Chapter 5 - Lecture 7 & 8.pdf

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Cell and Systems Biology
Ashley Bruce

From fertilization to cleavage  Fertilization activates protein synthesis, DNA synthesis, and the cell cycle  The activation of mitosis-promoting factor is one of the most important events in this transition to cleavage  MPF is the major factor responsible for the resumption of meiotic cell divisions in the ovulated frog egg  It continues to regulate the cell cycle of early blastomeres  Blastomeres generally progress through a biphasic cell cycle consisting of just 2 steps: M (mitosis) and S (DNA synthesis)  MPF activity is highest during M and undetectable during S  Shift between M and S in blastomeres is driven solely by the gain and loss of MPF activity  When MPf is made in these cells, they enter M phase o Nuclear envelope breaks down and chromatin condenses into chromosomes  After an hour, MPF is degraded -> return to S phase  MPF consists of 2 subunits: o Larger subunit, cyclin B, displays the cyclical behaviour that is key to mitotic regulation  Accumulates during S and degrades after the cells have reached M  Often encoded by mRNAs stored in the oocyte cytoplasm  If the translation of this message is specifically inhibited, the cell will not enter mitosis o Cyclin regulates the small subunit of MPF, cyclin dependent kinase  Activates mitosis by phophorylating several target proteins  Including histones, the nuclear envelope lamin proteins, and the regulatory subunit of cytoplasmic myosin  Responsible for chromatin condensation, nuclear envelope depolymerization, and the organization of the mitotic spindle  Without cyclin B, CDK will not function  Presence of cyclin B is controlled by several proteins that ensure its periodic synthesis and degradation  In most species, the regulators of cyclin B are stored in the egg cytoplasm so the cell cycle remain independent of the nuclear genome for a number of cell divisions  Early divisions tend to be rapid and synchronous  As the cytoplasmic components are used up, nucleus begins to synthesize them  In several species, the embryo now enters a mid-blastula transition -> several new properties are added to the biphasic cell divisions o the gap stages, G1 and G2, are added th  Xenopus add G1 and G2 to the cycle shortly after the 12 cleavage  Drosophila adds G2 during cycle 14 and G1 during 17 o The synchronicity of cell division is lost because different cells synthesize different regulators of MPF  Cells begin to “go their own way” o New mRNAs are transcribed  Many encode proteins that will become necessary for gastrulation  If transcription is blocked cell division will still occur at normal rates and times but the embryo will not be able to initiate gastrulation  Many of these new mRNAs are also used for cell specification  Interphase is subdivided into G1, S, and G2  Cells that are differentiating are usually taken “out” of the cell cycle and are in an extended G1 phase called G0  The cytoskeletal mechanisms of mitosis  Cleavage is the result of 2 coordinated processes: 1. Karyokinesis – the mitotic division of the cell’s nucleus o the mechanical agent of karyokinesis is the mitotic spindle made of tubulin 2. Cytokinesis – division of the cell itself o Mechanical agent is the contractile ring of the microfilaments made of actin o The mitotic spindle and contractile ring are perpendicular to each other and the spindle is internal to the contractile ring o The contractile ring creates a cleavage furrow, which eventually bisects the plane of mitosis, thereby creating 2 genetically equivalent blastomeres  Actin microfilaments are found in the cortex of the egg rather than in the central cytoplasm  This contractile ring is responsible for exerting the force that splits the zygote into blastomeres  The tightening of the microfilamentous ring creates the cleavage furrow  Microtubules are also seen near the cleavage furrow since they are needed to bring membrane material to the site of membrane addition  The placement of the centrioles is critical in orienting the mitotic spindle, and thus the division plane of the blastomeres  Depending on the placements of the centrioles, the blastomeres can separate either into dorsal and ventral daughter cells  The spindle can even be at an angle such that one daughter cell is clockwise or counterclockwise to the other  During the cleavage of insect eggs karyokinesis occurs several times before cytokinesis takes place so that numerous nuclei exist within the same cell  The OM of that one large cell eventually indents, separating the nuclei and forming individual cells Patterns of embryonic cleavage  Different organisms undergo cleavage in distinctly different ways  The pattern of embryonic cleavage peculiar to a species is determined by 2 major parameters: 1. The amount and distribution of yolk protein within the cytoplasm 2. Factors in the egg cytoplasm that influence the angle of the mitotic spindle and the timing of its formation  The amount and distribution of yolk determine where cleavage can occur and the relative size of the blastomeres: yolk inhibits cleavage generally  When one pole of the egg is relatively yolk-free, cellular divisions occur there at a faster rate than at the opposite pole  The yolk-rich pole is referred to as the vegetal pole; the yolk concentration in the animal pole is relatively low  The zygote nucleus is frequently displaced toward the animal pole  At one extreme are the eggs of sea urchins, mammals, and snails o These eggs have sparse, equally distributed yolk and thus isolecithal o In these species, cleavage is holoblastic, meaning that the cleavage furrow extends through the entire egg o With little yolk, these embryos must have some other way of obtaining food o Most will generate a voracious larval form, while mammals will obtain their nutrition from the placenta  Other extreme are the eggs off insects, fish, reptiles, and birds o Most of their cell volumes are made up of yolk which must be sufficient to nourish these animals throughout embryonic development o Zygotes containing large amounts of yolk undergo meroblastic cleavage – portion of the cytoplasm is cleaved o The cleavage furrow does not penetrate the yolky portion of the cytoplasm because the yolk platelets impede membrane formation there o Insect eggs have yolk in the center (centrolecithal) and the divisions of the cytoplasm occur only in the rim of cytoplasm, around the periphery of the cell (superficial cleavage) o The eggs of birds and fish have only one small area of the egg that is free of yolk (telolecithal eggs) and therefore the cell divisions occur only in this small disc of cytoplasm, giving rise to discoidal cleavage o Yolk is just one factor influencing a species’ pattern of cleavage  There are also inherited patterns of cell division superimposed on the constraints of the yolk o This can be seen in isolecithal eggs o In the absence of a large concentration of yolk, holoblastic cleavage takes place  4 major patterns of this cleavage type can be observed: radial, spiral, bilateral, and rotational holoblastic cleavage Gastrulation and Axis formation  The blastula consists of numerous cells, the positions of which were established during cleavage  During gastrulation, these cells are given new positions and neighbours, and the multilayered body plan of the organism is established  The cells that will form the endodermal and mesodermal organs are brought to the inside of the embryo, while the cells that will form the skin and nervous system are spread over its outside surface  Thus, the 3 germ layers are first produced during gastrulation  The stage is set for the interactions of these newly positioned tissues  Gastrulation usually proceeds by some combination of several types of movements  These movements involve the entire embryo, and cell migrations in one part of the gastrulating embryo must be intimately coordinated with other movements that are taking place simultaneously  Although patterns of gastrulation vary enormously throughout the animal kingdom, all of the patterns are different combinations of the 5 basic types of cell movements – invagination, involution, ingression, delamination and epiboly  Embryos must also develop 3 crucial axes that are the foundation of the body: the ant-post axis, the dorsal- ventral axis, and the right-left axis  The anterior-posterior axis is the line extending from head to tail or mouth to anus  The dorsal-ventral axis is the line extending from back to belly  The right-left axis is a line between the 2 lateral sides of the body Early Development in Snails  Snails are abundant along the shores of all continents, they grow well in the laboratory, and they show variations in their development that can be correlated with their environmental needs  Snails also have large eggs and develop rapidly, specifying cell types very readily  Snails have been used as examples of autonomous development, where the loss of an early blastomere causes the loss of an entire structure  In snail embryos, the cells responsible for certain organs can be localized to a remarkable degree Cleavage in Snail Embryos  The spiral is the fundamental theme of the molluscan organism  They are animals that twisted over themselves  Their larvae undergo a 180° torsion that brings their anus anteriourly above their head and the cleavage of the early embryos is spiral  Spiral holoblastic cleavage is characteristic of several animal groups: annelid worms, polyhelminth flatworms, and most molluscs  the cleavage planes are not parallel or perpendicular to the animal-vegetal axis but at oblique angles, forming a ‘spiral’ arrangement of daughter blastomeres  the cells are in intimate contact with each other, producing the most thermodynamically stable packing orientation  spirally cleaving embryos usually undergo relatively fewer divisions before they begin gastrulation, making it possible to follow the fate of each cell of the blastula  blastulae produced by spiral cleavage have no blastocoel -> called stereoblastulae  The first 2 cleavages are nearly meridional, producing 4 large macromeres: A B C D  In many species, these 4 are different sizes (D is the largest), a characteristic that allows them to be individually identified  In each successive cleavage, each macromere buds off a small micromere at its animal pole  Each successive quartet of micromeres is displaced to the right or to the left of its sister macromere, creating the characteristic spiral pattern  Looking down on the embryo from the animal pole, the upper ends of the mitotic spindles appear to alternate clockwise and counterclockwise  At the 3 cleavage, the A macromere -> macromere 1A and micromere 1a  The B, C, and D cells behave similarly, producing the first quartet of micromeres  In most species, these micromeres are to the right of their macromeres  At the 4 cleavage, macromere 1A -> macromere 2A and micromere 2a; micro 1a -> 2 micromeres: 1a¹ and 1a²  The micromeres of this second quartet are to the left of the macromeres  Further cleavages, macromere 2A -> blastomeres 3A and 3a; micromere 1a² -> cells 1a²¹ and 1a²²  In normal development, the first-quartet micromeres form the head structures  The second-quartet micromeres for the statocyst (balance organ) and shell  Theses fates are specified both by cytoplasmic localization and by induction Autonomous cell specification and the polar lobe  Molluscs: blastomeres are specified by morphogenetic determinants located in specific regions of the oocyte  Autonomous specification of early blastomeres is especially prominent in those groups of animals having spiral cleavage, all of which initiate gastrulation at the future anterior end after only a few cell divisions  In molluscs, the mRNAs for some TFs and paracrine factors are placed in particular cells by associating them with certain centrosomes  This association allows the mRNA to enter specifically into one of the 2 daughter cells  In many instances, the mRNAs that get transported together into a particular tier of blastomeres have very similar 3’ tails, suggesting that the identity of the micromere tiers may be controlled largely by 3’ UTRs of the mRNAs that attach to the centrosomes at each division  In other cases, the patterning molecules appear to be bound to a certain region of the egg that will form a unique structure called the polar lobe The Polar Lobe  E.B. Wilson and his student observed that certain spirally cleaving embryos extrude a bulb of cytoplasm – the polar lobe – immediately after the first cleavage  In some species of snails, the region uniting the polar lobe to the rest of the egg becomes a fine tube  The first cleavage splits the zygote asymmetrically, so that the polar lobe is connected only to the CD blastomere  In several species, nearly 1/3 of the total cytoplasmic volume is contained in this anucleate lobe, giving it the appearance of another cell  The resulting 3-lobed structure is often referred to as the trefoil stage embryo  Crampton (1986) showed that if one removes the polar lobe at the trefoil stage, the remaining cells divide normally but the resulting larva is incomplete -> lacking its endoderm and mesodermal organs as well as some ectodermal organs  Also demonstrated that the same type of abnormal larva can be produced by removing the D blastomere from the 4-cell embryo -> concluded that the polar lobe cytoplasm contains the endodermal and mesodermal determinants, and that these determinants give the D blastomere its endomesoderm-forming capacity  Also showed that the localization of the mesodermal determinants is established shortly after fertilization, thereby demonstrating that a specific cytoplasmic region of the egg, destined for inclusion in the D blastomere, contains whatever factors are necessary for the special cleavage rhythms of the D blastomere and for the differentiation of the mesoderm  Centrifugation demonstrated that the morphogenetic determinants sequestered in the polar lobe are probably located in the lobe’s cytoskeleton or cortex, not its diffusible cytoplasm  Van den Biggelaar removed the cytoplasm from the polar lobe with a micropipette -> cytoplasm replaces the portion he removed and subsequent development of these embryos was normal  Experiments have demonstrated that the nondiffusible polar lobe cytoplasm that is localized to the D blastomere is extremely important in normal molluscan development: o Contains the determinants for the proper cleavage rhythm and the cleavage orientation of the D blastomere o Contains certain determinants for autonomous mesodermal and intestinal differentiation o Responsible for permitting the inductive interactions leading to the formation of the shell gland and eye  Also, the material i
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