|DEVELOPMENTAL BIOLOGY 3230|
Cleavage refers to the stereotyped pattern of early mitotic divisions that divides up the large volume egg cytoplasm. The early zygote is unique in being so large. Most cells undergo a period of growth between cycles of mitosis, but this is not true for early cleavage stage blastomeres. With each division the cells get smaller. This rapid pattern of cell division without concomitant growth abruptly halts at the stage called the mid-blastula transition where the zygotic nucleus takes control of the cell cycle.
There is some evidence that a maternal factor, perhaps a transcriptional regulator, is responsible for this early rapid pattern of cleavage divisions. By artificially altering the cytoplasmic to nuclear DNA ratio you can change the time of the midblastula transition. The midblastula transition refers to the time when the major switch from expression of maternal to zygotic genes takes place.
Fertilization in some species leads to radical cytoplasmic movements that are essential for ensuring the cytoplasmic determinants are located in the correct positions relative to subsequent cleavage events.
The types of eggs based on yolk characteristics are described as:
RADIAL HOLOBLASTIC CLEAVAGE
Excellent movie of sea urchin cleavage from Rachel Fink's "A Dozen Eggs".
Sea urchins also have radial holoblastic cleavage, but with some interesting differences. First cleavage is meridional.Second cleavage is meridional. Third cleavage is equatorial Fourth cleavage is meridional, but while the four animal pole cells split equally to give rise to eight equal sized animal blastomeres termed MESOMERES, the vegetal cells divide asymmetrically along the equatorial plane to give 4 large MACROMERES and 4 much smaller MICROMERES at the vegetal pole. Fifth division the MESOMERES divide equatorially to give two tiers of eight MESOMERES an1 and an2 , the MACROMERES divide meridionally forming a tier of eight cells below an2, the MICROMERES divide to give a cluster of cells below the veg1 layer. The sixth divisions are all equatorial, giving a veg2 layer. The seventh divisions are all meridional giving a 128 cell blastula.
The conclusion from these experiments is that there is some factor in the vegetal pole of the egg that determines the formation of the micromeres and further that there must be a molecular clock that starts at egg activation. The clock is independent of the actual cleavage event.
Additionally, the cleavage planes are somewhat different from other animals. First cleavage is meridional just like sea urchin and frog. However, the second cleavage division sees one of the blastomeres dividing meridionally and the other equatorially! This type of cleavage is called ROTATIONAL HOLOBLASTIC CLEAVAGE.
Another unique feature of mammalian cleavage is that the blastomere cleavages are asynchronous. (compared with the synchrony of sea urchin and frog up till the midblastula transition). Cleavage of the mammalian embryo is regulated by the zyotic nucleus from the very start.
This COMPACTION results in part from the production of an novel adhesion molecule UVOMORULIN (E-Cadherin) and is stabilized by the formation of tight junctions between the outer cells which like in the sea urchin seals off the interior of the blastula from the exterior. The cells also form gap junctions among themselves that allows the passage of small molecules, such as ions and some second messenger molecules such as Ca++ and C-AMP. The compacted 16 cell morula consists of an outer rind of cells and a few cells (1-2) completely internal. Most of the external cells give rise to the TROBLASTIC OR TROPHECTODERMAL CELLS. These cells do not contribute to the embryo proper, but instead are necessary for implantation of the embryo in the uterine wall and form the tissues of the CHORIAN, an essential component of the placenta that we’ll talk about later.
FORMATION OF THE INNER CELL MASS
It's not until the equatorial cleavages that the cells of the blastoderm separate from the yolk. Further equatorial cleavages create a multilayered blastoderm three or four cells thick.
In birds a space forms between the blastoderm and the yolk called the SUBGERMINAL cavity. By the 16 division (60,000 cells) cells of the blastoderm migrate into the subgerminal cavity to form a second layer. The two layers are called the outer EPIBLAST and inner HYPOBLAST with the blastocoel between. We will study this in more detail latter when we discuss bird and mammal gastrulation
After several rounds of karyokinesis the naked nuclei migrate to the periphery of the egg. At this stage it is called the SYNCYTIAL BLASTODERM because all the nuclei share the same cytoplasm. Cellularzation occurs at about the 14th nuclear division to create the CELLULAR BLASTODERM. After this time cells divide asynchronously. This corresponds to the midblastula transition of frogs and sea urchins. (transition from maternal to primarily zygotic gene expression) Remember that the midblastula transition was thought to be triggered by the ratio of chromatin to cytoplasm. Evidence for this mechanism in flies is seen by examining mutant haploid embryos. These embryos undergo the midblastula transition and cellularization one division later 15th. Furthermore you can accelerate cellularization by ligating the egg and reducing the volume of cytoplasm. Although the syncytial blastoderm stage suggests that all the nuclei are equipotent in that there do not seem to be diffusional barriers to cytoplasmic determinants, in fact the cytoplasm is very regionalized and the nuclei have highly organized cytoplasmic domains around them.
MECHANISMS OF CLEAVAGE
Conversely if nuclei from non-dividing cells are put into fertilized enucleated eggs they start dividing. Artificially activated enucleated eggs without centrioles will undergo cortical contractions reminiscent of cleavage. Some of the cytoplasmic factors regulating cell division in the early embryo have been identified.
CELL FATE DETERMINATION
Cytoplasmic Localization of DETERMINANTS as a general and basic mechanism for early patterning (Examples Tunicate and Sea Urchin). A major question of developmental biology is when and how cell fates are determined during development. This is intimately related to the question of how pattern formation occurs during development. The embryo must not only generate the right number and type of differentiated cells, but they must be organized in the correct way relative to all the other cells in the embryo to form a functional animal. We will examine two possibilities of cell fate determination and pattern formation: 1. Cell fate could be determined by intrinsic factors placed into the egg during oogenesis and then parceled out to specific blastomeres during cleavage, 2. Extrinsic signals provided by the embryo's environment might provide the patterning information to regulate cell fate. As we will see most complex organisms use a combination of intrinsic and extrinic signals to regulate cell fate and embryonic pattern formation.
Autonomous cell fate specification by cytoplasmic determinants suggests that a cell's fate is entirely dependent on its lineage, whereas "regulative" development suggests that a cell's fate is determined by external signals from other cells. These two mechanisms of cell specification can be distinguished experimentally by isolation, ablation, and transplantation experiments. If a blastmere isolated from an embryo differentiates normally (as if it were still in its normal position in the embryo) we can say that it must have intrinsic determinants that specify its fate. However if it differentiates abnormally we can say that its cell fate is dependent on external signals. If we ablate a blastomere from an embryo and the embryo develops abnormally, missing all the cells fates that normally arise from the ablated blastomere, we say that development is cell autonomous and intrinsically specified. However, if the embryo develops normally we say that the remaining blastomeres can regulate their cell fate to compensate for the missing cells. If a transplanted cell maintains its cell fate based on its original position then we say its fate has been determined, if it takes on a new fate based on its newly transplanted position we say that its fate is regulated by external signals from nearby cells.
CYTOPLASMIC LOCALIZATION AND REGULATION IN THE TUNICATE EGG
Note the fate map correlates with the different colored cytoplasms of the tunicate embryo. Don't be confused by the different colors in two figures. The "orange" yellow crescent cytoplasm is correlated with muscle fates and the Yolky (yellow) cytoplasm is correlated with endodermal fates. The grey (white or bluish purple) cytoplasm above the yellow crescent is correlated with neural ectoderm.
This lineage map shows the invariant linage correlation with blastomeres parceled particular colored cytoplasms by the invariant cell cleavages. However, invariant cleavages and lineages do not necessarily prove autonomous cell specification by cytoplasmic determinants.
Experimental manipulations are required to test regulative versus cell autonomous determination of cell fate. The classic isolation experiments shown in the next three figures attempt to show that cell fate is determined by cytoplasmic determinants they acquire through stereotype cleavages. A glass needle is used to separate the B4.1 pair of blastomeres from the rest of the embryo. The B4.1 blastomeres normally acquire the yellow crecent cytoplasm correlated with muscle cell fate.
Here we can see the results of the isolation experiments. In each case the isolated blastomeres give rise to only that subset of cell fates they would normally produce in the intact embryo. The isolated blastomeres do not regulate their fate to compensate for their missing neighbors. Animal pole blastomeres, a4.2 and b4.2, give rise only to ectodermal cells. A4.1 gives rise to notochord and endodermal cells, while B4.1 gives rise to muscle and endodermal cells. None of the isolated blastomeres can give rise to all the cellular components of a normal embryo.
The next experiment below uses a needle to manipulate the equatorial cleavage plane so that it is more vegetal than normal and now the animal pole blastomeres, b4.2, acquire some of the "yellow crescent" cytoplasm. When these blastomeres are isolated they now give rise to some muscle cells. This nicely demonstrates that the "yellow crescent" cytoplasm can determine muscle cell fate and can do so in a cell autonomous manner.
A jelly canal defines the location of the animal pole and reflects the early polarity of egg. The early pattern of cleavages does not depend on the site of sperm entry, but are determined by the intrinsic polarity/asymmetry of egg. Boveri (1901) described a subequatorial band of pigment arranged orthongonally to animal-vegetal axis. These granules also indicated the location of cytoplasm that is later included in the cells of the archenteron. Horstadius (1928) separated animal and vegetal blastomeres and showed that only the vegetal blastomere would give rise to micromeres, gastrulate, and form skeleton. His conclusion was that cytoplasmic factors located in vegetal half are necessary for micromeres, gastrulation and archenteron fromation,and skeleton formation.
This shows the fate map of the 64 cell stage sea urchin blastula. Notice that the micromeres are the primary mesenchyme cells and give rise to the larval skeleton (the pluteus stage spicules).
At the four cell stage, if the blastomeres are isolated from each other they are able to "regulate" their fate and give rise to 4 small pluteus stage larvae.
In contrast, at later stages if you isolate animal half blastomeres you find that they only produce an "animalized" dauerblastula that does not express any mesodermal or endodermal cell fates. Isolated vegetal half blastomeres give rise to larva that express ectodermal, mesodermal, and endodermal cell fates showing that the fate of these cells can be regulated. Isolated micromeres (primary mesenchyme) undergo the correct number of cell divisions and ALWAYS give rise to spicules on schedule. Thus, micromeres are definitively specified as the precursors of the skeletogenic mesenchyme cells when they first appear at the 16 cell stage. The key experiments were putting micromeres together with animal pole blastomeres and showing that although micromere fate was "fixed or determined" at the time of their birth, micromeres were able to "induce" new cell fates in the animal pole blastomeres. The micromeres were able to induce endodermal and mesodermal fates in the animal pole blastomeres! Thus, the late experiment in "C" shows that when micromeres are added to an animal half blastula you can now induce the formation of a recognizable larva expressing endodermal, mesodermal, and ectodermal fates.
The final set of experiments demonstates that even in a normal embryo, if you transplant micromeres to the animal pole cap you can induce a secondary archenteron and alter the normal axial patterning. This again argues that the micromeres acquire a cytoplasmic derminant the specifics their cell fate and that they provide the inductive signal that patterns the axial structures of the sea uchin embryo. Micromere fate cannot be altered, but signals from the micromeres can alter the fate of all the other blastomeres.
Horstadius: (1928, 1935) showed experimentally that in a 16 cell stage embryo all tiers of blastomeres except the micromeres will take on different fates when transplanted into different positions in chimeric embryos. The archenteron will develop from veg 1 blastomeres if veg 2 cells are removed and the micromeres are placed in contact with the veg 1 layer. In the absence of micromeres, veg 2 blastomeres give rise to archenteron and skeletal structures. Classically, a duel animal-vegetal gradient has been invoked to account for these results. However these results only indicate that decisive inductive interactions occur between adjacent blastomere tiers.
GENERAL RESULT OF TRANSPLANTATIONS: the fate of given blastomeres is always found to be affected by the apposition of different neighboring cells that adjoin them in normal embryos.