Animal Developmental Biology - Lecture notes - Lecture 1 PDF

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Animal Developmental Biology Lecture 1 1. Fertilization  Zygote (single cell) Animal Development  Adult 2. Model systems (easy to study, yet representative of animal development more generally) a. The frog b. The fish c. The chick d. The mouse e. The worm f. The fly   

Conjoint twins Distinct from the shoulders up Died 8 months old

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Only adults in America joined at the head, 42 years old Pennsylvania Share bone, tissue and blood lines, joined at corner of left eye Share brain tissue, but have individual identities Siamese twins Comes from two siam twins that had joined livers Proclaimed their oneness Seen as having political or religious significance Signify political union… Conjoined twins as a natural phenomenon

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The heart apex is pointing down to the right Spleen is on the right Stomach is on the right All of ours are on the left Cytosinversis (left to right reversal of the internal organs; rare but in conjoined twins) Seen in Rita and Christina

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13 days after conception, embryo begins to organise itself (gastrulation) Gives the embryo its geometry Remove the membrane surrounding the egg, to it upside down, and excise the tissue, then graft into another embryo on the other side Would the transplanted cells behave differently from the hosts? Almost all of the embryos died Transplanted cells organised the host cells into an extra, but conjoined creature Individual cells don’t govern their own fates Organiser limited the potential of cells around it, was the origin of order

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Organiser = clump of cells

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Amsterdam = museum with collection of mutants Terrontolity? – study of monsters Most of the specimens in the museum are mutants,

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Mermaid syndrome Legs are fused together, feet protrude like flippers Usually fatal because it affects many organs, like the heart Midline disorder, lower half of body collapses in on itself Operations took plastic surgery to a new level, lower half of body had to be completely rebuilt Tiffany, only person in the world to survive with it to the age of 16

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Causes of it have been obscure, but knew it was the organiser Needed to find the substance secreted by cells in the organiser Discovery was called Corden – it was a signalling molecule Retinoic acid

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If you cut the tale off a tadpole it regenerates, but if you put it in retinoic acid you get a tangle of legs

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Last body part to form – Face Pig that has 2 faces, 2 snouts, 4 nostrils, 3 eyes (Called Ditto) Had an optic nerve that connected to the third eye, but unsure if he could see through it Chicken with 2 upper beaks Sonic gene – place a soaked bead in a different area in the embryo, but still in the face region

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Lecture 2 

Xenopus o Page 43 in Gilbert o Red part is the Deuterostomes which include the vertebrates

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Deuterostomes

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Page 27 Fertilization (0h. 1 cell)  Cleavage (rapid cell divison = 6h. 10,000 cells)  Gastrulation (re-arrangement of cells from interior to exterior, generates 3 germ layers; 10h. 30,000 cells)  Organogenesis/Neurulation (generates the organs, infold of cells from the dorsal surface to give rise to the CNS; 19h. 80,000 cells)  Larval stages (110h. 1,000,000 cells)  Maturity  …

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Cell division occurs precisely from the animal pole at the top, bottom is the vegetal pole? o Can be seen clearly as its pigmented o Higher concentration of yolk at vegetal pole o Yolk inhibits cytokinesis (division of the cell) Cleavage furrow moves down through the embryo to reach the vegetal pole Second cell division starts to take place, when first division is still incomplete Third cell division starts before second has finished, at right angles to first 2 divisions; embryo now divided up into 8 cells o 4 at the animal pole, 4 at the vegetal pole o Smaller cells at the animal pole and larger cells at the vegetal pole Blastula is the final stage o 10s of thousands of cells, with fluid filled cavity (blastocoel) towards animal pole Individual cells referred to through these stages are blastomeres Cells of the animal pole will give rise to the ectoderm, cells around vegetal pole gives rise to endoderm, and cells around equatorial region give rise to the mesoderm

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Cell division occur every 30 mins, very rapid Single cell zygote is over 1mm across, 1000 larger then a bacteria Genomes that need to be duplicated are so much larger than in the bacteria The embryo never grows in diameter, it just undergoes organisation Each cell generated all has its own copy of the genes

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Blastula then undergoes gastrulation Mid-blastular transition – when cell division stops and cells start to re-arrange = gastrulation The cells start to fold into the interior from the dorsal surface (back of the animal) o Cells left on outside are the dorsal-blastopore lip? Ectoderm gives rise to the outside surface, endoderm will line the inside/gut of the animal, mesoderm fills the middle Archenteron = fluid filled cavity  Gives rise to the lumen of the gut As it folds in, the mesoderm forms between the 2 Ectoderm cells move around the outside at the endoderm folds in Few endoderm cells still left on the outside, which is the yolk plug o ….

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As the endoderm folds in, it reaches the other side of the embryo, and where it comes into contact with the ectoderm with no mesoderm = where the mouth forms (secondarily) Anus forms at the connection between the archenteron and … Deuterostome comes from o Means mouth forming secondarily

Neurulation o To generate the CNS Small circle is the notochord, mesodermal tissue, runs from head to tail just below the surface o Send signals to overlying ectoderm that they’re going to give rise to the CNS o That region of the ectoderm are going to fold into the interior, which generates a neural groove as the cells fold up and close over o To pinch off the neural tube running all the way down the back

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Neuropore closure failure o The ends cant be sealed up o Not only in the frog, but in us as well o The ends are referred to as Neuropores o Has serious consequences (exposes brain the outside service so it degenerates = anencephaly; if tail end fails to close = spina bifila)

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Vertebrate cloning was achieved a long time ago King, 1952 o Blastula stage nuclei in Rana pipiens o Transferred those to anucelated zygotes (nucleus removed) o Embryo would develop fine through to adult stage Gurdon, 1962 o Differentiated tadpole intestinal nuclei in Xenopus laevis o Low success of the experiment o But could be increased by … o Shows a fully differentiated cell could be used to create a new individual, and could generate all the different cell types the animal needed  The information was still retained even after it was fully differentiated

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Fate Maps o A map of the fates of the cells that are present in the embryo at a particular time o Glastular stage (label cells on outside using a dye, follow through the development, and can see what cells are derived from the previously labelled cells)

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Highly regulative development If placed on the ventral side (belly rather than back), then the planted cell gives off signals telling the embryo… … Second embryo that formed is largely composed of cells from the host…

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1-cell stage o up to 80 mins

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2-cell, 4-cell, 8-cell stages o up to 25 mins As they change through the cell cycle, … Embryos are large, accessible (external fertilisation), robust and well-studied o Signalling molecules that direct development have been biochemically purified (KEY THING) But Xenopus laevis is an allotetraploid as a result of an evolutionarily recent hybridization, making the species far from ideal fro genetic approaches The advances in the field were made through a genetic approach Allotetraploid = recently during its evolutionary history, 2 species have come together and hybridized to generate this species o Its effectively a tetraploid Genetics is hard enough with a diploid

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Xenopus tropicalis is the only one in Xenopus species which is diploid o The adult is much smaller than X. laevis o Generation time is 4-6 months o Transgenesis procedures developed o Genome sequence completed 2010

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Desert tadpole mutants in tropicalis o Lack of circulating blood o Heart is there but no blood to pump o The blood cells are moving around in the tail of the wild type

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Key points o Classic model, very important in developmental biology o Robust to experimental manipulation o Identification of inducing signals o

Lecture 4 REVISION Disadvantages of mammals as model organisms for studying development  In viviparous mammals, embryonic development is entirely in utero  Genetic manipulation is more difficult  Long gestation and generation times  Ethical restrictions Advantage! – It’s a vertebrate mammal so closely related to use The sperm must fuse with the egg  Receptors on the surface of the sperm, bind specifically to the zona pellucida protein 3 (ZP3)  Binding triggers the acrosome reaction (releases enzymes from the head of the sperm)  Leading to digestion of the zona pellucida and then membrane fusion

 Mechanisms operate to prevent polyspermy (fusion of more than one sperm with an egg)  Fertilization has occurred with 2 sperm cells fusing with an egg  18 chromosomes from each sperm nuclei  Sperm centrioles are brought into the cell at fertilization (2 centrioles); they divide so we have 4 centrioles  These centrioles organise the spindle of cell division  Each centriole sets up the corner for the spindle  6 spindles would be set up, so 6 equators for the cells to form  Each cell has an inappropriate number of chromosomes, so development would fail

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Mammals – sperm fusion leads to the release of the contents of cortical granules from the egg surface These granules contain enzymes that modify ZP1, 2 and 3; so sperm cells no longer penetrate the zona pellucida In other animals the cortical granules lead to the lifting of the vitelline membrane (tight against the surface of the egg) from the egg surface forming the fertilization membrane Both of these aspects are very slow! When sperm fuses with egg, its initially a fast but temporary fusion So a fast but temporary block to sperm fusion comes from an immediate change in the electric potential of the cell membrane

Movement of pre-implantation embryo down the maternal reproductive tract

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Oocyte is released from the ovary into the oviduct (where fertilization occurs) Initial cleavage occurs during this process It reaches the uterus where it implants itself – subsequent development occurs

Mammalian cleavage is equal, asynchronous, rotational and slow

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Equal o The initially formed cells are equivalent in terms of developmental potential (can contribute to any of the cells subsequently formed) Asynchronous o Don’t just et 1,2,4,8,16.. stagesl you also get 3,5,7,9 stages Rotational o First cell division occurs in one direction, whereas in the next cell division, the next 2 cells both divide at right angles to the first stage, but also at right angles to each other Slow o Takes 24 hours to part

 Compaction occurs at the 8 cell stage

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Compaction = they appear to become stuck together Tight and gap junctions are forming between the cells Tight junctions (important for sealing up the embryo), generate an inside and outside Gap junctions (important for communication), allow small molecules to flow between cells Cell division carries on and fluid filled cavity forms (blastocoel)

Differentiation of the inner cell mass and the trophoblast, and development of the blastocoel, to form the blastocyst

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This stage is a blastocyst, not a blastular Consists of essentially 2 cell types o Outside cells (trophoblast or integrin proteins) – generate the placenta for interaction with the mother o Inside cells (inner cell mass or hypoblast) – give rise to the foetus

Primary cell lineages in the mammalian embryo

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Primitive streak – when the cells moves into the inside

The blastocyst has to hatch from the zona pellucida before implantation  Blastocyst produces enzymes that digest the ZP and allow it to hatch out  Important event that has to be carefully controlled  If escapes too early, will result in eptopic pregnancy (implant into any tissue its up against) The hatched blastocyst then implants into the wall of the uterus  The inner cell mass now consists of the epiblast and the hypoblast  After implantation, a second fluid filled cavity begins to form (amniotic cavity)  Forms on the inner side of the cell mast where the epiblast is The amniotic cavity grows  The extra embryonic endoderm forms the yolk sac (although there is no yolk in the mammalian embryo) Gastrulation  Occurs with cells moving inwards from what will be the dorsal surface, forming the endoderm then the mesoderm (around 16 days)  The node (equivalent of the frog organiser) is seen at the end of the primitive groove

Human neurulation, with closure of the neural tube at the dorsal surface

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The amniotic cavity wraps around the entire embryo, with the ectoderm, mesoderm and endoderm folding round to meet on the ventral midline

Neural crest cells (characteristic of vertebrates)  The last cells derived from ectoderm to move into the interior  Migrate individually to various locations forming the: cartilage, bone and muscle of the face, the peripheral nervous system, the pigment cells of the skin and adrenal medulla (modified sympathetic ganglion – in adrenal gland above kidney)  Equivalent to neural crest cells have been seen in other chordates in the past year  The shape of the face arises from neural crest cells that have migrated from the dorsal surface of the embryo

Formation of the amniotic cavity in the chick

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For birds, the chorion is important for gaseous exchange In mammals, its important for the interaction in the placenta with the mother Evolution of these events was crucial, allowed animals to escape the need to develop in water and invade the land Allantoic membrane is where toxic products develop away from the embryo

Lecture 5 REVISION Mammalian cells are very highly regulated  The cells of the inner cell mass of the blastocyst show totipotency (ability to differentiate into any type of body cell)  Particularly regulative due to the nature of the early development implanting in the uterus wall 1. Origins of identical human twins (1 in 400 births)

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Result of a single fertilization event generating a zygote that divides into 2 individuals 3 different scenarios can occur A – if separation occurs before day 5 of fertilization (1/3 of instances); end up with 2 blastocysts that implant into uterus wall, and develop 2 placentae B – between day 5 and 9 (2/3 of cases); single blastocyst but inner cell mass separated into 2 clumps of cells, single placenta generated, shared by 2 individuals; develop in own amniotic cavity, so effectively develop in own environment C – separation occurs after day 9 (very rare); derive from a single blastocyst, single clump; embryos develop sharing the amniotic cavity and risk being conjoined = developing in same fluid filled environment, cells can become intermingled, but development carries on as best it can Conjoined twins are joined through the same part of their anatomy Hagness (the wrong way) of the organs within twins, as well as the whorl on the head One twin has normal hagness the other has opposite hagness (left-right orientation)

2. Cloning by embryo splitting – here in a non-human primate (Rhesus)  Possibly derive a clone of individuals from early on in embryos  Take an 8-cell mammalian embryo split into 4 pairs of blastomeres, which can be replaced in empty zonae for culture

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Culture in vitro for a while, then implanted into a surrogate mother and will develop due to its high regulative nature A miscarried pregnancy and normal development resulted from transfer of two quadruplet embryos each to two surrogates

3. PGD – Pre-implantation Genetic Diagnosis  Cell is removed from an early human embryo for genetic testing  The remaining cells will give rise to a complete individual  After genetic analysis of the isolated cell, the remaining embryo is returned to the mother with implantation 4. Formation of chimaeras  Cells from different mouse blastocysts can be mixed together and all can contribute to all tissues

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Once the 2 embryos have been mixed up, the cells can still pass signals between each other and generate a single blastocyst Place into a surrogate mother and will develop fine Chimaera – ½ cells from one strain, ½ from the other (hence 2 tone mouse) Cells from multiple 4-cell Rhesus monkey embryos can be mixed together to create chimeric infants

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Single chimeric individuals produced

5. ES cells – embryonic stem cells  Inner cell mass can give rise to any cell types present in embryo  They can be isolated then cultured, as ES cells  These cells divide and are maintained in an undifferentiated state  By applying the right protein signals, these cells can be directed to differentiate into different cell types (e.g. blood cells, nerve cells, muscle cells), potentially for medical applications 6. Reverse genetic analysis in the mouse  ES cells can be genetically manipulated in vitro  Once these manipulated cells are returned to the host blastocyst, they can contribute to the germ line of the embryo and so genetically manipulated strains can be established  These cells can be maintained in culture in large numbers  Manipulate cells and change their DNA content o Indicated in the picture below

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Can use electric shock to electro berate the DNA into these cells This process ^^^ allows us to generate a transformed strain of mice The embryo will be chimeric, consisting of cells from host blastocyst and ES cells in culture

DNA introduced into ES cells can recombine homologously with the cell’s own genetic material

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DNA at the top is being introduced, contains an extra bit of DNA in red Now it can recombine with the chromosomal copy of the gene When recombination occurs the extra DNA is inserted into the chromosomal copy of the target gene Can select for the very rare events, and identify the specific cells where the event has occurred If inserted randomly, the blue bit of DNA will be inserted as well Can generate a strain of mouse that has a very precise modification

2007 Nobel Prize for Physiology or Medicine  Capecchi, Evans and Smithies  “Principles for introducing specific gene modifications in mice by the use of embryonic stem cells” Reverse genetics in the mouse to knockout sets of genes

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Top  wild type; Middle  hox10aaccdd knockout (lumbar to thoracic vertebrae); Bottom  hox11aaccdd knockout (sacral to lumbar vertebrae) Hox genes originally identified in D. melanogaster Hox10 exists in 3 different versions (a,...