Lecture: Blastogenesis PDF

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Lectures from Animal Science year 2 with Ioannis Vatsos...

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Embryology: Blastogenesis Early development Events in fertilized egg ● In vertebrates, except rodents, the centriole is paternally inherited. The paternal proximal centrosomal material is necessary in the formation of the spindle for correct mitosis. Duplication at pronucleus stage and migration around to pronuclei to the opposite pole and formation of the mitotic spindle. The first cell divisions ● First step of activation of the egg metabolism, replication of the centriole. ● MPF mitosis-promoting factor is responsible of completion of meiosis II and is active in the transition fertilisation - first cleavage. ● The 2 sets of chromosomes move in equatorial position in the spindle. ● First mitosis is precisely controlled and is important for further development of the embryo. ● Maternal factors are involved in the egg metabolism. Rapid divisions, G1 and G2 phases are omitted Frog: ≈ 15 divisions in 43 hours = 35 000 cells produced. The cells are named blastomeres. The blastomere size decreases gradually with increasing number of mitosis. Re-establish a normal nucleus/cytoplasm ratio. The early blastomeres are totipotent, they can regenerate a complete embryo, but rapidly they become more and more specialised and differentiate into different germ lines. ● ●

Viviparous animals: constant temperature, so the timing of divisions is constant Oviparous animal: ○ Constant developmental rate because of parental care in birds ○ In amphibian the developmental rate is dependent on the ambient temperature. Optimum temperature range, extreme temperatures result in abnormal development.

Main patterns of cleavage ● Holoblastic mesolecithal radial: amphibians ● Holoblastic isolecithal rotational: mammals ● Meroblastic telolecithal discoidal: birds Development stages All embryos go through the same developmental stages: ● Zygote, cell cleavages, morula (solid ball of blastomeres), development of an internal cavity (blastocoel) at blastula stage, then gastrula stage formation of the germs layers after rearrangement, and the subsequent development into the different tissues and organs organogenesis.

Fertilization events in frog Rotation is 30 ° Axis determination of the embryo: ● Animal pole: future anterior part ● Vegetal pole: future posterior part ● Grey crescent: future dorsal side ● Point of entry of sperm: future ventral side Early cleavages in amphibians ● Cleavage is holoblastic and unequal The 2 first divisions give equally sized blastomeres. Animal pole is pigmented and blastomeres divide rapidly (small). Blastomeres at vegetal pole containing yolk divide slowly resulting in bigger blastomeres. From the 5th division the regularity of division is lost. Morula = aggregate of 64 blastomeres. Short duration. From 128 cells-stage Formation of a fluid-filled cavity, the blastocoel, that develops in the middle of morula. Separates different kind of cells and will allow cell movements at gastrulation. no increase in size, cells are getting smaller. 2 layers:

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○ The epiblast ○ The hypoblast Before MBT ○ Synchronic and rapid divisions ○ Only maternal mRNAs are translated ○ Zygotic DNA is not yet expressed ○ MBT events ○ Progressive activation of zygotic genes ○ Cell cycle lengthens, G1 and G2 mitotic phases are added ○ Asynchronous cell divisions ○ Development of cell mobility ○ Initiated when Nuclear/Cytoplasmic ratio reaches a threshold (cells decrease in size, N/C ratio increases) Blastocoel floor expands, bringing presumptive neuroectoderm (B1) into contact with inductive mesodermal precursors (C1)

This zone is called the Spemann- Mangold organiser indicated by * Differentiate into dorsal mesoderm Induce the formation of paraxial (=lateral) mesoderm Induce the formation of the neural tube

● Initiate movements at gastrulation ● Epiboly: cells proliferate and cover the embryo ● ● Involution the movement through the blastopore lip ● ● Invagination process that forms the 3 germ layers and brings the endoderm inside The organiser initiates gastrulation movement, an extensive cell migration that gives rise to the 3 germ layers Organisation of the 3 germ layers 1. Ectoderm (epidermis, nervous system...) 1. 2. Mesoderm (skeleton, muscles, heart, kidney, blood..) 3. Endoderm (respiratory, digestive system and associated glands) Inductive interactions between the different cells of the germ layers will result in the development of the different organs ● Fertilisation in the upper part of the oviduct before albumen and shell are synthesised ● ● Cleavages start at once after fertilisation ● ● When the egg is laid the blastoderm has 50 000 cells Cleavage discoidal meroblastic and does not extend to the yolk The first cleavages are vertical: single layer blastoderm Thereafter equatorial and vertical cleavages until 5-6 layers of blastomeres Blastomeres absorb some of the albumen and secrete it between blastoderm and yolk ○ Sub-germinal space ○ The deep cells of the centre of the blastoderm are shed and die. One cell layer thick area pellucida The peripheral ring of the blastoderm that has not shed the deep cells forms the area opaqua Between areas pellucida and opaqua, marginal zone that is very important in determining cell fate (A) ● area pellucida cells have delaminated and migrated individually into the subgerminal cavity to form the polyinvagination islands = primary hypoblast(B). 5 to 20 cells in clusters ● ● a sheet of cells from the posterior margin of the blastoderm (Koller's sickle) migrates anteriorly to join the polyinvagination islands, thereby forming the secondary hypoblast (C) ● ● 2 layers blastoderm with blastocoel Formation of the primitive streak = thickening of the epiblast at the posterior region of the embryo

Just anterior to Koller’s sickle First cells migrate through Hensen’s node. Centre is a funnel- shaped depression Functional equivalent to the blastopore lip= the organiser 1. Streak elongates towards the future head region 2. 3. 1. Streak define the axis of the embryo: ant/post, dorsal/ventral and left/right First cells migrate through Hensen’s node, migrate deep and will give the endoderm of the foregut and most of the extra-embryonic membranes Displace the hypoblast cells to the anterior portion of area pellucida = germinal crescent, contain the precursors of the germ line, but don’t form the embryo Next cells moving will give the mesoderm ● Epiblast: ○ Embryo ○ Large part of the extra-embryonic membranes ● ● Hypoblast: ○ Does not contribute at all to the embryo tissues ○ Membranes of the yolk sac and the stalk that connects the yolk sac to the digestive tube ● While ingression of mesodermal cells continues the primitive streak start to regress ● Hensen’s node migrates backwards to give the anal region ● The 3 layers are now established ● ● Epiboly of ectoderm Epiblastic cells proliferate and migrate to surround the yolk sac. After app 4 days the yolk sac is completely enclosed Capacitation of spermatozoa in uterus Oocyte is ovulated in the abdominal cavity, wrapped in cumulus cells

Fimbria lined with hair-like cilia catches the oocyte

Fertilisation in the upper part of Fallopian tube, the ampulla

First cell division starts after 12-24 h, very slow development

Holoblastic isolecithal rotational: ● 1st cleavage meridional ● 2nd cleavage 1 blastomere divides meridionally, the other equatorially (some exceptions) ● ● Asynchronous cell divisions: odd numbers of cells ● ● Compaction: loose arrangement at 8-cell in mouse, so the cells form a compact ball ● ● Morula stage: 16 blastomeres ● ● Further divisions and formation of the blastocoel Transition starts early with variations between species: ○ Mouse: starts late 1-cell stage ○ ○ ○ ○ ○ Rabbit, cow, rhesus macaque and human : 4 to 8-cell stage. ○ Maternally encoded factors persist for some time and are essential for development Blastula contractions that end up with hatching from the zona pellucida Implantation: ○ Embryo development ○ Extra-embryonic membranes development that will give rise to the placenta for nutrient exchange between the mother and the growing embryo/foetus Formation of the primitive streak Epiblast in blue Hypoblast in yellow Fate of the cells depends on their position in the embryo ● XX in female and XY in male, but equal expression of X chromosome in male and female, regulation by dosage compensation ● ● 1 X chromosome express Xist a long non coding RNA, histones and DNA methylation. Very condensed heterochromatin = genes are not expressed. Pseudo autosomal part on the short arms are expressed at least in humans. Common area on X and Y that pairs up during meiosis ● ● Inactivated X replicates later ● ● ● Imprinted inactivation: Late morula in the cell lineage that will give the extra-embryonic membranes: Xp is inactivated ● ● Random inactivation in the epiblast. No further changes, inactivation is highly stable.

Chromosomes from a mouse cell. Red indicates the visible Xist RNA on an inactivated X chromosome (micrograph). (Image: Ng K et al. EMBO Reports 2007, 8: 34) ● Males ZZ and female ZW ● ● Mechanism that equilibrates expression of Z chromosome ● ● Dosage compensation does not affect the entire Z chromosome, no inactivation of Z chromosome ● ● Degree of dosage compensation varies between genes...