Developmental Bio 2

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periplasm: outermost layer of egg that is yolk free
endoplasm: inner, yolk rich cytoplasm
lateral inhibition: -example of cell determination
-important in neuroblast determination in the ventral neurogenic region of the drosophilia early gastrula
-cells wth potential to become neuroblasts make Achaete and scute proteins(both called proneural genes) (helix-loops-helix proteins, transcription factors)
-also make receptor protein Notch as well as its ligand Delta(not soluble but on the surface)...this is an example of so-caled juxtacrine signaling
-when a cell starts to produce slightly more Delta than a neighboring cell will gain an advantage
-Delta activates a Notch-mediated signaling cascade that which results in the inhibtion of neuroblast genes (req. for neuroblast differentiation)
-expressin of neuroblast genes are required for contined productin of Dela...so neighbring cells are at even bigger disadvantage cause not producing neuroblast genes...so they become epidermal cells by default
-the overproducer becomes a neuroblast and it leaes the epithelium and moves to the basal side of the future epidermis
notch/delta mechanism: -very fundamnetal and used in many tissues and many species as spacing mechanism...bristle spacing in drosophilia, hair cel spacing in inner ear and patterning of feathers in chicks
superficial meroblastic cleavage: -divisions limited to periplasm
-not true cleavage
-cytokinesis is delayed until afer multiple rounds of mitoses have occured and occurs by the proces of membrane furrowing
-begins with successive nuclear cycles (nuclear divisions)
-during the first 8 cycles, nuclei are deep within the endoplasm so not visible from outside
-by cycle 9, a few nuclei have moved to the posterior pole and become enclosed by plasma membranes-they are now called pole cells and are PGC
-by cycle 10 most of the nuclei have reached the periplasm
-cycles 10-3 are known as the preblastoderm stage, nuclei exist at the periphery but still a "syncytium"
-during this stage the yolk components become even more concentrated toward the center and the periplasm thickens
-by cycle 14, there are 5000 nuclei in the periplasm (syncytial blastoderm stage)
-cellularization until only small cyto. stalk exiss between endoplasm and blastoderm cells
Cellularization in superficial meroblastic cleavage: -plasma membrane furrows cut down in between the nuclei, the nuclei elongate and they become enclosed in a cage of microtubules
-these membrane furrows (underlying actin) deepen in a microtubule-dependent process.
-eventually the furrows reach deeper than the nuclei and broaden thereby separating the cells (except fro the cytoplasmic stalks)
control of cleavage orientations: -carried ou by the actin and myosin molecules of the conrtractile rings
-like a purse string in holoblastic cleavage
-in meroblastic, infoldings of plasm membranes advacne an cut like knives-interplay between acing and mircotubules
FOCUS ON HOLOBLASTIC
-contractile ring composes of rings of actin associated with myosin molecules-works in a similar manner to muscle contraction
-ring is parallel to the plane of ytokinesis and in the case of asymmetric divisinos, it is positioned closer to one end of he mitotic spindle
-spindle orientation deermines cleavage plane (which will be perpendicular to its long axis)
-specific portions of the spindle that are most important are the asters
-orientation of the spindle can be influenced by cell shape...spindles will orent themselves with their long axes paallel to the long axis of a cell
-in asymmetric divisions, ill-defined molecules in the cortexbind to one centrosome. Spindle pulled into an "eccentric" position (markedly toward one end of the cell), which leads to unequal cell division
control of timig of cleavages: -transition of from fertilization to cleavage is cause by MPF activity
-blasomeres geneally have modified 2 step cell cycle-M and S (some species have a short G2)
-MPF act. is highest during M and undetectable during S, due to the degradaion of the cyclin subunit by proteosomes
-the components nec. for MPF activiation (cyclin as well as a tyrosine phosphatase called cdc25, which is encoded by the string+ gene in Drosophilia, an dwhcih removs an inhibitory phosphate group at Try 15) are initially made from ample RNAs store in the cytoplasm of the oocyte.
-exception to the above rule occurs in drosophila, in which, during the first 7 cycles, MPF acvity (due to stored cyclin+cdc complexes) is constant and nuclear divisions occur as rapidly as DNA synthetic enzymes can permit
-at the mid blastula stage, cell cyclesslow and become less synhronous and there is a shift in reliance to zygotic mRNAs (must synthesize own string protein, cyclins)...occurs at around cleavage 11-14 in Drosophila, more abpt in xenopus, in which zygotic RNA snthesis and asnchronous divisions art to occur at cleavage 13 (seems as though all maternal RNAs are all selecively targeted for degradation in xenopus)
-mammals and sea urchins produce zygotic RNAs all along, so they dont have an MBT
fate: destiny of the cell or region and its descendens during the course of normal development...this is determined experimentally
potency: the total of all strucures that a cell or region or its descendens can form if placed under the appropriate conditions
-the potency for a given cell always includes its fate
fate mapping: -classic strategy for determining the fates of cells and regions
fate map: -a diagam of an organism at a particular stage of development, inicating the fate of its component cells or regions at a later stage
-on these maps, the wor primordia=rudiments refers to embyronic regions with a distinct fates
-comparing fate maps between normal embryos and embryos treated in certain ways (mutants)gives an idea about the function of a particular gene
-the more predictable the cleavage he better fate mapping works
-in mammals or xenopus in which cleavage is variant and there is a mixing of cells that contribue to strucures in unpredicable ways, fate mapping is pretty impossible
clonal analysis: analysis of suviving descendants of an original founder cell or region which then can be used to construct a fate map
-diff between this and lineage anaylysis is this involves the anaylysis of many diff cells at once
-clonal analysis of drosohilia at the cellular blastoderm stage resulted in the reproucible appearance of clones that overlapped between wings and legs on the same side of the body (thereofre, the clones overapped between dorsal and ventral structures)
-no clones were found to overlap between the mesothorax region an the head or abdomen
-this started to hint at what we now know is true..that blastoderm cells are determined to contribute to a single segment, but they are not yet determined to form certan structures along the dorsal-ventral axis within that segment
two ways clonal anaylsis can be done: 1. injection of large, visible, metabolically inert dye so it can't diffuse

or

2. genetic aleration of a subset of cells by, for example, using x-irradiation-but the problem is that x-ras cannot be focused onto single cells so the mapping is not precise...and clones arise at unpredictable locations and multiple locations
-x-rays induce somatic crossover...pieces of homologous chroms are swapped in somatic cells
-procedure: zap the cell, leads to crossing over, and it produces 2 progeny
-one homozygous recessive for the multiple wing hairs
-this homozygous recessive cels becomes the founder cell for a clone of visibly different cells
info gained from the tedious scoring or documenting of clones in clonal analysis: -so then they can document or score the clones in order to find the degree of cell mixing (deduced by smoothness of margins)
-also can estimate the number of cellsin an organ primoridium at the time of x-irradiation
-allows the defining of restriction lines and compartments
-these are boundaries which clones generate at a certain stage and do no cross
-these compartments are regulated by specific gene products or specific combinations of gene products
-if these producs or selector genes are mutated then the compartments can creae inapproprae cell types
cellular potency: -testing of potency usually involves ectopic transplantation and/or isolation
-isolatin test the potency in the absence of the influence of surounding parts of an organism
-ectopic transplantation indicated the potency of a cell/region when it is exposed to cells oher han its normal neighbors
totipotent: -capable of giving rise to all the cell types made by the zygote, including the placenta
-these are the zygote and the early blastomeres
pluripotent: capable of giving rise to most of the cell types represented in the adult
multipotent: several of the several cell types
oligopotent: capable of giving rise to a few of the cell types represented in the adult
unipotent: capable of giving rise to only one of the cell types....same as determied/committed
community effect: tendency of large numbers of cells that are either isolate or etopically transplanted to adhere to their original fate, whereas the same cells woul ten to depart from their fate if treated as individuals
-ths effect implies signaling as a group
determination: -second stage of commitment
-determined when it can be transplanted ectopically and still carry out its intended fate
-also can be said that a cell is determined when its potency=its fate
-a founder cell cannot be said to be determined to contribute to one structure at a certain stage if its clone crosses boundaries between structures
-clonal restriction cannot be used as a definitive indication that a founder cell was determined, though, because it hasnt been introduced to a new medium
Getting beyond specification to determination-cell uses 3 methods: 1. community effect-due to autocrine signaling, signaling in which a cell secretes signal molecules that act on itself and or on other cells of the same type
-stabalizes the determined state for a group of cells that are similarly specified based upon cytoplasmic determinants that they already inherited
-example is fibroblast growth factor FGF secretion an perception by mesoderm precursor cells in xenopus blastulas are both required for the maintenane of the mesodermal fate

2. lateral inhibition-allows cell to gain advantage over its neighbors

3. embryonic induction-generally due to local interaction of non-equivalent cells (paracrie signaling=short range signaling between neighboring cells of different type
example of induction: lens formaion
-inmany species (including amphibians and mammals)the optic vescile (exension of the developing brain) induces the overlying ectoderm to form the eye lens...so the optic vesicle is the inducer
-the ectoderm must be cmpetent to respond to the inductive signals from the optic vesicle
-Pax6 needs to be present...Pax6 mutant mice do no have lenses
-transplantation experiments indicate that pax6 protein is needed in the ectoderm but not in the optic vesicle in order for the lens to form...so pax6 is called a competence factor
-one of pax6 actions may be to upregulate the synthesis of BMP4 receptors
-the optic vesicle secretes BMP4 which is perceived by receptors on the membranes of the ectodemral cells
-this perceptions leads to the transcritpion of the Sox2 and Sox3 transcription factors, which allow the expression of lens-specific genes
-inductive relatinships are often sequential and reciprocal
-a previous interactionbetween the neural plate and the etoderm was required in order for the latter to start expressing pax6
-eventually, the lens will begin o serve as an inducer for the optic vesicle-stimulates the optic vesicle to become optic cups, the walls of which will differentiate into retinal tissue...soo pax6-mutants are also missing retina
Imaginal discs: really cool exmaples of determined tissue (packaged legs, wings, antennae, genialia, etc)
-are formed in pockets of larval epithelium
-begin as 20-50 cells, and divide by mitosis during the laval period until they consist of tens of thousands of cells by the end of the larval period
-during metamorphosis, they are hormonally stimulated to undergo eversion..they unfold and form cuticle
-determination of these discs during larval period as been proven by transplanting them into body cavity of host larva, where they will respond to the hormones of host and and results i formation of an extra adult structure in body cavity
-this proliferation in body cavity is by mitosis and no eversion because proper hormones are not present
-the discs can then be subdivided and assayed for determination after several rounds of transfers-determination of the disc is stable after many transfers, although eventually, fidelty decreases and the wrong structure may form from the disc...called transdetermination
transdetermination of imaginal discs: -different from homeoic mutations, in which mutation in transcription factor genes resuls in the formation of incorrect appendages in incorrect places
-the phenomenon does not appear to involve such mutations, but rather the cells start to accmulate the signaling protein Wingless (a secreted peptide that=Wnt)which is upsream of B-catenin (and therefore, can lead to transcriptional activation)
-Wnt has since also been shown to funcin in lung cell- intenstinal cell transformations and stem cell-skin cell transformations in mice
-Wnt is a huge player in colon cancer and epidermal cancers
chimeras: another method for stuing determination
-organisms composed of cells with diff genotypes
-two 8 cell stage embryos can acually be fused into single large blastocyst, which, when implanted into a surrogate mother, will form into a single mouse
-cels on the outside (usually from trophoblast) of one 16-celled embryo can be combined with cells from the inside of another (would normally form the ICM)and although they will usually en up carring out their predicted fates, they would occasionally contribute to the "other" portion
-the above experiment highlight just how regulative/flexible mammalian development is an that mammalian cells undergo a gradual and prolonged process of determination
-this method is used to determine cell-autonomy, also
definition of cytoplasmic determinant: any component of the cytoplasm that is unevenly distribute in the egg or embyro AND affects the fate of the cells that will conain it
-could be an organelle or group of organellles in theory, but mos ofen the term refers to an mRNA or protein
earliest experimental evidence for cytoplasmic determinants..: -came from ligation experiments
-in sea urchin, if the egg is divided such that each half retains some animal and some vegetal cytoplasm, 2 normal embryos will form
-but if the egg is cleaved equatorially then the animal half develops into a so called dauer blastula (abnormal ciliated blastual that never developes further) and the vegetal half forms a very abnormal embyro
-clearly, it is important for both halves to inherit both vegetal and animal cytoplasm
classic example of cytoplasmic deerminan sequestraion-the polar lobe in the snail (undergoes spiral cleavage): -an anucleate love of cytoplasm is formed at the vegetal pole of the zygote just before the first cleavage (kinda like a cellular extension)
-after the first cleavage, it is evident that the lobe remains attached to one blastomere (called the CD blastomere)...called the trefoil-stage embryo
-subsequently, this lobe is resobred back into the cd blastomere, which is now larger than the ab blastomere
-before the second cleavage, another polar lobe is extruded at the vegetal pole of the cd blastomere
-afer cleavage it is then resorbed into the d blastomere and now d is larger than a b and c
-it appears that the polar lobe contains cytoplasmic determinants ha are vital for the formation of mesodermally derived organs such as muscles, heart, mouth and foot
-evidence for this is when the polar lobe is revmoed from teh trefoil sstage it results in embryos lacking in the above structures
-similar embryos occur if the d blastomere is removed
-if the free flowing part of the cytoplams is removed and cytoplasm from other blastomeres is used to replace i, then a reasonably normal embyro results
-so basically ther formatino of the polar lobe is a method for sequestering these determinants such tat they are selectively allocated to the d blastomere and its descendents and that this allocation is necessary for mesodermal organ formation
cytoplasmic determinants in insect eggs-polar grandules an germ cell determnation: -the primordial germ cells of the fly are the pole cells
-one distinguishing feature of the pole cells are the polar granules, which are dark in color and contain RNAs necessary for pole cell formation as well as for the formation of some abdominal structures
-UV irradiation of the posterior end of the embryo prior to cellularization also prevents gamete formatin suggesting cyto. determs reside there (in the so called pole plasm)
-the uv-irradiated embryos an be rescued by injecting donor posterior ole plasm (donor anterior cyto. does not work)
-the pole plasm is sufficient to induce pole cell formation
several of the polar granule components are maternally encoded: -oskar+ is actually necessary and sufficient for pole cell formation
-injection at other sites can cause ectopic pole cell formation...the protein product functions by causing the localization of, or recruiting of, other proteins and RNAs necessary for germ cell formaion
-oskar+ mRNA is normally localized to the posterior pole, after it is trasferred from the nurse cells, by mircotubules (posterior localization is prevented by treatment with colhicine
-movement of oskar+ mRNA requires the Staufen proein, which appears to couple the RNA to kinesin
-Vasa+ encodes a protein that interacts with the oskar protein, once the latter has been prouced by translation
-oskar/vasa/staufen protein complexes are though to interact with the posterior pole cytoskeleton where they trap other RNAs(imp for pole cell formaion)that are brought into their proximity by cytoplasmic streaming
-thus, mature polar granules are formed
bicoid is another example of a localized cytoplasmic determinant: -embryos are missing a head and a thorax, but instead, have posterior structures where anterior structures shold be
-can be rescued by intro of bicoid+ anterior cytoplasm at the anterior end of mutant
-injection of bicoid+ cytoplasm laterally (essentially into the midle of the mutant oocyte) cause the embryos to form head structures in the middle flanked b thoracic structures
-injecion at the posterior end results in head structures at both ends
-in the wildtype oocyte, bicoid+ RNA is localized to the anterior end
-after it is synthesized in the nurse cells, trasnfered into oocyte by mircotubule depenent mechanism and is localized to anterior end
-other maternal effect genes imp for bicoid+ localization, including exuperantia+, swallow+ and staufen+.
-exu may help in the initial recruitment of bicoid+ mRNA into complexes with swallow and staufen proteins which mediate its transport
-the 3' unranslated region of bicoid+ is also necessar and sufficient for its proper localization
-this region has an elaborate secondary structure that mediates its interaction with swallow and staufen
-if the 3' end is spliced onto a diff mRNA, it too will be localized to the anterior end...referred to zipcodes
-so the drosohila oocye is now highly polarized with the bicoid+ mRNA being present at the anterior pole and oskar+ being present at the posterior pole
another example of localized cytoplasmic determinants-myoplasm in ascidian eggs (sea squirts): -diff colored regions of cyto that are segregate into diff embryonic cells which give rise to diff tissues and organs
-myoplasm is the yellow cyto that gets segregated into cells that will form the tail muscle, is initially found in a layer just beneath the plasma membrane
-ectoplasm is the clear cyto that is derived from he germinal vescle, starts out in the animal half
-cells that inherit the ectoplasm will mainly give rise to ectoderm
-grey yolky cyto will mainly contribute to the endoderm
2 phases of cytoplasmic rearrangements of egg activation in sea squirts: PHASE 1
-occurs while the egg is completing its meiotic divisions
-myoplasm streams down the egg periphery and accumulates as a yellow cap at the V pole
-meanwhile, the ectoplasm also flows toward the v pole an forms a clear layer above the yellow cap
-grey cyto gets displaced to a-pole

PHASE 2
-begins after the meioic divisions are complete
-male pronucleus starts to migrate toward the a ole and the myoplasm and ectoplasm seem to move with is some how
-much of the grey cyto moves back into the v hemi
-as a 4th region of cyto, the dark chordoplasm, forms roughly opposite of the myoplasm..it eventually contributes to notochord
-two axes of polarity hae now been established...A-V marked by the ectoplasm and polar bodies and grey cytoplasm and V will be future dorsal side of embyro...and the second is the anerior-posterior, marked by he yellow myoplasm
(future posterior end) and chorodoplasm (future anterior end)
3 main axes: anteropsterior
dorsoventral
left-right
proximal and distal: proximal is near point of attachment and distal is away from point of attachment....albow is proximal to wrist and distal to shoulder
medial: midsagital, divinds left and right side
paramedial: sagittal but off to one side
logitudinal: can be used for boh medial and paramedial
transverse: cross section, any plane perpendicular to the the anterior-posterior axis
coronal: any plan perpen. to the dorsal ventral axis
rhizoid-thallus axis is Fucus: -large, brown algae
-egg is spherical with no inherit polarization
-after fert, zygote forms bulge called germination
-one day after fert, cell div occurs to create one pointy cell and one round cell
-if not external signals then rhizoids grows at point of sperm entry
-if external clues then rhizoid grows out opposite of induction of light
-4-10 hours after fert, rhizoid site is revesible, adjuts to latest cue
-10-12 hours after, axis fixation occurs
-Ca influx from surroundig seawatergeneraes inward electri current, net efflux occurs at other sites
-Ca channels accumulate at future rhizoid site 6-8 hours after fert, but can change
-axis formatino depen upon actin bc treatment with cytochalasin B prevens outgrowth bu once drug removed rhizoid grows out in random directions
-patch of actin fil. seen at future rhizoid sie
axis fixation in fucus: -seems to involve cell wall, protoplasts can retain reverisibilty unless permited to form cell wall
-at future rhizoid site there is a sulfated polysaccharide, fucoidan
-fucoidan exocytosed by F-granules, microfilament depenedent, which may be the step in which cytochalasin B inhibits axis fixation
-brefedlin A inhibis this exocytosis again causes retention of ability to repolarize, until drug washes out
-brefeldin a causes first cleavage to be randomly oriented intead of perp to budding rhizoid
-during reversible period, Ca channels accumulate in response to external cue and this increase in Ca stimulates accumulation of actin
-axis fixed when cell wall is modified via exocytosis
-modificed cell wall signals back to cell, provides orienting cues for mitotic spindle
what do the animal-half blasotmeres form?: ectoderm and CNS
what do the vegetal half blaosomeres form waht? what about in the intermediate zone?: endoderm

mesoderm and CNS

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