for the spinal cord. You can transplant it to induce another floorplate/motor neurons in a dorsal region. SHH is the signaling molecule. High concentration of SHH = floor plate, low concentration (5x less) = motor neurons. Most SHH-N (the inducing form of SHH) remain on the notochord cells to induce floor plate cells in adjacents, a smaller diffusible portion helps to form SHH-N triggers ventral marker expression: HNF3beta (floor plate cells), Nkx 2.1, 2.2, (ventrolateral neural tube cells), Is1 (motor neurons) Is1 is needed for proper motor neuron development. No Is1 also negatively affects neurons that express En1 (through indirect induction). Dorsal marker expression in spinal cord depends on BMP 2 and BMP 4 mediated planar signaling from ectoderm of neural plate stage. Directly affects roof plate and neural crest development. Lateral neural plate has highest levels of BMPs, these edges fold up to close neural tube/form roof plate. Pax 3 (needed for caudal neural tube closure and Dorsal root ganglion cells) and Pax 6 (needed for eyes, olfactory epithelium) are expressed in dorsal half and dorsal 2/3 respectively of neural tube. SHH-N blocks expression of dorsal markers like Pax 3, Pax 6, Msx1. Notochord blocks dorsal markers like Lmx1 (roof plate), Slug (neural crest), and LH2 (dorsal interneurons). SHH expression is transferred to floor plate and BMP expression is transferred to roof plate during development. Sept 27

SHH is expressed by ventral forebrain cells since notochord doesn’t run this far up. High concentrations of Is1 via SHH induce medial ganglionic eminence (globus pallidus), low concentrations induce lateral ganglionic eminence (striatum) Coordination of anterior-posterior patterning and ventral/dorsal patterning: anteriorposterior position determines what type of neuron SHH induces at a particular ventral point (responding neuron must have competence). In drosophila, all cells begin as multipotent precursors, fate determined by series of binary choices. Clusters of proneural cells compete via lateral specification (inhibition of neighbors) to become the neuron. Notch/delta signaling – notch is the receptor, when bound by delta, it reduces proneural gene expression. When bound, also reduces that cell’s expression of delta, which means that it inhibits its neighbors less. Least inhibited cells become CNS neuroblasts and migrate into the middle of the cell, undergo asymmetric divisions. Second round of inhibition follows, these cells become PNS precursors. Cells that don’t become neural become epidermal. Formation of the dorsal-ventral axis. From my first midterm: Early in development, there is a factor called decapentaplegic which has the function of “dorsalizing” the embryo. There is also a factor called sog (short gastrulation) which inhibits the action of dpp. Spatzle, a factor expressed very early in development, binds a receptor called toll, which is distributed equally dorsoventrally. The spatzle toll complex degrades the protein cactus, which up to this time has been holding another protein called dorsal hostage via the mechanism of a bound complex. Dorsal must enter the nucleus to have its proper effect and is free to do so after being freed from cactus. Dorsal enters ventrally and subsequently activates sog. Sog inhibits dpp in a gradient with the highest inhibiton occurring ventrally where dorsal did its work. The highest activity of dpp occurs dorsall, and thus dorsal/ventral patterning occurs. Dpp binds TGF-beta receptors while doing its work. Results in an intracellular cascade dependent on other proteins like mad (mothers against dpp) Prepatterning genes like Lozenge, wingless, and Iroquois complex genes determines expression of proneural genes. Proneural genes are basic helix-loop-helix transcription factors. Include: AS-C: needed for external sensory organs and CNS development Atonal: needed chordotonal organs, internal stretch receptors Amos: needed for solo MD neurons and olfactory cells

No AS-C or Atonal = Most PNS gone None of the 3 = All PNS gone Products of proneural genes function as heterodimes. More protein factors that interact with basic helix-loop-helix transcription factors: Pro-regulators: Daughterless – dimerizes with other proneural genes Negative regulators: Extromacrochaetae, Hairy Mechanism for inhibition of neurogenesis: Kuzabanian cleaves delta from the notch receptor to allow notch activation. Notch activation frees notch intracellular domain (NICD), which binds suppressor of hairless, which then can travel to nucleus to activate transcription of enhancer of split complex. Products of enhancer of Spl inhibit neurogenesis. In drosophila, sensory organ precursor cells undergo 2 divisions to form 2 non-neural cells and 2 neural cells (provided notch is activated). Asymmetric divisions aid the cytoplasmic localization of proteins. Asymmetric divison: -Mitotic spindle oriented perpendicular to neuroepithelium Spindle forms perpendicular to cells. Allows proteins to determine daughter cell fate. - Numb protein specifies neural fate for a neuroblast. It interferes with the Notch signaling pathway. Notch normally activates tramtrack gene to inhibit neurogenesis. Notch interferes with this pathway. - Partner of numb anchors numb to the basal membrane pole. -Prospero, limits proliferation of ganglion mother cells via repression of cell cycle genes, must enter nucleus to have effect. -Miranda, anchors Prospero to basal membrane pole. -Inscuteable protein acts upstream of Prospero/Numb to orient mitotic spindle perpendicularly. -Bazooka anchors inscuteable to apical membrane. With no lateral specification, all neural plate cells become neural and the whole plate invaginates. Sept 29: Lateral inhibition in vertebrates:

Homologues have been found, similar notch-delta signaling. Inhibited cells become glial rather than epidermal. Neurogenin is the proneural gene inhibited by delta expression. Neurogenin leads to Neuro D. Homologues include Notch 1 and m-Numb (which binds the notch intracellular domain). Asymmetric divisions still used. Vertebrate homologues of Drosophila bHLH genes: Homologues of Atonal: Neurogenin family – act early in development, before delta business. area of expression defines 3 regions of neurogenesis on the neural plate. If you get rid of Delta1 (homologue of delta) you get neurons in-between these 3 regions. Lateral region becomes sensory neurons, medial region interneurons, and medial region motor neurons. Three regions correspond to neurogenin expression. NeruoD – can cause ectoderm cells to form neurons. Expressed transiently in many neurons, expressed by differentiated CNS neurons. Math1 - expressed in ventricular zone of dorsal neurotube and cranial sensory ganglia as well as rhombic lip of hindbrain, forms granule cells of germinal cerebellar layer AS-C homologues: Mash1 – required for early neuron development including olfactory, telecenphalic, and autonomic Xash3 – overexpression grows neural plate at expense of epidermis Hairy homologue: Hes 1 and 5 – negative regulators of neuronal development Expression of proneural genes in complimentary regions: Dorsal telecenphalic cells express proneural gene Ngn2 Ventral telecenphalic cells express proneural gene Mash1 Without proper expression, ventral markers appear dorsally, etc. Chordin and noggin activate the Sox family of transcription factors @ time of neural induction (from blastula stage) SoxD causes expression of neurogenin / anterior neural differentiation Zic genes generally upregulate expression of neurogenins, but Zic2 inhibits development of neurons in regions surrounding 3 stripes.

In neurogenesis, early cell divisions are symmetric. Beginning of neurogenesis is marked by asymmetric cell division in which one cell remains in the ventricular zone while the other differentiates/migrates. Division and differentiation can be influenced by glial proteins, neurotransmitters, FGFs, SHH, WNT1 Motor neurons – large cell bodies on ventral aspect of neural tube, growth cones leave neural tube. Influenced by SHH, if SHH concentration gets too high, won’t develop into motor neurons. SHH induces expression of MNR2 in post-mitotic neurons, MNR2 has positive feedback loop. Leads to increased expression of Isl1, Isl2. Isl1 and 2 produce motor neuron differentiation. Subtypes of motor neurons are specified by a specific set of LIM-homeobox genes. Different motor neurons have different morphologies and different peripheral targets. More caudal motor neurons express the Lhx3 and 4 exit ventrally. More rostral motor neurons exit dorsally. Three motor neurons innervate each muscle segment: rostral, mid, and caudal. Fate of neuron depends on position relative to other neurons. Ant/Post variation in motor neurons is a function of Hox genes, which pattern brachial vs thoracic regions Mesoderm also helps signal motor neurons via retinoic acid and other molecules Each muscle segment innervated by motor and sensory neurons, these sensory and motor neurons express same ETS genes. Neurons must coordinate Neurotransmitter elements including NT synthetic enzymes, NT storage mechanism, and transporter proteins to coordinate expression of NT apparatus. Cholinergic neurons control these aspects under same cis-acting DNA regulatory element. The express of NT apparatus must match the target cell recognition molecules.