Chapter 8 - Lecture 10&11.pdf

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Department
Cell and Systems Biology
Course
CSB328H1
Professor
Ashley Bruce
Semester
Fall

Description
Chapter 8 – Early Development in Vertebrates: Amphibians and Fish  Despite vast differences in their adult morphology, the early development of each of the vertebrate groups is very similar  Fish and amphibians are 2 of the most easily studied vertebrates  In both, hundreds of eggs are laid externally and fertilized simultaneously  They are anamniotic vertebrates – do not form an amnion that permits embryonic development to take place on dry land  They generate body axes and organs employing manyof the same processes and genes used by other vertebrates, including humans Early Amphibian Development  Salamander and frog embryos - Large cells and rapid development  But they undergo a long period of growth before they become fertile and chromosomes are often found in several copies -> precluding easy mutagenesis Fertilization, Cortical Rotation, and Cleavage  Frogs usually have external fertilization, with the male fertilizing the eggs as the femaleis laying them  Even before fertilization, the frog egg has polarity: o The dense yolk is at the vegetal end o Animal part of the egg has very little yolk  Certain proteins and mRNAs are already localized in specific regions of the unfertilized egg  Fertilization can occur anywhere in the animal hemisphere of the amphibian egg  Point of sperm entry -> determines dorsal-ventral polarity o Point of sperm entry marks the ventral side of the embryo o 180° opposite -> dorsal side  The sperm centriole enters with the sperm nucleus o Organizes the microtubules of the egg into parallel tracks in the vegetal cytoplasm -> separating the outer cortical cytoplasm from the yolky internal cytoplasm o These tracks allow the cortical cytoplasm to rotate with respect to the inner cytoplasm o These parallel arrays are first seen immediately before rotation starts, and disappear when rotation ceases  In the 1-cell embryo, the cortical cytoplasm rotates about 30° with respect to the internal cytoplasm at 80% into the 1 cleavage  In some eggs, this exposes gray-coloured inner cytoplasm directly opposite the sperm entry point  this region, the gray crescent is where gastrulation will begin o Even in xenopus eggs, which do not expose a gray crescent, cortical rotation occurs and cytoplasmic movements can be seen  Gastrulation begins at the part of the egg opposite the point of sperm entry -> region will become the dorsal portion Unequal radial holoblastic cleavage  Cleavage in most frog and salamander embryos is radially symmetrical and holoblastic  The amphibian egg contains a lot of yolk which impedes cleavage  1 division begins at the animal pole and slowly extends down into the vegetal region st  In those species with a gray crescent, the 1 cleavage usually bisects the gray crescent st  While the 1 cleavage furrow is still cleaving the yolky cytoplasm of the vegetal hemisphere, the 2 cleavage has already started near the animal pole st o This cleavage is at right angles to the 1 one and is also meridonal rd  3 cleavage is equatorial o But because of the vegetally placed yolk, the rd 3 cleavage furrow is not at the equator but is displaced toward the animal pole o It divides the amphibian embryo into 4 small animal blastomeres (micromeres) and 4 large blastomeres (macromeres) in the vegetal region  Despite their unequal sizes, the blastomeres continue to divide at the same rate until the 12 cell cycle with only a small delay of the vegetal cleavages  As cleavage progresses, the animal region becomes packed with numerous small cells, while the vegetal region contains a relatively small number of large, yolk-ladenmacromeres  An amphibian embryo containing 16-64 cells is commonly called a morula  At 128 cell stage, the blastocoels becomes apparent, and the embryo is considered a blastula  Numerous cell adhesion molecules keep the cleaving blastomeres together o One of the most important: EP cadherin  The mRNA for this protein is supplied in the oocyte cytoplasm  If this message is destroyed by antisense oligonucleotides so that no EP-cadherin is made, the adhesion between blastomeres is dramatically reduced, resulting in the obliteration of the blastocoel  Although amphibian development differs from species to species, in general, the animal hemi cells -> ectoderm; vegetal cells -> endoderm; the cells beneath the blastocoels cavity will become mesoderm  The cells opposite the point of sperm entry will become the neural ectoderm, the notochord mesoderm, and the pharyngeal (head) endoderm  The amphibian blastocoels serves 2 major functions: 1. It can change its shape o Such that cell migration can occur during gastrulation 2. It prevents the cells beneath it from interacting prematurely with the cells above it  When Nieuwkoop took embryonic newt cells from the roof of the blastocoels in the animal hemisphere (animal cap) and placed them next to the yolky vegetal cells from the base of the blastocoels prevents the premature contact of the vegetal cells with the animal cap cells, and keeps the animal cap cells undifferentiated The mid-blastula transition: preparing for gastrulation  Important precondition for gastrulation: activation of the zygotic genome th  In xenopus, nuclear genes are not activated until late in the 12 cell cycle  At that time, the embryo experiences a mid-blastula transition  Different genes begin to be transcribed in different cells, the cell cycle acquires gap phases, and the blastomeres acquire the capacity to become motile  It is thought that some factor in the egg is being absorbed by the newly made chromatin because the time of this transition can be changed experimentally by altering the ratio of chromatin to cytoplasm in the cell Amphibian Gastrulation  There is no single way amphibians gastrulate  Different species employ different means to achieve the same goal Vegetal rotation and the invagination of bottle cells  Amphibian blastulae need to: o Bring inside the embryo those areas destined to form the endodermal organs o Surround the embryo with cells capable of forming the ectoderm o Place the mesodermal cells in the proper positions between the ecto and endo  The cell movements of gastrulation that will accomplish this are initiated on the future dorsal side of the embryo, just below the equator, in the region of the gray crescent  Here the cells invaginate to form the slitlike blastopore  These bottle cells change their shape dramatically  The main body of each cell is displaced toward the inside of the embryo while maintaining contact with the outside surface by way of a slender neck  The bottle cells will initiate the formation of the archenteron  Gastrulation in the frog begins in the marginal zone – the region surrounding the equator of the blastula, where the animal and vegetal hemisphere meet o Here the endodermal cells are not as large or as yolky as the most vegetal blastomeres  Cell involution is not a passive event  At least 2 hours before the bottle cells form, internal cell rearrangements propel the cells of the dorsal floor of the blastocoels toward the AC  This vegetal rotation places the prospective pharyngeal endoderm cells adjacent to the blastocoels and immediately above the involuting mesoderm  These cells then migrate along the basal surface of the blastocoel roof, traveling toward the future anterior of the embryo  The superficial layer of marginal cells is pulled inward to form the endodermal lining of the archenteron, merely because it is attached to the actively migrating deep cells  Although experimentally removing the bottle cells does not affect the involution of the deep or superficial marginal zone cells into the embryo, removal of the deep involuting marginal zone cells stops archenteron formation  Involution at the blastopore lip o After the bottle cells have brought the IMZ into contact with the blastocoels wall, the IMZ cells involute into the embryo o As the migrating marginal cells reach the lip of the blastopore, they turn inward and travel along the inner surface of the outer animal hemisphere cells o The order of the march into the embryo is determined by the vegetal rotation that abuts the prospective pharyngeal endoderm against the inside of the AC tissue o The animal cells undergo epiboly, producing a stream of cells that converge at and become the dorsal blastopore lip o The first cells to compose the dorsal blastopore lip and enter the embryo are the prospective pharyngeal endoderm of the foregut o These cells begin to transcribe the hhex gene, which encodes a TF that is critical for forming the head and heart o As these first cells, pass into the interior of the embryo, the dorsal blastopore lip becomes composed of cells that involute into the embryo to become the prechordal palte (head meso precursors) o These cells transcribe goosecoid gene -> product is a TF that activates numerous genes controlling head formation o The next cells involuting through the dorsal blastopore lip are the chordamesoderm cells o These cells will form the notochord, the transient mesodermal rod that plays an important role in inducing and patterning the nervous system o Chordamesoderm cells express tha Xbra gene -> product is a TF critical for spinal cord formation o Dorsal blastopore lip cells are constantly changing as the original cells migrate into the embryo and are replaced by cells migrating downward, inward, and upward o As the new cells enter the embryo, the blastocoels is displaced to the side opposite the dorsal lip o Meanwhile, the lip expands laterally and ventrally as bottle cell formation and involution continue around the blastopore o The widening blastopore “crescent” develops lateral lips and, finally, a ventral lip over which additional mesodermal and endodermal precursor cells pass o With the formation of the ventral lip, the blastopore has formed a ring around the large endodermal cells that remain exposed on the vegetal surface o This remaining patch of endoderm is called the yolk plug – eventually internalized o At that point, all endo precursors have been brought in, ecto has encircled, and meso has been brought between them o The first cells into the blastopore become the most anterior  Convergent extension of the dorsal mesoderm o Involution begins dorsally, led by the pharyngeal endoderm and head mesoderm o These tissues will migrate most anteriorly beneath the surface ectoderm o Meanwhile, as the lip of the blastopore expands to have dorsolateral, lateral and ventral sides, the prospective heart, kidney, and ventral mesodermal precursor cells enter into the embryo Epiboly of the prospective ectoderm  During gastrulation, the AC cells and noninvoluting marginal zone cells expand by epiboly to cover the entire embryo o These will form the surface ectoderm  One important mechanism of epiboly in xenopus gastrulation appears to be an increase in cell number coupled with a concurrent integration of several deep layers into one  a second mechanism of Xenopus epiboly involves the assembly of Fn into fibrils by the blastocoels roof o This fibrillar fn is critical in allowing the vegetal migration of the AC cells and enclosure of the embryo  In xenopus and many other amphibians, it appears that the involuting mesodermal precursors migrate toward the animal pole by traveling on an extracellular lattice of fn secreted by the presumptive ectoderm cells of blastocoels roof  Confirmation of fn’s importance for the involuting mesoderm came from experiments with a chemically synthesizedpeptide fragment that was able to compete with fn for the binding sites of embryonic cells  If fn were essential for cell migration, then cells binding this synthesized peptide fragment instead of extracellular fn should stop migrating  unable to find their “road” these prospective mesodermal cells should cease involution o precisely what happened and the mesodermal precursors remained outside the embryo, forming a convoluted cell mass  thus fn-containing ECM provides both a substrate for adhesion as well as cues for the direction of cell migration Progressive Determination of the Amphibian Axes Specification of the germ layers  The unfertilized amphibian egg has polarity along the animal-vegetal axis  Thus germ layers can be mapped onto the oocyte even before fertilization o Animal hemisphere blastomeres -> cells of the ectoderm (skin and nerves) o Vegetal hemisphere cells -> gut and associated organs (endoderm) o Internal cytoplasm around the equator -> Mesodermal cells  This general fate map is thought to be imposed on the embryo by the vegetal cells which have 2 major functions: 1. To differentiate into endoderm 2. To induce the cells immediately above them to become mesoderm  The mechanism for this “bottom up” specification of the frog embryo resides in a set of mRNAs that are tethered to the vegetal cortex o Includes the mRNA encoding the TF VegT, which becomes apportioned to the vegetal cells during cleavage  VegT: critical in generating both the endodermal and mesodermal lineages o When VegT transcripts are destroyed by antisense oligonuc, the entire embryo becomes epidermis, with no mesodermal or endodermal components o Its mRNA is translated shortly after fertilization o Product activates a set of genes prior to the mid-blastula transition  Another set of early genes activated by VegT -> encodes Nodal paracrine factors that instruct the cell layers above them to become mesoderm o Nodal secreted from the vegetal cells in the nascent endoderm and signal the cells above them to express phosphorylated Smad2 o Smad2 helps activate the eomesodermin and Brachyury genes in those cells, causing the cells to become specified as mesoderm o The Eomesodermin and Smad2 proteins working together can activate the zygotic genes for the vegT proteins, thus creating a positive feedforward loop that is critical in sustaining the mesoderm o In the absence of such induction, cells become ectoderm  Vg1 mRNA that has been stored in the vegetal cytoplasm is also translated  The production of Vg1 (Nodal-like protein) is needed to activate other genes in the dorsal mesoderm o If either Nodal or Vg1 signaling is blocked, there is little or no mesoderm induction  Thus, by the late blastula stage, the fundamental germ layers are becoming specified o Vegetal cells are specified as endoderm through TFs such as Sox17 o Equatorial cells are specified as mesoderm by TFs such as Eomesodermin o The animal cap – which has not received signals yet – becomes specified as ectoderm  The critical factor in this partition of the embryo into the 3 germ layer regions appears to be Nodal-like paracrine factors, whose actions are stimulated by T-box Tfs such as VegT and Eomesodermin The Work of Hans Spemann and Hilde Mangold Autonomous Specification versus inductive interactions  In 1903, Spemann demonstrated that early newt blastomeres have identical nuclei, each capable of producing an entire larva o Shortly after fertilizing a newt egg, Spemann used a baby’s hair to “lasso” the zygote in the plane of the first cleavage o He then partially constricted the egg, causing all the nuclear divisions to remain on one side of the constriction o Eventually – often as late as the 16 cell stage – a nucleus would escape across the constriction into the non-nucleated side o Cleavage then began on this side too, whereupon Spemann tightened to the lasso until the 2 halves were completely separated o Twin larvae developed, one slightly more advanced than the other o Spemann concluded that early amphibian nuclei weregenetically identical and that each cell was capable of giving rise to an entire organism  However, when Spemann performed a similar experiment with the constriction still longitudinal, but perpendicular to the place of the 1 cleavage, he obtained a different result altogether o The nuclei continued to divide on both sides of the constriction, but only one (future dorsal side) gave rise to a normal larva o Other side -> unorganized tissue mass of ventral cells which Spemann called the Bauchstück – the belly o This tissue mass was a ball of epidermal cells containing blood and mesenchyme and gut cells but it contained no dorsal structures such as NS, notochord, or somites  Why the different results? o One possibility: when the egg was divided perpendicular to the 1 cleavage plane, some cytoplasmic substance was not equally distributed into the 2 halves  Fortunately, the salamander egg was a good place to test that hypothesis o There are dramatic movements in the cytoplasm following the fertilization of amphibian eggs, and in some amphibians, these movements expose a gray, crescent-shaped area of cytoplasm in the region directly opposite the point of sperm entry  This 1 cleavage plane normally splits the gray crescent equally between the 2 blastomeres o If these cells are then separated, 2 complete larva develop o Should this cleavage plane be aberrant, the gray crescent material passes into only 1 of the 2 blastomeres  Spemann’s work revealed that when 2 blastomeres are separated such that only 1 of the 2 cells contains the crescent, only the blastomere containing the gray crescent develops normally  It appeared then that something in the region of the gray crescent was essential for proper embryonic development  Fate maps showed that the GCR gives rise to those cells that form the dorsal lip of the blastopore o These cells are committed to invaginate into the blastula, initiating gastrulation and the formation of the head endomesoderm and notochord  Because all future amphibian development depends on the interaction of cells that are rearranged during gastrulation, Spemann speculated that the importance of the gray crescent material lies in its ability to initiate gastrulation, and that crucial changes in cell potency occur during gastrulation  In 1918, performed experiments that proved both statements o He found that the cells of the gastrula were uncommitted but late gastrula cells were determined  Spemann’s demonstration involved exchanging tissuesbetween the gastrulae of 2 species of newts whose embryos were differently pigmented  When a region of prospective epidermal cells from an early gastrula of one species was transplanted into an area in an early gastrula of the other species and placed in a region where neural tissue normally formed, the transplanted cells gave rise to neural tis
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