BIO 325 Lectures 1 - 5 Review

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Bio 325 Lecture 1 Basic Concepts Embryology- the study of biological processes that happen from egg to birth Body organization, cell differentiation, cell/tissue movement, organogenesis Developmental Biology- the study of embryonic processes that happen in embryos and after birth- what happens after birth Juvenile and adult cell replacement, stem cell renewal, differentiation, regeneration, healing. Developmental Biology 1. Differentiation 2. Morphogenesis-process where cells and tissue are organized 3. Growth mass of tissue, cell growth 4. Reproduction- producing next generation 5. Evolution 6. Environmental Integration Major approaches in Developmental Biology 1. Anatomical Approaches look and see physical characteristics o Comparative Embryology- look and see o Evolutionary Embryology-comparing different species o Teratology-birth defects o Mathematical modeling 2. Experimental embryology o Manipulation of embryos and their molecular components Examples. Removal or grafting of embryo pieces/cells Ablation of cells Treating tissue with chemical substances. 3. Genetic o Classical genetics: randomly mutagenize genome (drsosphila flies) Observe Phenotype Analyze function of gene at molecular level. o Reverse genetics: knock out gene of interest Analize phenotype o Modern molecular genetics Geneome analysis, DNA sequencing Gene expression analysis Transgenesis Popular animal used to study development: Nematode work, Furit fuly, Sea urchin, Zebrafish, Chicken , Frog, mouse Anatomical Approaches
Comparative embryology- a traditional approach that unites with
Aristotle- first embryologist of record o Used chicken embryo, egg. 1651: William Harvey - Concludes all organisms come from eggs 1672: Marcello Malpighi -Publishes microscopy of chicken development - Described a head/body blastodisk in center Germ Layers Mesodermal tissue-red Epidermis tissue- blue Endoderm tissue- yellow Lecture 2- evolution and embryology frog development germ layers Anatomical Approaches cont. Comparative embryology- a traditional approach that unites with Evolutionary embryology o Phylogenetic relationships Dueterostomes (vertebrates) (triploblasts) Protostomes (triploblasts) Diploblasts o Triploblatic- three germ layers o Diploblastic- two germ layers Frog life cycle o Gametogenesis- oocyte (pre-fertalized egg) and sperm (gametes o Fertilization o Cleavage cutting up the egg into smaller units Morula (multi cell blastula) into blastula o Gastrulation- organizes cells into germ layers Turns Blastula into Blastocoel and then splits into 3 germ layers: Ectoderm, Mesoderm, Endoderm o Organogenesis o Larval Stages (metamorphosis) o Maturity Amplexus- Male squeezes eggs out of female, ends into egg mass Eggs orient so that the dark part is on top. To protect from sun. Egg cleaves into blastomeres (large cells present in cleavage stages of embroyes) talks about cleavage stage. Blastomeres turns into a Blastocoel Cell divisions are synchronous at first, dont coordinate their division. Try to divide as fast as possible. Mass and size of the embryo is not increasing.
then become asynchronous, mid blastula transition. Dividing process become slower. 12th division. Will reorient to form germlayers in process called Gastrulation. Will create a pinch called the Dorsal Blastopore lip. start moving inside, Will create a yolk plug on top of blastopore Neural plate forms Brain and CNS develop from a neural groove. At this point the egg is divided into Endodorm, Mesoderm, and ectoderm.
An old question: Epigenesis or Preformation Epigenesis- parts of an embryo arise de nova or from scratch at each generation Preformation- miniature adults were preformed in the egg or sperm o Nicholas Hartsoeker Epigenesis- embryos increase in complexity over time Organization of embryo o Epidermal ectoderm o Neural groove o Lateral plate Mesoderm (heart, Blood,vessels)- arises later than other two o Intermediate mesoderm (source of kidneys, gonads) o Somites (source of muscles, spine, ribs) o Notochord o Endoderm (gut, lungs) Christian Pander in 1817 o Described the fine structure of the chick embryo over time o First suggested the germ layers Ectoderm-blue Endoderm-yellow Mesoderm-red- arises after the other two Zygote Blastula Gastrulao ectoderm (outer layer) forms the epidermis cells of skin, CNS and neural crest. o Mesoderm (middle layer) forms the notochord, bone tissue, cell of kidney, red blood cells, and facial muscle, connective tissues, gonads o Endoderm (inner layer) forms the cells of digestive tract, liver, Lung cells, thyroid cells. o Germ cells forms the sperm and egg. Combine with gonad cells that come from the mesoderm. Neural crest- evolution of pharyngeal arch structures in vertebrate head. o Structures from our head are developed from the neural crest, it can differentiate into a lot of cells. Diploblasts
o Diploblasts egg gastrulate to form mesoderm just like it happens in triploblasts. This process has been cserved because it allows for a complex morphology. o Unlike deutrostomes that have mouth and anus, diploblasts only have one opening. o Later stages are different, although early stages are similar Evolutionary embryology 1. seek to understand mechanisms that define groups of animals. 2. Evolutionary relatioshops reflected by morphology of their embryos 3. Genetic connectedness between animclas is reflected by common embryonic features 4. The degree of divergence from ancestor is reflected by appearance of divergent structures. Karl Erst von Baer 1820s also described the existence of germ layers and extended panders generalizations. 1. General features appear earlier than specialized features 2. Less general characteristics develop from more general. 3. Embryo of a given species depart more and more from a simpler bodied species 4. The early embryo of a higher animal is never like that of a lower animal. Lecture 3 Karl Erst von Baer 1820s also described the existence of germ layers and extended panders generalizations. 1. General features appear earlier than specialized features 2. Less general characteristics develop from more general. 3. Embryo of a given species depart more and more from a simpler bodied species 4. The early embryo of a higher animal is never like that of a lower animal Homology: same or similar function that share a common evolutionary origin. Related developmental mechanisms. A fate map informs us about what various regions or blastomeres of the early embryo contribute to in the later embryo, larvae or adult. It does not tell us how that happens.
How do you make a fate map?

o Vital dye- Take chips of ager that is a dye and put it on embryo. Stains membrane, sees its fate. Some of the dots went inside the embryo at gastrulation. 3,4 elongated. o Label and follow o Lineage tracing to make fate map- using flourecent dyes to dye one cell in the embryo, slice up the frog larvae and see where the label went. Fate mapping with transgenic DNA shows that the neural crest is critical I making the bones of frog jaw. If you label surface cells, marginal cells or deep cells in nueral plate or epidermis, it will have the same fate. If you were to label In the mesodermal part, the outer part is part of the mesoderm but the inner part is part of them endoderm
Fate mape tells us where cells are heading, how do cells get to where they are going? Reorganization of the cytoplasm and the cortical rotation produce the gray crescent. Sperm entry contribute a centriole, acticate microtubules, this will move the cortical cytoplasm to the inner cytoplasm called the cortical rotation. This creates the gray crescent following by cleavage. Future dorsal side. Lecture 4 gastrulation movements/epithelial cell adhesion/ localized mRNA as fate determinants. What happens after cleavage? Cell movements during from gasrulation stage 10.5 Invagination happens at the dorsal blastopore lip. Will go all the way around the marginal cells. Four movements of gastrulation o Epiboly
o Involution o Invagination o Convergent-extension Invagination- Bottle cells are contracting, pulling in, squeezing, creating a blastopore lip. Moves from posterior to anterior. (part1) A and B Bottle cells form at the dorsal blastopore lip, invagination movements. The bottle cells are pulling in, at their apex, squeezing, contracting at the surface and pigment comes to the surface. Migrate toward the blastocoel, to the anterior. Part 2, pull in endodermal and mesodermal cells. Bottle cells start to invaginate. Elongation of the dorsal mesoderm. Part C Mesoderm is coming down and making a U turn. That is involution.
Involution cell move down, turn the corner and go up. Inward movement of the outer layer. Part 1 and part 2 of gastrulation. Happens mostly on dorsal side. Part 3. Extension to drive dorsal mesoderm to head postion. Blastocoel will get squeezed out of existence. Stage 12. Part D and E Blastopore has gotten smaller. Dorsal side you have involution, what will move that mesoderm at the head is convergence-extension movement. Part 4- completion of gastrulation. Part F Ventral side of mesoderm is filling up to connect mesoderm layer. Mesenchyme- cells not touching very closely. Cells in embryo are holding together as epithelia. Layers are where they are supposed to be. Anus is formed on dorsal side. Mouth and pharynx forms on ventral side.
A,B) Early gastrulation. The bottle cells of the margin move inward to form the dorsal lip of the blastopore, and the mesodermal precursors involute under the roof of the blastocoel. AP marks the position of the animal pole, which will change as gastrulation continues. (C,D) Mid-gastrulation. The archenteron forms and displaces the blastocoel, and cells migrate from the lateral and ventral lips of the blastopore into the embryo. The cells of the animal hemisphere migrate down toward the vegetal region, moving the blastopore to the region near the vegetal pole. (E,F) Toward the end of gastrulation, the blastocoel is obliterated, the embryo becomes surrounded by ectoderm, the endoderm has been internalized., and the mesodermal cells have been positioned between (he ectoderm and endoderm 1. invagination- outer surface of the layer pinches and becomes more narrow. Vegital rotation of endoderm- first movement, sight of blastopore. Invagination of bottle cells. Involution movement where mesoderm moves in and the exoderm stretches. Bottle cells pushed out of existence. 2. Involution starts. Cells crawl to animal pole. Blasocole is pushed and eliminated.
Archenteron is created MZ- marginal zone, inside of embryo. middle IMZ, involuting marginal zone, extends vegitally. Mediolateral intercalation drives convergence- extension Cells have podia to climb up the blastocoel roof, which is ectoderm. 3. Convergent-Extension of the equatorial presumptive mesoderm. Moving toward the dorsal and anterior 4. Epiboly of the ectoderm- close the yolk plug and create the blastopore. Epiboly of the ectoderm is accomplished by cell division and intercalation go in between eachother and cause spreading. Epiboly Radial intercalation- cells stack on eachother and flatten out, whereas in convergent-extension Medio-lateral intercalation, the cells create a one cell line from a plane of cells. Bottle cells have invaginated, the mesoderm is extending, marginal zone is involuted (IMZ). NIMZ , ectoderm that doesnt go in the embryo but it covers up using epiboly. Radial interaction of the ectoderm to spread it. In the mesoderm, it is undergoing medio-lateral intercolation to enlongate the layer. A bit of it (medio-lateral intercolation) happens in the ectoderm that will be the notochord.
Morphogenesis depends on changes in tissue cohesiveness and integrity. Two general ways tissues are organized: 1. Epithelium o Layered, cohesive cells, regularly arranged, mostly immobile but can move as a group. Most tissues move as epithelia during gastrulation. 2. Mesenchyme o Loosely arranged, even single cells can be highly motile. Epithelial cells o Dispersal- epithelim becomes mesenchume (entire structure) Epitheliam-Mesenchymal Trnasition = EMT o Shape change or growth cells remain attached as morphology is altered. Like a group of bottle cells. Some long some short. Microillaments o Cell Migration (intercalation) rows of epithelia mege to form fewer rows. o Migration formation of free edges. Cell surface properties, affected by cell differentiation, determine how cells interact and organize to form tissue They hold together because they have specific adhesion proteins. They can reorganize by binding to eachother via cell adhesion molecules. Key process: Cadherin-mediated cell adhesion o Two cells cadherins bind. Calcium is a co factor to make this work. o Creates a connection between the two cells. o Bind calcium and the other cadherin from the other cell. o Homophilic, express same cadherins; they bind. Blocking cadherin expression causes tissue at animal pole to disperse. Depleation of EP-cadherin mRNA results in the loss of adhesion between blastomeres o Protocadherin expression seperates axial (notochord) mesoderm and paraxial mesoderm ,somites etc. o Each major tissue express specific cadherins.
Determinants to tell cells what to do Molecules When one cell divides, once cell gets something and the other doesnt Localized RNA, only in one part o Animal Pole: An-1 RNA helicase An-2 Mitochondrial ATPase subunit Etc. o Vegital Pole o Vgt o VegT o Xwv11 First they are everywhere, over the course of development, they become localized.
Lecture 5 1. Maternal mRNAs- VegT loss of function 2. Dorsal axis induction by Wnt signal 3. Induction of the germ layers: TGF-betas Vg1, wnt11, VegT all found in the vegetal pole, close to the membrane. Vg1 is a growth factor, VegT is a transcription factor that binds to DNA. Wnt11 is also a growth factor Testing the embryonic functions of localized mRNAs. Can be tested by blocking their activity with antisense oligonucleotide inhibition of protein synthesis in eggs. Two ways commonly used ways to block mRNA function o Inject chemically modified antisense DNA oligonucleotide (e.g. Morpholino) into fertilized egg or cleaving blastomres to block translation o Inject antisense DNA oligonucleotide into oocyte to destroy the mRNA through endogenous RNAseH. Antisense = Complementary orientation to mRNA sequence. Morpholino oligo inhibits mRNA translation in egg sterically blocks ribosomes from initiating at the AUG start codon. DNA oligo triggers mRNA destruction by RNAseH in the oocyte. Will break up the mRNA Host transfer, take the oocyte, manipulate it and then inject it into the female VegT is needed to develop normally. Loss of VegT inhibits expression of endoderm genes. Expression of nodal (Xnr-) and derriere genes are lost in embryo once VegT is inhibited. Overview: maternal determinants on the dorsal side are influenced by cortical rotation. 4 signal model. Secreted signals. All are extracellular signaling. 1 and 2 are in blastula stages. o endoderm and mesoderm on vegetal side. 3 and 4 and in the gastrula stages Signal 2 Dorsal axis formation by a maternally-loaded wnt signal o Cortical rotation moves dorsal determines o Wnt11 activates those dorsal determinants o The wnt pathway causes beta-catenin to stabilize and move to the nucleus
o Beta catenin + TCF/LEF activates particular genes on the future dorsal side of the embryo Fertilization triggers cortical rotation Exposure of UV light after fertilization blocks the cortical rotation. Blocks microtubules fibers from forming because UV blocks tubulin subunit polymerization. Blocking CR causes loss of dorsal and head structures Cortical roation o Molecules move in the cortical rotation o Dsh binds to GBP which binds to Kinesin which moves along microtubules. o At the same time, Wnt11 mRNA moves slowly by cortical rotation. o These molecules reach dorsal side, causes inhibition of GSK3 and Bcatenin. o Beta-Catenin in the nuclei on the dorsal but not ventral side of midblastula embryo. Proteins that function in Wnt growth factor signaling o Wnt11 growth factor o Wnt receptors frizzled and LRP o Dishevelled (Dsh) o GSK3, APC, Axin = the destruction complex o GSK3 binding protein (GBP) o Beta catenin and LEF/TCF The proteins in italic are considered dorsal determinants o Wnt growth factors are a paracrine type of factor- affects cell next to it. Induction modes o Paracrine signaling signaling between cells. o Juxtacrine signaling needs cell contact. Wnt signaling pathway
Wnt signaling attaches to frizzled protein receptor which activates disheveled protein which inactivates GSK3 and APC, axin which allows beta catenin to go in the nucleus and inteact with LEF/TCFF which activates transcription. When the pathway is off, the molecule complex GSK3 and APC, axpin will degrade Beta catenin which will never reach the nucleus. To activate the pathway you have to inactive and inactivator. By inactivating GSK3, you activate Beta catenin. Wnt turns on specific genes Wnt11 growth factor is part of signal 2 Dorsal determinants activate the wnt pathway Enrichment of beta-catenin mRNA induces an extra head Depletion of maternal wnt11 blocks dorsal axis formation. (dorsal tissues organized along the anterior to posterior) Beta-cathenin activates: o Goosecoid o Siamois o Chordin o Xnr3 These genes are important for creating head and dorsal side. Signal 2 Signal 1 which is part of Signal 2 Induction to create mesoderm and part of endoderm
Key experiment: Animal cap pushed against vegetal cells, doesnt become ectoderm, its destined fate, but mesoderm. One experiment took different cells of ectoderm from fate map, and were connected to animal cap to see what part of mesoderm it created. Found cell 1 to be very different. Called Nieuwkoop center. Formation of head due to wnt11 signal. Signal 1 Mesoderm inducing signals. Signal 2- dorsal mesoderm inducing signals. Includes organizer and niewkoop center What are mesoderm inducers? o Took animal cap (skin) and did an assay on it. The right type of substance, it will create mesoderm o Animal cap in Activin turned into an embryo head structure. Activin B is a TGF-Beta member Members of TGF-beta and FGF growth factor families are mesoderm inducers o TGF-beta superfamily Activin not real inducer, signal molecules. Doesnt ccause loss of mesoderm with oligo. The ones below are. Vg1 Nodals Derriere Bone morphogenetic proteins Fibroblast Growth Factor family (FGF) Vg1 growth factor is in the vegetal cell in blastula stages. It is a mesodermal inducer Nodal genes are expressed on a higher level on the dorsal side than ventral side Nodal Pathway for vg1, derrier, and nodal o Smad2 or Smad3 bind Smad4 goes to the nucleus and causes gene transcription or repression FGF genes are expressed in the marginal zone. Helps out mesoderm formation process VG1 destruction causes head and A-P axis loss. No mesoderm forms if Vg1 mRNA is not expressed. Specific genes are not expressed Signal 1 = FGFs + TGFB mix (activin, Vg1, Nodals, Derriere, Bone morphogenetic proteins) Signal 2 = FGFs + TGFB mix + Wnt VegT is a transcription factor that activates Nodal genes. o VegT is a protein in the T-box family of transcription factors characterized by a conserved sequence that binds DNA. o Depletion of VegT mRNA blocks mesoderm and endoderm formation. No gastrulation o Nodal and derrierer genes o Veg1 activates nodal and derrier genes. Signal 1 = Nodals, Derriere and Vg1 Signal 2 = Vg1 + nodals+ derriere + Wnt11 making mesoderm and organizer.
LOF experiments show that these growth factors regulate mesoderm induction
Summary of experimental evidence: Depletion of Vg1 maternal mRNA inhibits mesoderm formation Blocking expression of nodal genes inhibits mesoderm formulation Dominant negative receptors for nodal, activin and Vg1 block mesoderm formation Expressing Proteins that bind and block nodal (e.g. Cerberus short) block mesoderm induction. VegT maternal mRNA activates transcription of nodal genes. Blocking VegT expression (using oligo-mediated depletion) inhibits mesoderm formation. Blocking FGF receptor activity reduces mesoderm induction. Chapter 7 notes Morula- an embryo containing 16 to 64 cells
The amphibian blastocoel serves two major functions. First, it permits cell migration during gastrulation; and second, it prevents the cells beneath it from interacting pre- maturely with the cells above it. EP-cadherin RNA acts as adhesion, keeping cells together. mid-blastula transition activation of the genome. Modification of certain promoters regulate gene expression- methylated DNA Gastrulation movements in frog embryos act to position the mesoderm between the outer ectoderm and the inner endoderm. marginal zonethe region surrounding the equator of the blastula, where the animal and vegetal hemispheres meet. The dorsal side.
Bottle cells initiate gastrulation. contraction of these form the blastopore.
Vegetal rotation places the prospective pharyngeal endoderm cells adjacent to the blastocoel end 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
Removal of the involuting marginal zone (IMZ) cells stops archenteron formation. Next, Involution at the blastopore lip. o When the migrating marginal cells reach (and become) the dorsal lip of the blastopore, they turn inward and travel along the inner surface of the outer animal hemisphere cells, toward anterior. Thus, the cells constituting the lip of the blastopore are constantly changing. First is the pharyngeal cells
The Blastocoel is displaced, the lip expands which forms the yolk plug. Endoderm goes inside. Epiboly of the ectoderm to spread the external layer. Involution of the mesodermal cells Convergent extension of the dorsal mesoderm. o Involution begins dorsally, led by pharyngeal endoderm and the head mesoderm o The IMZ is originally serveral layers thick, before involution through the blastopore lip, it thins out, called convergent extension. What drives convergent extension? o Polarized cell cohesion. As mesodermal movement progresses, convergent extension continues to narrow and lengthen the involuting marginal zone. The involuting cells contain the prospective endodermal roof of the archenteron in its superficial layer and the prospective mesodermal cells, including those of
the notochord, in its deep region. Toward the end of gastrulation, the centrally located notochord separates from the somitic mesoderm on either side of it, and the notochord elongates separately as its cells continue to intercalate
Epiboly of the prospective ectoderm During gastrulation, the animal cap and noninvoluting marginal zone cells expand by epiboly to cover the entire embryo
Determination
The vegetal cells have two major functions, one being to differentiate into endoderm, the other to induce the cells immediately above them to become mesoderm.
VegT is appointed to the vegetal cells during cleavage. When removed, the embryo becomes epidermis. No mesodermal or endodermal componenets.
VegT activates the zygotic transcription of genes encoding sever- al members of the TGF-p paracrine factor family, Once the dorsal portion of the embryo is established (opposite the point of sperm entry), the movement of the involuting mesoderm establishes the anterior-posterior axis The first endomesoderm to migrate over the dorsal blastopore lip will induce the ectoderm above it to produce anterior structures, such as the forebrain; mesoderm that involutes later through the dorsal blastopore lip allows the ectoderm to form more posterior structures, such as the hindbrain and spinal cord. This process, whereby the central nervous system forms through interactions with the underlying mesoderm, has been called primary embryonic induction and is one of the principal ways that the vertebrate body becomes organized. Spemanns experiment
The experiments of Spemann and Mangold showed that the dorsal lip of the blastopore, along with the dorsal mesoderm and pharyngeal endoderm that form from it, consti- tuted an "organizer" able to instruct the formation of embryonic axes. It appeared, then, that something in the region of the gray crescent was essential for proper embryonic development. It initiates gastrulation Gray crescent forms the dorsal blastopore lip
When gray crescent was split unevenly, only one part would develop into a larva.

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Comparative embryology- a traditional approach that unites with

How do you make a fate map?

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