Comparative Neurodevelopment and Neural Stem Cells PDF - Neurobiology Master Degree

Here is the converted text from the images you sent, structured in Markdown format: # COMPARATIVE NEURODEVELOPMENT AND NEURAL STEM CELLS 1st year of Neurobiology Master Degree ## NEURAL INDUCTION Metazoans are animals and their tissue is in layers. Layers are important for differentiation. The l...

Here is the converted text from the images you sent, structured in Markdown format: # COMPARATIVE NEURODEVELOPMENT AND NEURAL STEM CELLS 1st year of Neurobiology Master Degree ## NEURAL INDUCTION Metazoans are animals and their tissue is in layers. Layers are important for differentiation. The layers give rise to the various organs and tissues, including the nervous system, and they are generated from the egg cell through a series of cell divisions and rearrangements. Once the three primary germ layers are established the development of the nervous system begins. The development does not take place in a vacuum. * Zygote -> thanks to cleavage we arrive at the eight-cell stage * blastula: ball of cells hollow inside (blastocele) * gastrulation: invagination of cells on one side, migrating into the hollow part. * gastrula, with blastocoel, endoderm, ectoderm, archenteron and blastopore. Blastula - animal embryo at the early stage of development when it is a hollow ball of cells. Gastrulation -> process by which the three germ layers are formed during embryonic development Gastrula -> embryo at the stage following the blastula when it is a hollow cup-shaped structure having three layers of cells Germ layer -> each of the three layers of cells: ectoderm, mesoderm ad endoderm, that are formed in the early embryo ## CAENORHABDITIS ELEGANS Studied because it presents: a simple structure (only a thousand cells), rapid generation time, and transparency. It presents a rigid structure outside: hypodermis, similar to other animals' epidermis but it is a syncytium of nuclei. Simple NS. Composed of 302 neurons and 56 glial cells; neurons are organized into nerve cords in the dorsal and ventral sides of the animal, but some run along the lateral side. Simple digestive system and they move by a series of longitudinal muscles. It is possible to predict the number of neurons because of the stereotypical position of the cells and their division. Egg fertilized, from the zygote (PO) in the first division there will be 2 different populations: AB (for hypodermis and nervous system) and P1 (body, muscle, gut, somatic gonad). AB is bigger than P1. In 4 cell embryo, there will be $ABa$ e $ABp$ (anterior and posterior), $P2$ and EMS (mesendo). In 8 cell embryo, the mesendo is divided into mesoderm and endoderm. Upon gastrulation, AB progeny will spread out over all the embryo, producing the hypodermis. After gastrulation there is the indentation, here the embryo starts developing as a worm and it presents only 556 cells, then it will add the remaining (arriving at 959). Upon indentation, there is the enlargement of cells that out of the ectoderm are squeezed into the animal and here the neurons are created -> specialization, motor neurons will be located ventrally, whereas sensory neurons are located dorsally. ## DROSOPHILA MELANOGASTER It is easy to bread and with short lifespan. In this animal, there is a complete nervous system made of the brain and spinal cord, instead of only neurons as in C. Elegans. The NS is located ventrally to the structures of the animal. From the early stages, there is an anterior-posterior and a dorsal-ventral axis. The ectoderm is sitting dorsally to the embryo, the two neurogenic regions are located to the sides and the mesoderm is ventral to the animal. In the beginning, there are no cells, indeed it is a syncytium: nuclear divisions are not accompanied by corresponding cell divisions. Cellularization will occur in gastrulation. In gastrulation there is also an increase in the size of the embryo, pushing the ectoderm downward and the neurogenic regions too and the mesoderm will be pushed inside. With cellularization, there is the creation of the cells that will compose the exterior (epidermis) and the interior (guts from endoderm and mesoderm) and neuroblast. The nervous system is ventral to the axis because of the push of the lateral side to the ventral part; there will be the creation of neuroblasts in the intern and they will fuse into the ventral nerve cord -> laminization. Differentiation will also occur into the anterior-posterior axis, to create the brain and spinal cord. Neurogenesis -> cells of ectoderm (neurogenic progenitors) increase their size and are squeezed after cellularization -> delamination. Each squeeze gives rise to a neuroblast and from each neuroblast (through waves of cell cycles) to ganglion mother cells (GMCs), numbered starting from 1 to..... Each GMC generates a pair of neurons or glia. Very regulated in time and space. It changes over time. Along the anterior-posterior axis, many molecules and signalling will dictate the delamination and anteriorly there is the need to split the tube into the two parts: the two encephalic vesicles, ## VERTEBRATES XENOPUS LAEVIS -> there is an egg with an animal and vegetal pole. The former is where the embryo will develop, and the latter provides nutrients and energy. Formation of the blastula and the blastocoel, the fundamental hole. After gastrulation, with the formation of the blastopore, there is also the creation of the involuting marginal zone (IMZ), part of the ectoderm that will migrate into the blastocoel. The blastopore is created at the opposite side of the entrance of the spermatocyte. The first cells that migrate from the blastopore and will migrate further, they will determine the ventral part of the embryo we can already predict the anterior-posterior axis. The migration of the cells will also determine the establishment of the mesoderm and is crucial for the induction, on the overlaying ectoderm, of the formation of the neural plate. The first IMZ cells will form the anterior part (the head), and then mesoderm will lead to the differentiation of the brain and the later IMZ cells will form the posterior part (the spinal cord). The neural plate is induced by mesoderm, triggered and controlled by the notochord. It starts invaginating and will finish when the pinkish part is fused together, forming the neural tube. The last few cells that migrate in the groove are the neural crest cells which will give birth to the neurons and glial cells of the peripheral nervous system. The neural tube is created dorsally to the endoderm. NS develops dorsally because the induction of the neural plate takes place dorsally in the embryo. The ectoderm will give rise to the skin and cover all the embryo. Anteriorly to the neural tube, there is the notochord, which will give rise to bone structures, including vertebrae which will include the neural tube Blastopore is an inducer but also an organizer. ## ZEBRAFISH yolkier. At the animal pole, there will be blastomeres that will continue dividing, forming the epiboly. The epiboly tends to include the vegetal pole (the direction of the growth of the cells is to crawl on the side of the vegetal and embrace all of it). At 50% of the formation of the epiboly, there is the creation of a shield and the start of the gastrulation. Upon gastrulation, there is the beginning of migration and the formation of the mesoderm. In this animal, there is the presence of an enveloping layer, epiblast (the equivalent of the ectoderm) and yolk syncytial layer (the equivalent of the endoderm). Migration occurs below the epiblast and upon the yolk syncytial layer, so the cells will form the mesoderm (hypoblast). The direction is back toward the animal pole, In the junction between the mesoderm (blu) and the neural plate (red), there is the blastopore. The first migrating cells are the ones that will form the ventral part, so the head. On the shield, there is the formation from the ectoderm of the neural plate. Then the rest of the ectoderm will continue the migration, until the vegetal pole is completely enveloped by 100% epiboly. ## CHICKEN -> yolkiest We don't talk about blastula but about blastodisc and there is no migration, differentiation, and proliferation of the yolk because it is too thick, so blastodisc will navigate on the top of the yolk. The processes are the same, so there is the migration of the cells within the blastula to produce the primitive streak, the groove that is the site of active migration within the blastodisc. The mode of migration is the same, starting from the lateral part of the blastodisc, cells migrate into the groove and backward to generate all the tissues, comprehending the neural plate and then the neural tube upon the complete covering by the ectoderm. Endoderm is the site of contact between the embryo and the yolk. Hensen's node is at the posterior end of the primitive streak, analogous to the dorsal lip of the blastopore, it is the organizer for the migration from the outside to the inside and forming the mesoderm, which will induct the neuronal differentiation of the ectoderm. There is the appearance of somites from early stages. ## MAMMALIAN There is no poles or nutrient, indeed nutrients and energy are provided by the mother already in the original egg. In the blastula, there is the inner cell mass, for the development of the embryo, whereas the rest of the blastula is for placenta and implantation within the uterine wall. After implantation, there is gastrulation with the formation of the three germ layers, the primitive streak for the anterior-posterior axis, and the neural tube (for the head and spinal cord surrounded by somites). ## INDUCTION OF NEURONAL DIFFERENTIATION To comprehend mechanisms of differentiation many experiments were used. From two embryos at different stages, pre-gastrulation and after-gastrulation were transplanted pieces of ectoderm that were supposed to migrate into the blastopore upon gastrulation. Cultivating the transplanted tissue from the pre-gastrula, differentiation occurred only to the epidermis, whereas waiting after gastrulation, some areas were committed to differentiating into neural tissue. Studies to understand molecular mechanisms -> transplantation of dorsal lip of the blastopore into another frog embryo. This induced the creation of a new fully formed animal with its anterior-posterior axis. So, the blastopore is considered an organizer because it can organize an entire anterior-posterior axis and an entire neural tube with eyes, head, brain and spinal cord. In addition, it was found that the nervous system of the animal was produced by both cells from the introduced tissue and the host animal -> it is also an inducer, because it induces a new nervous system outer from cells that are from the host origin. Transplanting the dorsal lip, what was transplanted was the mesoderm -> mesoderm is able to organize an anterior-posterior axis and to induce the ectoderm of the host into a new neural plate and then the brain and spinal cord. 3 germ layers, no complete embryo if we cultivate only one layer -> is the interaction of the different layers to form the embryo. Mesoderm drives the differentiation of the rest. ## POLARITY AND SEGMENTATION During the nervous system development, there is specialization and regionalization. ### ANTERIOR-POSTERIOR AXIS Drosophila is a very good model because even in mice and humans the same molecular pattern is driving the differentiation. The first step of differentiation is when there is the anterior-posterior axis, and it occurs early, as soon as the fly egg is polarized. The anterior pole expresses Bicoid and the posterior Nanos. The first is a transcription factor, whereas the latter is an RNA-binding protein. These are present as mRNAs that have to be translated after fertilization. Both are involved in the control of the expression of other genes (positive or negative control of the expression) binding to their site on the DNA or sequestrating the mRNA. The levels of Bicoid and Nanos determine whether the "gap genes", are expressed in a particular region of the embryo and split the gradient formed by Nanos and Bicoid in two, The gap genes, in turn, control the striped pattern of the "pair-rule genes", splitting in smaller chunks the anterior-posterior axis. Finally, the pattern of expression of the pair-rule genes controls the segment-specific expression of the "segment polarity genes". At this point, the anterior-posterior axis is defined. This developmental hierarchy progressively divides the embryo into smaller and smaller domains with unique identities. The next step requires the expression of segment-specific genes: the homeotic (HOX) genes. ### HOX GENES Homeotic genes were discovered by studying mutants. Mutations in Ultrabithorax lead to the creation of two pairs of wings, one from the second segment and an additional one from the third thoracic segment. Mutations of Antennapedia cause the transformation of a leg into another antenna. Knocking out all the HOX genes, all the parts of the animal look nearly identical. HOX genes are important for the anterior-posterior axis and segmentation. The homeobox genes are arranged in two clusters, both in linear arrays on the chromosomes in the order of their expression along the anterior-posterior axis. These genes are activated by other genes, specifically to differentiate into the deputed region. They are 8 genes in two complexes: antennapedia (ANT-C) e Bithorax (BX-C) clusters. 5 in the first and 3 in the other. They encode for proteins of the homeodomain class of transcription factors, binding to consensus sequences of DNA in promoters of other genes (85-170) and activating the pattern of the transcription factor for the function, so the HOX genes do not provide the differentiation directly. Homebox clusters similar to those of flies have been identified in vertebrates -> conservation. However, in high-order animals, there will be some similar genes in addition (on other chromosomes) in order to compensate if mutations happen. ## HINDBRAIN DEVELOPMENT -> give rise to: * Cerebellum -> motor coordination, posture, balance and cardiac, respiratory and vasomotor centers * Brainstem -> breathing, heartbeat and blood pressure * Pons -> biting, chewing, and swallowing; as part of the brainstem, it controls the intensity and frequency of breathing * Medulla oblongata -> regulating of breathing, swallowing and heart rate The hindbrain undergoes a pattern of "segment formation" that resembles that occurring in flies. The hindbrain is composed of rhombomeres, from R1 to R8. Each gives rise to a repeated pattern of neuron differentiation and from each, a unique set of motor neurons arises (1-3 for trigeminal; 4-5 for facial; 5 for abducens; 6 for glossopharyngeal; 7-8 for vagus). The rhombomeres are generated by paralogous groups of genes; the number of groups is 4. Group 4 is for 7-8; the 3 from 5-8; the 2 for 2-5. The pattern is similar in all vertebrates. In Hoxal null mice, the rhombomere 4 domain is reduced and does not form a clear boundary with r3. R5 is lost or fused with r4 into a new region called rx. The abducens (in the r5) is lost and also the facial is defective. R3 is bigger and the trigeminal is also modified. In mice, a single mutation does not produce a dramatic phenotype as the one in Drosophila and this is due to the presence of the gene in multiple copies. The larger of the number of genes mutated or nulled, the larger the phenotype. In zebrafish, the same happens. ## ORGANIZER AND NEURAL INDUCERS Induction -> some cells evoke a specific developmental response in others. An example of a piece of dorsal blastopore lip from gastrula transplanted into a ventral/lateral position of another gastrula -> it invaginates and develops notochords and somites. It also induces the host ectoderm to form a neural tube, to then grew into a nearly complete secondary embryo. It is an organizer because organizes the axis of the creature. The axis itself induces the host creature to produce a nervous system for the new creature. The animal half will ultimately give rise to neural tissue and ectodermal tissue, while the vegetal half will give rise primarily to endoderm. The mesoderm, which will ultimately go on to make muscle and bone and blood, arises in between these two tissues, from the cells around the embryo's equator. In order to discover the molecules involved, experiments were made. When the animal pole is cultivated alone, it doesn't develop into mesoderm. When you take an animal pole and an additional vegetal pole and stuck them together, the mesoderm will develop -> vegetal pole drives the differentiation of the junction with the animal pole into mesoderm. Studies thus concentrated on identifying factors that would increase the expression of neural genes without the induction of mesoderm-specific gene expression. These led to the identification of Noggin, Chordin, and Follistatin, which produce primarily anterior brain structures. Further down the line of the identification of the role of inducers, many studies then led to the discovery of retinoic acid, Wnts, and FGFs. They are transformers. ## RETINOIC ACID It is teratogen -> birth defects, craniofacial and brain abnormalities. Crosses the cell membrane to bind a cytoplasmatic receptor. RAR complex (retinoic acid receptor) goes into the nucleus and binds to the RARE (RAR retinoic acid response element) in the promoter of target genes. High RA concentrations fail to develop the head -> the expression of anterior Hox genes is inhibited upon RA exposure. Low concentrations of RA drive the expression of Hox genes normally expressed in the anterior embryo, while at higher concentrations more posteriorly expressed Hox genes are induced -> there is an increasing gradient of RA