Chapter 5 Flashcards
The egg cytoplasm plays a major role in determining
patterns of cleavage, gastrulation, and cell specification (by interacting with the nuclear genome established at fertilization).
During cleavage and gastrulation, the major axes of the embryo are determined and the embryonic cells begin to acquire their respective fates. Three body axes:
- Anterior-posterior (head-tail)
- Dorsal-ventral (back-belly)
- Left-right
Different species specify these aces at different times, using different mechanisms.
Metazoans Sponges – develop completely different from any other animal group.
- Has three major types of somatic cells, one of which, the archeocyte, can differentiate into all the other cell types in the body.
- Individual cells can reaggregate to form new sponges, in some instances, species-specific (thought that the motile archeocyte collects its own species).
- Contain no mesoderm => no true organ systems, no digestive tube, circulatory system, nerves, or muscles.
- Do undergo gastrulation and pass through an embryonic and larval stage however.
- Share many features of development with all the pther metazoan phyla; including gene regulatory proteins and signalling cascades.
Metazoans Diploblasts – animals that have two germ layers, ecto and endoderm. Includes jellyfish etc.
- Most have no mesoderm and radial symmetry.
- Some however, have mesoderm like derived tissues, but it seems to have evolved independently from traditional mesoderm (evolutionary convergence)
Metazoans Triploblasts – animals with three germ layers, bilateral symmetry (anterior-posterior)
- Protostomes – (Greek, “mouth first”), includes molluscs and arthropods, mouth is formed first, anus later at a different location.
- Deuterostomes – (Greek, “mouth second”), includes chordates (includes vertebrates, chord = notochord) and echinoderms, mouth is formed second, anus first
Blastomere
cell derived from cleavage in an early embryo
Blastula
embryonic stage composed of blastomere (mammalian = blastocyst)
Blastocoel
fluid filled cavity within the blastula
Stereoblastula
blastula that lacks blastocoel
Blastopore
invagination where gastrulation begins
Cleavage is?
a series of mitotic divisions after fertilization, whereby the enourmous volume of egg cytoplasm is divided into numerous smaller, nucleated cells.
In most species (mammals chief exclusion), both the initial rate of cell division and the placement of the blastomeres with respect to one another are under the control of the proteins and mRNAs stored in the oocyte. Only later does these come under the control of the newly formed genome.
Cleavage and cytoplasmic volume
Initially, cytoplasmic volume does not increase instead the zygote cytoplasm is divided into increasingly smaller cells.
Cleavage is so rapid in beginning in vertebrates probably to quickly restore the somatic ratio of nuclear volume to cytoplasmic volume, often accomplished by abolishing gap periods of the cell cycle (G1 + G2). Fx. Frog egg – 37k cells in 43 hrs
Blastomere cell cycle
generally biphasic – M (mitosis) and S (DNA synthesis).
- MPF highest during M, undetectable during S.
- Shift between driven solely by the gain and loss of MPF activity.
Mitosis-promoting factor (MPF)
regulates cell cycle of early blastomeres.
- Triggers entry into M phase (nuclear envelope breaks down, chromatin condenses into chromosomes), lasts about an hour, then it’s degraded and chromosomes return to S phase.
- Consists of two subunits: larger = cyclin B, smaller = cyclin-dependent kinase (CDK) (fx cdc2)
- Most regulators of cyclin B (and thus MPF) is stored in egg cytoplasm => cell cycle independent of nuclear genome in beginning, until used up
Mid-blastula transition (MBT)
When MPF is no longer controlled by regulators present in egg cytoplasm, but nucleus begins to synthesize them by itself.
- Gap stages (G1 + G2) are added to biphasic cell cycle; xenopus at 12th cleavage, drosophila at 14th +17th respectively.
- Synchronicity of cell cycle lost, pga different cells synthesize different regulators of MPF.
- New mRNAs are transcribed, many encode proteins necessary for gastrulation
Cytoskeletal mechanisms of mitosis - Mitotic cleavage is the result of two coordinated processes:
- Karyokinesis: mitotic division of cell nucleus, mechanical agent is mitotic spindle, with its microtubules composed of tubulin
- Cytokinesis: the division of the cell itself, mechanical agent is contractile ring of microfilaments made of actin.
Division plane is controlled by
the placement of the centrioles, pga controls orientation of mitotic spindle.
Depending on placement, daughter cells can separate along ventral/dorsal axis, anterior/posterior or left/right axis, and symmetrical / asymmetrical.
Embryonic cleavage patterns determined by:
- The amount and distribution of yolk protein within the cytoplasm – determines where cleavage can occur and the relative size of the blastomere. In general, yolk inhibits cleavage.
- Factors in the egg cytoplasm that influence the angle of the mitotic spindle and timing of its formation
Embryonic cleavage patterns
- Holoblastic – (Greek holos, “complete”), cleavage furrow extends through the entire egg, little yolk = other ways to obtain food (most through voracious larval form, mammals through placenta)
- Meroblastic – (Greek meros, “part”), only a portion of the cytoplasm is cleaved, cleavage furrow does not penetrate the yolky portion, which serves as sufficient food to nourish these animals throughout embryonic development
Embryonic cleavage patterns - Holoblastic (Greek holos, “complete”), cleavage furrow extends through the entire egg, little yolk = other ways to obtain food (most through voracious larval form, mammals through placenta)
- Isolecithal – (Greek, “equal yolk”), eggs with sparse, equally distributed yolk
- Mesolecithal – moderate vegetal yolk disposition
Embryonic cleavage patterns - Meroblastic (Greek meros, “part”), only a portion of the cytoplasm is cleaved, cleavage furrow does not penetrate the yolky portion, which serves as sufficient food to nourish these animals throughout embryonic development
- Telolecithal – dense yolk throughout most of the cell, only small area is free
- Centrolecithal – yolk in the center of the egg
Gastrulation
the numerous cells of the blastula are given new positions and new neighbours, and the multi-layered body plan of the organism is established.
Usually proceeds by some combination of several types of movements; these involve the entire embryo and migration in one part must be intimately coordinated with simultaneous movements in other parts.
Five basic types of cell movement:
- Invagination: infolding of a sheet (epithelium) of cells, much like the indentation of a soft rubber ball then it is poked. Fx sea urchin endoderm.
- Involution: inward movement of an expanding outer layer so that it spreads over the internal surface of the remaining external cells. Fx amphibian mesoderm.
- Ingression – migration of individual cells from the surface into the embryo’s interior. Individual cells become mesenchymal (i.e. separate from one another) and migrate independently. Fx sea urchin mesoderm.
- Delamination – splitting of one cellular sheet into two more or less parallel sheets. Resembles ingression on a cellular level, but result is formation of a new (additional) epithelial sheet of cells. Fx hypoblast formation in birds and mammals.
- Epiboly – movement of epithelial sheets (usually ectoderm cells) spreading as a unit to enclose deeper layers of the embryos. Can occur by cells dividing, changing shape, or by several layers intercalating into fewer layers (often, all three are used). Fx ectoderm formation in sea urchins.
Type I embryogenesis – snails and C.elegans nematode worm
- Immediate activation of the zygotic genes
- Rapid specification of the blastomeres by the products of the zygotic genes and by maternally active genes
- A relatively small number of cells (a few hundred or fewer) at the start of gastrulation.
The nematode C.elegans
Caenorhabditis elegans is a small, free-living (nonparasitic) soil nematode with relatively few cell types.
Also has a relatively rapid period of embryogenesis (ca 16 hrs), which it can accomplish in a petri dish.
Moreover, its predominant adult form is hermaphroditic, with each individual producing both eggs and sperm (can reproduce by self-fertilization or normal cross-fertilization).
The nematode C.elegans - random notes, why useful as model organism?
- Early development – gonades produce sperm that is stored in the Spermatheca. Later development – oocytes are produced in the oviduct => pass through the Spermatheca => get fertilised and arrive to the Uterus => Embryos leave through the Vulva
- Adult contains 959 somatic cells (558 cells present in newly hatched larva).
- The entire cell lineage for each cell has been traced through its transparent cuticle.
- C.elegans lineage is almost entirely invariant from one individual to the next; there is little room for randomness.
- Genome: 18K-20k genes (humans 20k-25k), but only 3% the number of nucleotides compared to humans. Lacks alternatively spliced RNAs like human genes => few / 1 protein pr gene. Plus each protein has only one function.
- Displays the rudiments of nearly all the major types of bodily systems (feeding, nervous etc, except no skeleton).
- Exhibits an aging phenotype before it dies.
- DNA injected into C.elegans is rapidly incorporated into their nuclei, and they can take up antisense RNA from its culture medium.
C.elegans fertilization:
egg becomes fertilized by rolling through a region of the embryo (the spermatheca) containing mature sperm.
When a sperm fuses with the egg cell membrane, polyspermy is prevented by the rapid synthesis of chitin (the protein comprising the cuticle) by the newly fertilized egg.
It undergoes early divisions and is extruded through the vulva.
C. elegans cleavage
zygote exhibits rotational holoblastic cleavage.
Early cleavage: each asymmetrical division produces one founder cell (denoted AB, E, MS, C and D) that produces differentiated descendants; and one stem cell (P1-P4 lineage).
C. elegans axis determination - AP axis
AP axis is determined before the first cell division, cleavage furrow is located asymmetrically along this axis of the egg, closer to what will be the posterior pole. First cleavage forms an anterior founder cell (AB) and a posterior stem cell (P1).
C. elegans axis determination - DV axis
DV axis is determined during the second division.
The AB cell divides equatorially (90° to AP), while the P1 cell divides meridionally (transversely) to produce another founder cell (EMS) and a posterior stem cell (P2).
Meridional division with founder cell and posterior stem cell to continue lineage always happens for stem cell lineage. EMS cell marks ventral region.
C.elegans axis determination - RL axis
RL axis is seen at the transition between 4- and 8-cell stage. Two “granddaughters” (ABal, ABpl) of AB cell are on the left side, while two others (ABar, ABpr) are on the right side.
C. elegans AP axis formation PAR proteins
“partitioning-defective”, distinctly arranged in oocyte.
- PAR-3 and PAR-6: interacts with PKC-3 (protein kinase), uniformly distributed in the cortical cytoplasm. PKC-3 phosphorylates PAR1- and PAR-2 restricting them to the internal cytoplasm.
- The sperm centrosome contacts the cortical cytoplasm through its microtubules and initiates cytoplasmic movements that push the male pronucleus to the nearest end of the oblong oocyte, this end becomes the posterior pole.
- These microtubules locally protect PAR-2 from phosphorylation, allowing it (and its binding partner PAR-1) into the cortex nearest the centrosome.
- Here PAR-1 phosphorylates PAR-3 causing it (and its binding partner PKC-3) to leave the cortex.
- At the same time, the sperm microtubules induce the contraction of the actin-myosin cytoskeleton toward the anterior => clears PAR-3, PAR-6 and PKC-3 from the posterior end.
- During first cleavage the metaphase plate is closer to the posterior, and the fertilized egg is divided into an anterior cell (AB) with PAR-3 and PAR-6, and a posterior (P1) with PAR-1 and PAR-2.
C. elegans DV axis formation
AB cell divides becoming longer than the eggshell is wide => sliding.
One cell becomes anterior (ABa), another posterior (ABp). Sliding also causes ABp cell to take a position above the EMS cell => ABp = dorsal, EMS = anterior
C. elegans LR axis formation
Not readily seen until 12-cell stage, when the MS blastomere (from division of EMS cell) contacts half the “granddaughters” of the ABa cell, distinguishing the right from the left side.
This asymmetrical signalling sets the stage for several other inductive events that make the right side of the larva differ from the left.
First indication of LR symmetry probably occurs at the zygote stage, where, just prior to the first cleavage, the embryo rotates 120° inside its vitelline envelope, always in the same direction to already established AP axis => indicating LR awareness.
Cell specification modes - C.elegans
C.elegans demonstrates both conditional (AB needs P1 descendants) and autonomous (P1 can develop without presence of AB). Details – see p. 173-176
C.elegans - Gastrulation
- Starts extremely early, just after the generation of the P4 cell in the 26-cell embryo.
- The two E daughter cells migrate from the ventral side into the centre of the embryo, where they divide to for, a gut consisting of 20 cells.
- Their inward migration creates a tiny blastopore; the next cell to migrate through (P4) is the precursor of the germ cells. Mesodermal cells (descendants of MS cell from anterior and C- and D-derived muscle precursors from posterior) move in next.
- Ca. 6 hrs after fertilization, the AB-derived cells that contribute to the pharynx are brought inside. The hypoblast cells (precursors of the hypodermal skin cells) move ventrally by epiboly, eventually closing the blastopore. And the two sides of the hypodermis are sealed.
- During the next 6 hrs, the cells move and develop into organs, while the ball-shaped embryo stretches out to become a worm with 556 somatic cells and 2 germline stem cells.
- 115 cells undergo apoptosis during development into adult.
- After four moults, the worm is a sexually mature, hermaphroditic adult, containing 959 somatic cells + numerous sperm and eggs.
Cell fusion - C.elegans
One characteristic of C.elegans that distinguishes it from most other well-studied organisms is the prevalence of cell fusion.
During gastrulation about 1/3 of all cells fuse together to form syncytial cells containing many nuclei. This fusion prevents individual cells from migrating beyond their normal borders.
In the vulva, cell fusion prevents hypodermis cells from adopting a vulval fate and making an ectopic (and non-functional) vulva.
35 metazoan phyla (patterns of animal development) Four major branches:
- the sponges
- diploblasts (two embryonic cell layers, endo and ectoderm)
- protostomes (worms, molluscs, arthropods)
- deuterostomes (echinoderms, vertebrate).
Last two are triploblastic, bilateral (anterior-posterior) animals.
Schizocoelous formation?
The protostome coelom (body cavity) forms from the hollowing out of a previously solid cord of mesenchymal cells (schizocoelous formation).
Enterocoelous formation?
Most deuterostomes form their body cavity from mesodermal pouches extending from the gut
Embryonic cleavage
Isolecithal – (Greek, “equal yolk”), eggs with sparse, equally distributed yolk
- Radial cleavage
- Spiral cleavage
- Bilateral cleavage
- Rotational cleavage
Radial cleavage
Holoblastic - isolecithal
echinoderms, amphioxus
splits along axes one at a time
Spiral cleavage
Holoblastic - isolecithal
Annelids, molluscs, flatworms
Cytoplasm unevenly distributed between daughter cells => spiral pattern
Bilateral cleavage
Holoblastic - isolecithal
Tunicates
Splits only along two axes (?)
Rotational cleavage
Holoblastic - Isolecithal
mammals, nematodes
Very similar to radial but end results different, pga asymmetric (?)
Telolecithal – dense yolk throughout most of the cell, only small area is free
- Bilateral cleavage
- Discoidal cleavage
Bilateral cleavage
Meroblastic - Telolecithal
Cephalopod molluscs
Along two axes “in” surface of yolk
Centrolecithal – yolk in the center of the egg
Superficial cleavage – most insects, division occur only in the rim of the cytoplasm, around the periphery of the cell
Discoidal cleavage
Meroblastic - Telolecithal
Fish, reptiles, birds
Cells divide “on” surface of yolk
Mesolecithal – moderate vegetal yolk disposition
Displaced radial cleavage
Holoblastic - Mesolecithal
Amphibians
Yolk displaces dividing cells (?)
