# What is the total number of rounds of cleavage during mammalian embryonic development?

That for frog is 12, but what about mammalian embryos? I cannot find the exact number anywhere.

It's not a totally answerable question, since some types of cells are going to divide more times than others. But for an estimate, take as a starting proposition that there are 1 trillion cells in the adult human body. [1] The average weight for a human is 62kg. [2] Average birth weight is about 3.4 kg. [3]

So that implies roughtly (3.4/62)* 1 trillion = 55 billion cells in a newborn.

You then take the log base 2 of 55 billion, which gives you the exponent you have to hang on 2 in order to get 55 billion, which is about 35. Then add one for that additional cell division to get from one to two cells == 36 divisions.

Of course I'm just using math, not biology, so your actual reality may vary. Certainly some cells will reproduce more often than others, maybe cells actually grow in mass instead of dividing (i.e., baby cells might have less mass than adult cells) so the baby-cell-count could be off, lots of possible sources of error.

[1] http://www.nichd.nih.gov/publications/pubs/fragileX/sub3.cfm

[2] http://en.wikipedia.org/wiki/Body_weight#Average_weight_around_the_world

[3] http://en.wikipedia.org/wiki/Infant#Weight

## Cleavage (embryo)

In developmental biology, cleavage is the division of cells in the early embryo. The process follows fertilization, with the transfer being triggered by the activation of a cyclin-dependent kinase complex. [1] The zygotes of many species undergo rapid cell cycles with no significant overall growth, producing a cluster of cells the same size as the original zygote. The different cells derived from cleavage are called blastomeres and form a compact mass called the morula. Cleavage ends with the formation of the blastula.

Depending mostly on the concentration of yolk in the egg, the cleavage can be holoblastic (total or entire cleavage) or meroblastic (partial cleavage). The pole of the egg with the highest concentration of yolk is referred to as the vegetal pole while the opposite is referred to as the animal pole.

Cleavage differs from other forms of cell division in that it increases the number of cells and nuclear mass without increasing the cytoplasmic mass. This means that with each successive subdivision, there is roughly half the cytoplasm in each daughter cell than before that division, and thus the ratio of nuclear to cytoplasmic material increases. [2]

## What is the total number of rounds of cleavage during mammalian embryonic development? - Biology

The product of fertilization is a one-cell embryo with a diploid complement of chromosomes. Over the next few days, the mammalian embryo undergoes a series of cell divisions, ultimately leading to formation of a hollow sphere of cells known as a blastocyst. At some point between fertilization and blastocyst formation, the embryo moves out of the oviduct, into the lumen of the uterus.

The images below demonstrate major transitions in structure during early embryogenesis in cattle. Note that in all of the the early stages, the embryo is encased in its zona pellucida. Embryos from other mammals have a very similar appearance, and the general sequence of stages is seen in all mammals.

Unfertilized oocytes typically fill the entire space inside the zona pellucida, but after fertilization, the one-cell embryo usually is somewhat retracted from the zona pellucida surrounding it. Although not visible in this image, one or two polar bodies are often visible in the perivitelline space , the area between the embryo and the zona pellucida.

The one cell embryo undergoes a series of cleavage divisions , progressing through 2-cell, 4-cell, 8-cell and 16 cell stages. A four cell embryo is shown here. The cells in cleavage stage embryos are known as blastomeres . Note that the blastomeres in this embryo, and the eight-cell embryo below, are distinctly round.

Early on, cleavage divisions occur quite synchronously. In other words, both blastomeres in a two-cell undergo mitosis and cytokinesis almost simultaneously. For this reason, recovered embryos are most commonly observed at the two, four or, and seen here, eight-cell stage. Embryos with an odd number of cells (e.g. 3, 5, 7) are less commonly observed, simply because those states last for a relatively short time.

Soon after development of the 8-cell or 16-cell embryo (depending on the species), the blastomeres begin to form tight junctions with one another, leading to deformation of their round shape and formation of a mulberry-shaped mass of cells called a morula . This change in shape of the embryo is called compaction . It is difficult to count the cells in a morula the embryo shown here probably has between 20 and 30 cells.

Formation of junctional complexes between blastomeres gives the embryo and outside and an inside. The outer cells of the embryo also begin to express a variety of membrane transport molecules, including sodium pumps. One result of these changes is an accumulation of fluid inside the embryo, which signals formation of the blastocyst . An early blastocyst, containing a small amount of blastocoelic fluid, is shown to the right.

As the blastocyst continues to accumulate blastocoelic fluid, it expands to form - you guessed it - an expanded blastocyst . The blastocyst stage is also a landmark in that this is the first time that two distinctive tissues are present. A blastocyst is composed of a hollow sphere of trophoblast cells , inside of which is a small cluster of cells called the inner cell mass . Trophoblast goes on to contribute to fetal membrane systems, while the inner cell mass is destined largely to become the embryo and fetus. In the expanded blastocyst shown here, the inner cell mass is the dense-looking area at the botton of the embryo.

Eventually, the stretched zona pellucida develops a crack and the blastocyst escapes by a process called hatching . This leaves an empty zona pellucida and a zona-free or hatched blastocyst lying in the lumen of the uterus. Depending on the species, the blastocyst then undergoes implantation or elongates rapidly to fill the uterine lumen.

As mentioned, the developmental progression depicted above for bovine embryos is essentially identical to what all mammalian embryos go through, including humans. For example, the image to the left shows an expanded blastocyst from a dog. This embryo was stained to accentuate the trophoblast and inner cell mass.

The length of time required for preimplantation development varies somewhat, but not drastically, among species. The zona-intact bovine blastocysts shown above were collected 5-6 days after fertilization. The same stages would be seen in mice at about 3.5 days after fertilization.

In addition to the morphological changes in the embryo described here, preimplantation development is associated with that might be called an awakening of the embryonic genome. There is, for instance, little transcription in the embryos of most species prior to the 8 cell stage, but as embryos develops into morulae, then blastocysts, a large number of genes become transcritionally active and the total level of transcription increases dramatically.

## What is the total number of rounds of cleavage during mammalian embryonic development? - Biology

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.

PATTERNS OF EMBRYONIC CLEAVAGE
Pattern of embryonic cleavage is determined both by the position of the mitotic spindles and by the amount and distribution of yolk. Yolk tends to inhibit cleavage. It slows it down or actually prevents complete cleavage. Yolk is an adaptation of those animals that go through more or less of embryogenesis isolated from any food supply. Some animals, like sea urchin, have relatively little yolk because they rapidly develop into a free swimming larval form that acquires nutrients from their environment. Other animals such as marsupials are born prematurely, but are provided nourishment in a parental pouch. Placental mammals develop a specialized organ through which the embryo is nourished throughout development and so also have little yolk.

The types of eggs based on yolk characteristics are described as:
Isolecithal: sparse evenly distributed yolk, eg., sea urchin, mouse
Mesolecthal: moderate amount of yolk, often unevenly distributed, eg., frog
Telolecithal: dense yolk concentrated at one end, eg., bird, reptile
Centrolecithal: yolk concentrated at the middle of the egg, eg. fly

Many eggs are polarized with a yolk rich pole, termed the vegetal pole and a yolk poor pole termed the animal pole, eg., frog. The zygotic nucleus is generally displaced towards the animal pole. Zygotes with relatively little yolk (isolecithal and mesolecithal) cleave HOLOBLASTICALLY. The cleavage furrow extends all the way through the egg. While telolecithal and centrolecithal zygotes undergo MEROBLASTIC cleavage where the cleavage plane extends only to the accumulated yolk. In centrolecithal eggs (many insect eggs) cleavage is meroblastic and superficial, while in telolecithal eggs (birds and fish) cleavage is discoidal

There are several types of cleavage symmetry seen in nature: radial(echinoderms, amphibians), spiral (mollusks, annelids), Bilateral (ascidians,tunicates), Rotational (mammals). The two figures below show examples of holoblastic and meroblastic cleavage symmetries.

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.

What determines these cleavage patterns? Are they dependent on the previous cleavage and played out like a tape or are they determined by some intrinsic clock? In 1939 Horstadius inhibited one or two of the first three cleavages and found the appearance of the micromeres occurred at the right time regardless of the history of cleavages

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.

The 128 cell blastula is a rather loose ball of cells surrounding a hollow blastocoel. The ball is one cell layer thick with all cells in contact with the external hyaline layer and the internal fluid of the blastocoel. At this stage in development the cells begin to form the tight junctions characteristic of an epithelium. The central blastocoel is now isolated from the external environment. The blastomeres continue to divide with their axis parallel to the hyaline layer, remaining a epithelium one cell thick. The blastocoel continues to enlarge.

Two theories attempt to account for the pattern of enlargement of the blastocyst
1. The osmotic theory suggests that ions and proteins are secreted into the blastocoel by the blastomeres and this results in a pressure buildup due to the osmotic flow of water. This pressure would then be responsible for aligning the axis mitosis of the blastomeres and the enlargement of the blastocoel.

2. The alternate theory by Wolpert and his colleagues suggests that it is really the adhesive interactions among the blastomeres and between the blastomeres and the hyaline layer that aligns the mitotic axis's. That is the adhesion to the hyaline is greatest, the adhesion to other blastomeres is next, and finally the interaction with the blastocoel wall is least. The dominant adhesion with the hyaline layer forces the expansion of the blastocyst and blastocoel.

The cells of the blastula grow cilia on their outer surface, secrete a “hatching enzyme” (hyalinase) and become free swimming.

AMPHIBIAN CLEAVAGE
Cleavage in many amphibians is holoblastic with radial symmetry, however the large volume of yolk (its mesolecithal) interferes with cleavage. At the animal pole first cleavage proceeds at about 1mm/min, while through the vegetal pole is proceeds 50-100 times slower (.02mm/min). While the first cleavage is still incomplete in the yolky vegetal region of the egg the second meridional cleavage begins to take place.

The third cleavage is equatorial, but because the nuclei and asters are displaced “animal-ward” the cleavage plane although perpendicular to the animal vegetal axis is also displaced towards the animal pole and does not equally divide the blastomeres. The result is four smaller animal blastomeres (termed MICROMERES) and four large vegetal pole blastomeres (termed MACROMERES). This unequal holoblastic cleavage gives rise to a more rapidly dividing animal pole made up of smaller micromeres and a slower dividing vegetal pole made up of macromeres. The animal pole soon is composed of many small micromeres and the vegetal pole a few yolk filled large macromeres. Although the formation of the blastocoel begins with the first cleavage, it does not become obvious until the 128 cell stage.

WHAT FUNCTION DOES THE BLASTOCOEL SERVE?
The blastocel spatially separates cells so they do not touch one another. Cells at the roof of the blastocoel normally become ectoderm. If you transplant cells from the roof of the blastocoel next to the yolky cells at the base of the blastocoel they will differentiate as mesoderm. Mesodermal derivatives are normally produced from cells adjacent to the endodermal precursors. One possibility that we will thoroughly explore is that the vegetal cells “induce” via cell-cell interactions the adjacent cells to become mesodermal. Thus the formation of the blastocoel may be necessary to prevent inappropriate "inductive" interactions among early cells of the blastocyst. The second obvious need for the blastocoel may be during the subsequent stage of development, GASTRULATION, where cells migrate into the interior of the blastocoel.

MAMMALIAN CLEAVAGE
The mammalian egg is released from the ovary into the oviduct where it is fertilized. First cleavage begins about a day after fertilization within the oviduct. In sharp contrast to most animals, cleavage in mammals can be very slow---1/day.

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.

Through the third cleavage the blastomeres form a ball of loosely associated cells just like the other animals we&rsquove studied. Before the fourth cleavage the cells of the blastula dramatically change their behavior towards one another. They rapidly try to maximize their contacts with the other blastomeres and in doing cause the blastula to compact.

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&rsquoll talk about later.

The cells of the embryo are derived from the inner few cells of the 16 cell stage blastula. These cells generate the inner cell mass of cell from which the entire embryo develops. By the 6th cleavage, the 64 cell stage the inner cell mass and trophoblastic layer are completely separate. The trophoblasts secret fluid internally to create the blastocoel. The embryo is now call a blastocyst.

FORMATION OF THE INNER CELL MASS
How are these inner cell mass cell created? Are there certain blastomeres fated by intrinsic factors to become inner cell mass progenitors? The answer seems to be no. All the early blastomeres seem to be totipotent and the determination of which cells will contribute to the trophoblastic layer and which to the inner cell mass simply a matter of chance position. Cells from a 4 cell stage embryo, which will normally give rise to both inner cell mass and trophectoderm cells, transplanted to the outside of a 32 cell stage embryo only give rise to trophectoderm. They do not contribute to the embryo proper. Remember from the earlier lecture on cloning that fusion of two 8 cell stage mouse embryos results in a normal embryo, suggesting that all the cells at that stage are totipotent.

MEROBLASTIC CLEAVAGE
In telolecithal and centrolecithal eggs the large dense yolk prevents cleavage. Telolecithal eggs are characteristic of birds, fishes, and reptiles while centrolecithal eggs are characteristic of insects. Telolecithal eggs result in meroblastic discoidal cleavage. Cleavage is restricted to the blastodisc at the animal pole of the egg. At early cleavages, because cleavage cannot proceed through the yolk, the blastomeres are continuous at their vegetal margins.
This movie of zebrafish development byRolf Karlstrom is excellent. (Movie by Paul Myers)

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

Centrolecital eggs of arthropods undergo a SUPERFICIAL CLEAVAGE. The large central mass of yolk confines the cleavages to the cytoplasmic rim of the egg.

An interesting and informative variation is seen in insects. The zygotic nuclei divide with out cleavage. That is the nuclei undergo karyokinesis----mitotic division of the nucleus--- without cytokinesis---the division of the cell. These naked nuclei are called ENERGIDS. The nuclei divide at an amazing rate---every 8 minutes (all of embryogenesis takes only 22 hrs).

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
Cell Cycle

M-mitosis
G1- pre-replication gap
S- DNA synthesis
G2-premitotic gap

In cleavage stage embryos such as frogs and flies the blastomeres go directly from M to S without intervening G1 or G2 stages. After the midblastula transition cells in both animals have a G1 and G2. Elegant transplant experiments have demonstrated that it is the cytoplasm that regulates both karyokinesis and cytokinesis. If nuclei from dividing cells are transplanted into oocyte they immediately stop dividing.

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.

CYTOSTATIC FACTOR (CSF) is elevated after the first meiotic division and arrests the oocyte in the second meiotic metaphase. Upon fertilization the Ca inactivates CSF, meiosis is completed and the pronuclei fuse.

MITOSIS PROMOTING FACTOR (MPF) causes cells to enter M phase. MPF activation causes: 1. chromosome condensation by H1 histone phosphorylation, 2. nuclear envelope breakdown by hyperphosphorylation of 3 nuclear lamins, 3. RNA polymerase inhibition to shut down transcription, 4. Myosin regulatory subunit phosphorylation to inhibt cytokinesis.

Suggested model for cyclic regulation of cell cycle during cleavage stages of embrogenesis. MPF induces cell to proceed from S to M. CSF binds to MPF and prevents its inactivation. The cell remains in M. Ca increases and causes the inactivation of CSF which in turn leads to the inactivation of MPF and the cell proceeds through M to S and the cycle is repeated. MPF is made up of two subunits, Cyclin B and cdc2. It is cyclin B that undergoes a cell cycle specific synthesis and degradation regulated by the cells nucleus to control the cell cycle in normal somatic cells. However, during oogenesis the egg is loaded with "regulators" of cyclin B and cyclin B mRNA so that its syntheis is regulated by maternal factors independent of the zygotic nucleus. Thus it is not untill the maternal components "run out" that the zygotic nucleus takes over and a normal cell cycle (M, G1, S, G2) returns.

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
At the end of oogenesis the tunicate egg has a clearly distinguished animal and vegetal pole. There is a yellow cortical cytoplasm that surrounds a grey yolky inner cytoplasm. The oocyte nucleus is displaced towards the animal pole. Sperm entry in the vegetal hemisphere fertilizes the egg and initiates development. A dramatic rearrangement of the egg cytoplasm occurs after fertilization giving rise to regionally colored cytoplasms that seem to correlate with subsequent blastomere fates.

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.
Remember the pattern of early cleavages. The micromeres arise during the fourth cleavage (16 cell stage) from an unequal equatorial division of the vegetal pole blastomeres.

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.

Implanted individual micromeres near the animal pole inhibit apical tuft formation and in some cases induce a new embryonic axes. Veg 2 blastomeres will also induce changes similar to micromeres when transplanted next to animal pole blastomeres.

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.

HYPOTHESIS: Localized maternal cytoplasmic determinants specify certain cells in the normal embryo, in particular the micromeres and the archenteron precursors near the vegetal pole. These cells then determine inductively the fates of neighboring blastomeres, which interact in turn with their neighbors. Many of the blastomeres retain potentialities other than those they normally express, and for some time these blastomeres are only reversibly specified, as required for a developmental system that depends to a large extent on induction.

The following figure is from a recent study ⎖] using video and genetic analysis of in vitro human development during week 1 following fertilization.

• EGA - embryonic genome activation
• ESSP - embryonic stage–specific pattern, four unique embryonic stage–specific patterns (1-4)

## RESULTS

### Establishment of an automated culture system for mouse preimplantation development in space

We developed an automated mini incubator to enable mouse early development in space that was equipped with programmable controls for temperature, automatic micrography and fixation of samples, for cultivating mouse embryos (Fig. 1A, and Fig. S1A and B). We first asked whether the growth of preimplantation mouse embryos in this mini incubator was feasible under sealed conditions (Fig. S2). To do so, we cultured frozen-thawed 2-cell embryos for 64 h under conventional culture (CC) in the microdroplet CO2 incubator or sealed culture (SC) in developed automated mini incubator conditions and compared the experimental outcomes. Under the CC conditions, most of those 2-cell embryos reached the morula/blastocyst stage. Likewise, the SC conditions allowed the 2-cell embryos to achieve a very similar developmental outcome (88.16% for CC vs 85.09% for SC) (Fig. S3A). Furthermore, 130 blastocysts from SC conditions and 90 blastocysts from CC conditions were transferred into pseudopregnant mice (9 to 12 embryos into one recipient female) to observe the potential to develop to full-term offspring (Fig. S3B). The recipients gave birth to 42 (SC) and 35 pups (CC), respectively. The rate of live offspring production from SC embryos (32.31%), albeit slightly lower than that from CC embryos (38.89%), was not significantly different (Fig. S3D). Additionally, the body weight (1.61 g vs. 1.58 g) and the sex of the pups (51% vs 47.6% female) were similar between SC and CC groups (Fig. S3E and F), while all of the pups were capable of growing to adulthood and delivering litters. Thus, the automated mini incubator allows the preimplantation mouse embryos to develop normally under the SC conditions and is therefore suitable for subsequent experimentation in spaceflight. Two automated mini incubators were developed, one for experiments in space and one on Earth with consistent composition.

In vitro development of mouse pre-implantation embryos in space. (A) The embryonic culture incubator used in space experiments. The incubator consists of four imaging culture chambers (indicated by the yellow dotted line and the numbers 1, 2, 3 and 4), two groups of culture fixation units (yellow dotted lines), a microscope (red arrow), and two module reservoirs of fixative solutions (red arrow). This experimental apparatus provides temperature stability inside the incubator, the ability to replace culture medium and automatic image acquisition. (B) The imaging culture chamber was filled with gas-saturated medium, and it consisted of a sample culture cavity with a red O-ring and a cover with a window. (C) The fixation culture unit is a cylindrical perfusion chamber with a top part of the chamber, main body and bottom part of the chamber. (D) Timeline of the SJ-10 satellite space mission showing the time points for embryos loading, payload transferring, embryos fixation, sample recovery and arrival to the laboratory. (E) Representative time-lapse images of embryonic development in imaging culture chambers during spaceflight, with highlighted images showing key stages of pre-implantation development. Scale bars, 100 μm.

In vitro development of mouse pre-implantation embryos in space. (A) The embryonic culture incubator used in space experiments. The incubator consists of four imaging culture chambers (indicated by the yellow dotted line and the numbers 1, 2, 3 and 4), two groups of culture fixation units (yellow dotted lines), a microscope (red arrow), and two module reservoirs of fixative solutions (red arrow). This experimental apparatus provides temperature stability inside the incubator, the ability to replace culture medium and automatic image acquisition. (B) The imaging culture chamber was filled with gas-saturated medium, and it consisted of a sample culture cavity with a red O-ring and a cover with a window. (C) The fixation culture unit is a cylindrical perfusion chamber with a top part of the chamber, main body and bottom part of the chamber. (D) Timeline of the SJ-10 satellite space mission showing the time points for embryos loading, payload transferring, embryos fixation, sample recovery and arrival to the laboratory. (E) Representative time-lapse images of embryonic development in imaging culture chambers during spaceflight, with highlighted images showing key stages of pre-implantation development. Scale bars, 100 μm.

The SJ-10 recoverable satellite was launched on 6 April 2016. It had an orbital attitude of ∼252 km, a microgravity level of 10 −4 −10 −6 g0 and an average dose of radiation of ∼0.15 mGy/day [ 19]. To assess mouse preimplantation embryonic development during spaceflight, 3400 morphologically normal mouse embryos at the 2-cell stage were placed into four imaging culture chambers (Fig. 1A and B) and six culture fixation units (Fig. 1A and C), respectively, 12 hours before launching of the satellite. The embryos in imaging culture chambers were monitored using microscopy to observe the morphology of live embryos every 4 hours, while the embryos in culture fixation units were either fixed with 4% paraformaldehyde (PFA) or treated with RNA protection reagents after 64-hour cultivation in orbit (Fig. 1D). Time-lapse imaging demonstrated that 2-cell embryos cultured in space underwent key stages of mammalian preimplantation development, including cleavage, compaction of blastomeres, cavitations and formation of expanded blastocysts (Fig. 1E and movie S1 to movie S4). However, the developmental defect of the embryo was significantly observed under the sealed culture conditions in space (SS) when compared with that under the SC conditions on Earth (movie S5 to movie S8).

### The rate of blastocyst formation and blastocyst quality are compromised in space

A limitation of the time-lapse imaging analysis is the low number of observable embryos due to floatation, aggregation and a lack of accessibility. Thus, we further assessed the morphology and rate of development of the embryos in fixation culture units returned from space. Of the 1500 embryos loaded, 1184 were recovered and consisted of compacted morulas and expanded blastocysts (Fig. S4). Among them, the majority (856/1184, or 72.3%) developed into morula/blastocysts, but the ratio of the blastocyst-stage (34.3%) was significantly lower than that of the CC (60.24%, P = 0.0023) and the SC group (56.87%, P = 0.0024) (Fig. 2A), indicating impaired developmental ability. Thus, 2-cell mouse embryos can develop into blastocysts in space, but the ratio of embryos developed to the blastocyst stage is greatly compromised.

Embryonic development and blastocyst quality are compromised in space culture condition. (A) Analysis of embryonic development in space in fixation culture units after satellite return to Earth. Data represent means of all embryos acquired from three independent units of fixative (1: indicates unit 1 2: indicates unit 2 and 3: indicates unit 3). CC: indicates conventional culture, SC: indicates sealed culture and SS indicates space sealed culture. (B) The total cell number in blastocysts under CC, SC and SS conditions calculated from 3D reconstruction analysis. Two-tailed Student's t-tests were used for statistical analysis. The SS group had significantly fewer cells than the CC (P = 0.047) and SC groups (P = 0.021). Each dot represents one embryo, and black bars indicate the mean cell number for each group. (C) Representative 3D images of Cdx2 and Oct4 immunofluorescence in blastocysts (>64-cell stage) developed under CC, SC and SS conditions. Oct4 stains the ICM (green), while Cdx2 stains the TE (red). Nuclei were stained with Hoechst33342 (blue). Arrowheads denote colocalization of Oct4 and Cdx2 (yellow). Scale bar, 20 μm. The percentage of Oct4-positive cells (D), Cdx2-positive cells (E) and double-positive cells (F) in CC, SC and SS embryos at the 16–32 cell, 32–64 cell and >64 cell blastocyst stages, respectively. The results are shown as the means ± SEM. P values are determined by Student's t-test (two-tailed) (SS vs. CC and SS vs.SC). n.s., not significant (P > 0.05).

Embryonic development and blastocyst quality are compromised in space culture condition. (A) Analysis of embryonic development in space in fixation culture units after satellite return to Earth. Data represent means of all embryos acquired from three independent units of fixative (1: indicates unit 1 2: indicates unit 2 and 3: indicates unit 3). CC: indicates conventional culture, SC: indicates sealed culture and SS indicates space sealed culture. (B) The total cell number in blastocysts under CC, SC and SS conditions calculated from 3D reconstruction analysis. Two-tailed Student's t-tests were used for statistical analysis. The SS group had significantly fewer cells than the CC (P = 0.047) and SC groups (P = 0.021). Each dot represents one embryo, and black bars indicate the mean cell number for each group. (C) Representative 3D images of Cdx2 and Oct4 immunofluorescence in blastocysts (>64-cell stage) developed under CC, SC and SS conditions. Oct4 stains the ICM (green), while Cdx2 stains the TE (red). Nuclei were stained with Hoechst33342 (blue). Arrowheads denote colocalization of Oct4 and Cdx2 (yellow). Scale bar, 20 μm. The percentage of Oct4-positive cells (D), Cdx2-positive cells (E) and double-positive cells (F) in CC, SC and SS embryos at the 16–32 cell, 32–64 cell and >64 cell blastocyst stages, respectively. The results are shown as the means ± SEM. P values are determined by Student's t-test (two-tailed) (SS vs. CC and SS vs.SC). n.s., not significant (P > 0.05).

After assessing the rate of development, we examined the quality of the blastocysts developed in space and found that consistent with the reduced rate of embryonic development, the total number of cells in the blastocysts (41.5 cells) developed in space (i.e. the SS condition) decreased significantly when compared with that of the CC (51.6 cells) and the SC (53.9 cells) condition (Fig. 2B). We performed immunostaining of Cdx2 (a trophectodermal (TE) marker) [ 20, 21] and Oct4 (an inner cell mass (ICM) marker) in blastocyst-stage embryos [ 22, 23], allowing us to find an expression pattern in SS embryos that is highly distinguishable from that in the CC or SC embryos (Fig. 2C). During normal mouse embryonic development, Oct4 was widely expressed in both the inner and outer layers of cells in blastocysts with 16–32 cells, while Cdx2 + cells were mainly located in the outer layer (Fig. S5A). In the blastocysts with 32–64 cells, Oct4 expression was diminished in a subset of TE cells, while a few Oct4 and Cdx2 double-positive cells still existed (Fig. S5B). In blastocysts with >64 cells, Oct4 expression was restricted to cells in the ICM and was largely absent in Cdx2 + trophectoderm cells (Fig. S5C). Using the Imaris cell imaging software, we quantified the number of nuclei (blue), Oct4 + (green), Cdx2 + (red), and Oct4 + and Cdx2 + cells (yellow) in blastocysts developed under the CC, SC and SS conditions. Strikingly, there was a significant increase in the percentage of Oct4 + cells in SS embryos at both the 32–64 and >64 cell stages (Fig. 2D). In contrast, the percentage of Cdx2 + cells was reduced in SS embryos at these stages (Fig. 2E). In keeping with these findings, the percentage of Cdx2 + /Oct4 + cells was significantly higher in the SS embryos than in the SC and CC embryos (Fig. 2F).

The increase in undifferentiated (i.e. Oct4 + ) cells and transitional Oct4 + /Cdx2 + cells, and the concomitant decrease in lineage-specific Cdx2 cells suggest that cellular differentiation from the pluripotent state was compromised under the SS condition, consistent with earlier reports [ 17]. To further confirm this notion, we examined the epiblast (EPI) and primitive endoderm (PrE) cell fate specification in various types of embryos at the blastocyst stage by using the EPI-marker Nanog and the PrE-marker Gata6. In normal embryos under the CC and SC conditions, Nanog and Gata6 were co-expressed within the uncommitted ICM at the stage of <32 cells (Fig. S6A), but they displayed a ‘salt and pepper’ pattern at the stage of 32–64 cells (Fig. S6B). Notably, in the SS embryos, many Nanog-positive cells still expressed Gata6 in hatching blastocysts (Fig. S6C). Together, these results suggest that although the environment in space does not affect polarization and the establishment of the ICM and TE, growth and cellular differentiation are severely compromised in preimplantation mouse embryos.

### DNA damage and DNA methylome alterations in mouse embryos in space

After revealing the developmental defects of SS embryos during development, we investigated the potential underlying mechanisms. Previous studies suggested that radiation in space can increase the frequency of DNA damage in Drosophila Melanogaster and human cells during spaceflight [ 24–26]. We therefore examined the formation of γH2AX and 53bp1 foci by using these two biomarkers for double-strand breaks (DSBs) of DNA repair [ 27, 28]. In sharp contrast to the ground-cultured blastocysts, many blastomeres of the SS blastocysts were positive for γH2AX and 53bp1, showing stronger γH2AX and 53bp1 signals in the nuclei (Fig. 3A and B). In addition, experiments conducted using XRCC1, a maker for efficient repair of DNA single-strand breaks (SSBs) [ 29], showed that XRCC1 was localized in the nuclei of the SS blastocysts (Fig. 3C). Expectedly, quantification of fluorescence intensity confirmed a significant increase in γH2AX, 53bp1 and XRCC1 expression in SS blastocysts in comparison with the ground-cultured embryos (Figs 3D–F). These results indicate that the exposure of mammalian preimplantation embryos to the space environment causes severe DNA damage in the cells.

DNA damage and DNA methylome alterations in mouse embryos in space. (A–C) Representative images of CC, SC and SS blastocysts stained with γH2AX, 53bp1 and XRCC1 antibodies, respectively. Distinct staining patterns of cells were observed in blastocysts developed in space (white arrowheads). Scale bars, 50 μm. (D–F) Quantification of γH2AX (D), 53bp1 (E) and XRCC1 (F) immunofluorescence intensity normalized to DNA staining by Hoechst and analysed with ImageJ software. One imaging section was selected for each embryo for quantification, and the pooled data from embryos were plotted in the graphs. n = number of embryos. The results are shown as the means ± SEM. P values are from two-tailed unpaired Student's t-test. (G) The levels of genome-wide DNA methylation in CC, SC and SS blastocysts are shown. Three blastocysts were used for each group. The result are shown as the means ± SEM. Asterisks indicate the significance level of P < 0.05 (t-test). (H) Distribution of DNA methylation in the CpG context among various genomic element regions, including promoters, 5'UTR, exons, introns and 3'UTRs in CC, SC and SS blastocysts. The numbers denote the ID of specific blastocysts. (I) The number of differentially methylated regions (DMRs) in each pair-wise comparison of groups are shown. Blue and red bars indicate the proportion of hypermethylated and hypomethylated DMRs.

DNA damage and DNA methylome alterations in mouse embryos in space. (A–C) Representative images of CC, SC and SS blastocysts stained with γH2AX, 53bp1 and XRCC1 antibodies, respectively. Distinct staining patterns of cells were observed in blastocysts developed in space (white arrowheads). Scale bars, 50 μm. (D–F) Quantification of γH2AX (D), 53bp1 (E) and XRCC1 (F) immunofluorescence intensity normalized to DNA staining by Hoechst and analysed with ImageJ software. One imaging section was selected for each embryo for quantification, and the pooled data from embryos were plotted in the graphs. n = number of embryos. The results are shown as the means ± SEM. P values are from two-tailed unpaired Student's t-test. (G) The levels of genome-wide DNA methylation in CC, SC and SS blastocysts are shown. Three blastocysts were used for each group. The result are shown as the means ± SEM. Asterisks indicate the significance level of P < 0.05 (t-test). (H) Distribution of DNA methylation in the CpG context among various genomic element regions, including promoters, 5'UTR, exons, introns and 3'UTRs in CC, SC and SS blastocysts. The numbers denote the ID of specific blastocysts. (I) The number of differentially methylated regions (DMRs) in each pair-wise comparison of groups are shown. Blue and red bars indicate the proportion of hypermethylated and hypomethylated DMRs.

Epigenetic regulation such as DNA methylation plays a vital role in early mammalian embryonic development [ 30]. The developmental defects in SS embryos led us to ask whether they are linked to changes in DNA methylation during preimplantation development. We performed genome-wide DNA methylation profiling of individual blastocysts using bisulfite sequencing (BS-seq) [ 31]. Three expanded blastocysts from each group were collected and analysed (Fig. S7A). Methylome analysis showed that the percentage of global cytosine-guanine (CG) methylation in SS blastocysts (4.70%±0.2%) was significantly lower than that in CC (7.77%±1.3%) and SC (7.82%±2.0%) embryos (Fig. 3G and Fig. S7B and C), and the decrease was accompanied by a reduction in methylation density (Fig. S8). Additionally, we discovered a high extent of hypomethylation for DNA elements, including promoters, untranslated regions (UTRs), exons, introns and intergenic regions (Fig. 3H, and Fig. S9A–F) in the SS embryos compared with the CC and SC embryos. We further analysed DMRs, allowing us to identify 12 517 DMRs between the SS and CC conditions and 11 498 DMRs between the SS and SC conditions. There were fewer DMRs with high methylation between SS and CC (n = 2730) and between SS and SC (n = 2996) than between SC and CC (n = 7464), consistent with the low-level global methylation in SS blastocysts (Fig. 3I, and Fig. S7D). We then annotated overlapping DMRs with hypermethylation or hypomethylation (‘exclusive’ DMRs) for each group. Functional enrichment analyses indicated that the low-methylation exclusive DMRs in the SS blastocysts were related mainly to histone modifications, responses to radiation, regulation of chromosome organization, cytoskeleton organization and others (Fig. S7E and F), while high-methylation exclusive DMRs for CC blastocyst were associated with embryonic development, regulation of RNA metabolic processes and regulation of intracellular protein transport. These results suggest that in response to the environmental factors in space the genes responsible for histone modifications and radiation are hypomethylated, leading to their activation and subsequent repair of DNA damage.

### Effects of ground-based radiation on embryonic development

The mouse preimplantation embryos in space are exposed to two main environmental factors: radiation and microgravity. To ask what factors might have caused the developmental, genetic and epigenetic abnormalities in the embryos, we employed ground-based low-dose radiation to mimic the environment inside the SJ-10 satellite (cumulative dose of radiation: ∼0.15 mGy/day) (Fig. 4A). We found that most 2-cell embryos (66.01%) exposed to 0.1 mGy for 64 h can develop to the blastocyst stage. However, when the dose was increased to 0.5 mGy (which is equivalent to the dose in the satellite), only 58.44% of the embryos reached the blastocyst stage, and the percentage decreased to 45.69% when the dose was increased to 2 mGy (Fig. 4B and Fig. S10). Consistent with the developmental defects, we detected fluorescent foci positive for γH2AX in embryos exposed to 0.5 mGy or 2 mGy, with the foci more intense with 2 mGy exposure (Fig. 4C).

Effects of ground-based radiation on embryonic development. (A) To investigate the responses of mouse embryos to the accumulated low- doses of radiation, we irradiated 2-cell embryos with Cs-137 gamma at the doses of 0.1 mGy, 0.5 mGy and 2 mGy in ground-based experiments. (B) The percentage of 2-cell embryos that successfully developed to the blastocyst stage during in vitro cultivation with radiation exposure. Data are presented as the means ± SEM from four independent experiments. * P < 0.05 ** P < 0.01 n.s., not significant (P > 0.05). (C) Representative fluorescence images of γH2AX in blastocysts that developed from 2-cell embryos with or without exposure to different doses of radiation for 64 h. Scale bars, 50 μm. (D) The level of genome-wide DNA methylation in blastocysts exposed to 0, 0.1, 0.5 and 2 mGy radiation. The results are shows as the means ± SEM. * P < 0.05 n.s., not significant (P < 0.05) (t-test). (E) The number of differentially methylated regions (DMRs) in each pair-wise comparison of groups are shown. Blue and red bars indicate the proportion of hypermethylated and hypomethylated DMRs.

Effects of ground-based radiation on embryonic development. (A) To investigate the responses of mouse embryos to the accumulated low- doses of radiation, we irradiated 2-cell embryos with Cs-137 gamma at the doses of 0.1 mGy, 0.5 mGy and 2 mGy in ground-based experiments. (B) The percentage of 2-cell embryos that successfully developed to the blastocyst stage during in vitro cultivation with radiation exposure. Data are presented as the means ± SEM from four independent experiments. * P < 0.05 ** P < 0.01 n.s., not significant (P > 0.05). (C) Representative fluorescence images of γH2AX in blastocysts that developed from 2-cell embryos with or without exposure to different doses of radiation for 64 h. Scale bars, 50 μm. (D) The level of genome-wide DNA methylation in blastocysts exposed to 0, 0.1, 0.5 and 2 mGy radiation. The results are shows as the means ± SEM. * P < 0.05 n.s., not significant (P < 0.05) (t-test). (E) The number of differentially methylated regions (DMRs) in each pair-wise comparison of groups are shown. Blue and red bars indicate the proportion of hypermethylated and hypomethylated DMRs.

Next, we analysed genome-wide DNA methylation profiling in mouse embryos with radiation exposure (Fig. S11A and B), which, as expected, induces global changes in DNA methylation. There was a statistically significant decrease in genome-wide DNA methylation in embryos with 2 mGy exposure when compared with the control (Fig. 4D and Fig. S11C), but not with the 0.1 or 0.5 mGy exposure. Analysis of DNA-methylated genomic elements also revealed hypomethylation after exposure of embryos to 2 mGy radiation (Fig. S11D), including a reduction in the methylation of genomic elements (Fig. S12) and in the density of methylation distribution of CG (Fig. S13). We also examined DMRs between the control and the embryos with radiation, allowing us to identify 5429 DMRs between the control and 2 mGy group, of which 4916 (90.5%) were hypomethylated, and 5365 DMRs between control and the 0.5 mGy group, of which 2986 (55.7%) were hypomethylated (Fig. 4E, and Fig. S11E).

Additionally, genes with differential methylation were enriched in cellular processes such as responses to radiation, responses to stress and cytoskeletal organization, regulation of histone modifications, regulation of histone acetylation and regulation of protein ubiquitination (Fig. S11F and G). Disruption of these processes might cause developmental defects of embryos and reduced offspring birth.

To examine the long-term consequences of radiation, we determined whether the embryos with radiation exposure could develop to full-term mice. We transferred the irradiated and control blastocysts into pseudopregnant females at day three (Fig. S14A) and compared the birth rates. Although live offspring were obtained from embryos with radiation exposure (Fig. S14B), the overall rates of production from embryos with 0.5 mGy (21.07%) and 2 mGy exposure (7.45%) were strikingly lower than that from embryos without radiation exposure (32.61%) (Fig. S14B to D). Thus, mouse embryos exhibit hypersensitivity to extremely low doses of γ-radiation during preimplantation development in vitro.

### Effects of ground-based microgravity simulation on embryonic development

Having assessed the role of low-dose radiation in mouse embryonic development, we asked whether microgravity could also cause defects in preimplantation embryos. We cultured 2-cell mouse embryos in a rotary cell culture system (RCCS), which was developed by NASA for the purpose of simulated microgravity (SMG), and subsequently examined the effects (Fig. 5A). We found that most 2-cell embryos (65.4%) exposed to SMG for 64 h could develop to the blastocyst stage, without statistically significant differences (65.4% vs 72.9%) when compared with normal gravity (NG) (Fig. 5B and Fig. S15). Furthermore, we failed to observe any differences in DNA damage between control blastocysts and those developed with SMG (Fig. 5C). We also investigated genome-wide DNA methylation profiling in mouse blastocysts developed with SMG exposure (Fig. S16A). The heatmap of the percentage of DNA methylation at differentially methylated CpG sites for each blastocyst showed a similar methylation pattern (Fig. S16B). No statistically significant difference was found between blastocysts with SMG and with NG in the genome-wide DNA methylation level (Fig. 5D and Fig. S16C), including the distribution of DNA methylation in the CpG context among various DNA elements (Fig. S16D and Fig. S17) and in the density of methylation distribution of CG in chromosomes (Fig. S18). Intriguingly, 6130 DMRs were identified between the SMG and the NG group and were composed of 4563 DMRs with hypermethylation and 1567 DMRs with hypomethylation (Fig. 5E). Thus, a lower proportion of hypomethylated DMRs was discovered.

Effects of simulated microgravity on preimplantation embryonic development. (A) An image of the experimental apparatus, in which the embryos were inoculated into 10 mL of the culture vessel of RCCS for the simulated microgravity culture and static culture in CO2 incubators. (B) The rates of development of embryos to the blastocyst stages under normal gravity (NG) and simulated microgravity (SMG) conditions. Data are presented as the means ± SEM from four independent experiments (embryos analysed: n = 899 for NG n = 886 for SMG). (C) Representative fluorescence images of γH2AX in blastocysts that were developed from 2-cell embryos exposed to NG and SMG conditions for 64 h. Scale bars, 50 μm. (D) The levels of genome-wide DNA methylation in NG and SMG blastocysts are shown. Data are presented as the means ± SEM from four blastocysts. (E) The number of differentially methylated regions (DMRs) between SMG and NG group are shown. Blue and red bars indicate the proportion of hypermethylated and hypomethylated DMRs. Throughout, a Student's t-test (two-tailed) was used for statistical analysis n.s., not significant.

Effects of simulated microgravity on preimplantation embryonic development. (A) An image of the experimental apparatus, in which the embryos were inoculated into 10 mL of the culture vessel of RCCS for the simulated microgravity culture and static culture in CO2 incubators. (B) The rates of development of embryos to the blastocyst stages under normal gravity (NG) and simulated microgravity (SMG) conditions. Data are presented as the means ± SEM from four independent experiments (embryos analysed: n = 899 for NG n = 886 for SMG). (C) Representative fluorescence images of γH2AX in blastocysts that were developed from 2-cell embryos exposed to NG and SMG conditions for 64 h. Scale bars, 50 μm. (D) The levels of genome-wide DNA methylation in NG and SMG blastocysts are shown. Data are presented as the means ± SEM from four blastocysts. (E) The number of differentially methylated regions (DMRs) between SMG and NG group are shown. Blue and red bars indicate the proportion of hypermethylated and hypomethylated DMRs. Throughout, a Student's t-test (two-tailed) was used for statistical analysis n.s., not significant.

## What is the total number of rounds of cleavage during mammalian embryonic development? - Biology

Introduction

Is it a boy or a girl? It’s one of the most common questions asked of pregnant women. The suspense around learning the baby’s sex never fails to excite family and friends. For centuries, members of the elder generations have offered advice on how to predict the outcome or even plan it&mdashDangle a needle over your belly by a thread. Does it swing side to side or in circles?

In the modern world, needle dangling doesn’t carry the same credibility as technological advances like ultrasonography. This radiographic technique is performed by placing a probe that emits high-frequency sound waves near the tissue to be examined. The probe transduces an image onto a computer screen, which can be measured to determine gestational age, screen for multiple pregnancies or anomalies, and identify the baby’s sex. So how early can we determine a baby’s sex? All embryos begin female by default&mdashthat is, for a male fetus to develop, it must undergo not only masculinization via the gene product of SRY, but also defeminization. These processes occur (or don’t) around six to eight weeks postfertilization. Don’t expect any answers before 16 to 17 weeks, however, because ultrasonography equipment does not have high enough resolution yet to give immediate answers.

In this chapter, we’ll continue the discussion from the previous chapter by beginning with fertilization, the formation of a diploid zygote from the union of a sperm and an ovum. We’ll then follow development from this point until the birth of an autonomously breathing baby. We’ll examine how the cells of a developing human divide and differentiate. We’ll also explore some specific system differences that exist between developing fetuses and adults as we present an overview of the stages of pregnancy and childbirth.

### 3.1 Early Developmental Stages

In this first section, we explore development from the formation of a diploid zygote until neurulation, or the formation of the neural tube that will differentiate into the nervous system.

As discussed in Chapter 2 of MCAT Biology Review, a secondary oocyte is ovulated from the follicle on approximately day 14 of the menstrual cycle. The secondary oocyte travels into the fallopian tube, where it can be fertilized up to 24 hours after ovulation. Fertilization, shown in Figure 3.1, usually occurs in the widest part of the fallopian tube, called the ampulla. When the sperm meets the secondary oocyte in the fallopian tube, it binds to the oocyte and releases acrosomal enzymes that enable the head of the sperm to penetrate through the corona radiata and zona pellucida. Once the first sperm comes into direct contact with the secondary oocyte’s cell membrane, it forms a tubelike structure known as the acrosomal apparatus, which extends to and penetrates the cell membrane. Its pronucleus may then freely enter the oocyte once meiosis II has come to completion. After penetration of the sperm through the cell membrane, the cortical reaction, a release of calcium ions, occurs. These calcium ions depolarize the membrane of the ovum, which serves two purposes: depolarization prevents fertilization of the ovum by multiple sperm cells, and the increased calcium concentration increases the metabolic rate of the newly formed diploid zygote. The now depolarized and impenetrable membrane is called the fertilization membrane.

Figure 3.1. Fertilization

Twins can occur by two different mechanisms. Dizygotic, or fraternal twins, form from fertilization of two different eggs released during one ovulatory cycle by two different sperm. Each zygote will implant in the uterine wall, and each develops its own placenta, chorion, and amnion&mdashthese structures are discussed later in the chapter. If the zygotes implant close together, the placentas may fuse. Fraternal twins are no more genetically similar than any other pair of siblings.

Monozygotic, or identical twins, form when a single zygote splits into two. Because the genetic material is identical, so too will be the genomes of the offspring. If division is incomplete, conjoined twins may result, in which the two offspring are physically attached at some point. Monozygotic twins can be classified by the number of structures they share. Monochorionic/monoamniotic twins share the same amnion and chorion. Monochorionic/diamniotic twins each have their own amnion, but share the same chorion. Dichorionic/diamniotic twins each have their own amnions and chorions. Which type of twinning occurs is a result of when the separation occurred. As more gestational structures are shared, there are more risks as the fetuses grow and develop.

After fertilization in the fallopian tubes, the zygote must travel to the uterus for implantation. If it arrives too late, there will no longer be an endometrium capable of supporting the embryo. In the process of moving to the uterus for implantation, the zygote undergoes rapid mitotic cell divisions in a process called cleavage. The first cleavage officially creates an embryo, as it nullifies one of the zygote’s defining characteristics: unicellularity. Although several rounds of mitosis occur, the total size of the embryo remains unchanged during the first few divisions, as shown in Figure 3.2. By dividing into progressively smaller cells, the cells increase two ratios: the nuclear-to-cytoplasmic (N:C) ratio and the surface area-to-volume ratio. Thus, the cells achieve increased area for gas and nutrient exchange relative to overall volume. There are two types of cleavage: indeterminate and determinate. Indeterminate cleavage results in cells that can still develop into complete organisms. In fact, monozygotic twins have identical genomes because they both originate from indeterminately cleaved cells of the same embryo. Determinate cleavage results in cells with fates that are, as the term implies, already determined. In other words, these cells are committed to differentiating into a certain type of cell.

Figure 3.2. An 8-Cell Embryo The embryo has undergone three cleavage events at this point.

Several divisions later, the embryo becomes a solid mass of cells known as a morula, as shown in Figure 3.3. This term comes from the Latin word for mulberry, which might help us grasp what an embryo at this stage looks like.

Figure 3.3. Morula The morula is a solid ball of cells.

Once the morula is formed, it undergoes blastulation, which forms the blastula, a hollow ball of cells with a fluid-filled inner cavity known as a blastocoel. The mammalian blastula is known as a blastocyst and consists of two noteworthy cell groups, as shown in Figure 3.4: the trophoblast and inner cell mass. The trophoblast cells surround the blastocoel and give rise to the chorion and later the placenta, whereas the inner cell mass protrudes into the blastocoel and gives rise to the organism itself.

Figure 3.4. Blastula The blastula contains a fluid-filled cavity called the blastocoel.

Remember that an embryo with a blasted-out cavity is a blastula.

Implantation

The blastula moves through the fallopian tube to the uterus, where it burrows into the endometrium. The trophoblast cells are specialized to create an interface between the maternal blood supply and the developing embryo. These trophoblastic cells give rise to the chorion, an extraembryonic membrane that develops into the placenta. The trophoblasts form chorionic villi, which are microscopic fingerlike projections that penetrate the endometrium. As these chorionic villi develop into the placenta, they support maternal–fetal gas exchange. The embryo is connected to the placenta by the umbilical cord, which consists of two arteries and one vein encased in a gelatinous substance. The vein carries freshly oxygenated blood rich with nutrients from the placenta to the embryo. The umbilical arteries carry deoxygenated blood and waste to the placenta for exchange.

Sometimes the blastula implants itself outside the uterus, a situation known as an ectopic pregnancy. Over 95% of ectopic pregnancies occur in the fallopian tube. Ectopic pregnancies are generally not viable because the narrow fallopian tube is not an environment in which an embryo can properly grow. If the embryo does not spontaneously abort, the tube may rupture, and a considerable amount of hemorrhaging may occur. In fact, a suspected ectopic pregnancy is often a surgical emergency.

Until the placenta is functional, the embryo is supported by the yolk sac. The yolk sac is also the site of early blood cell development. There are two other extraembryonic membranes that require discussion: the allantois and the amnion. The allantois is involved in early fluid exchange between the embryo and the yolk sac. Ultimately, the umbilical cord is formed from remnants of the yolk sac and the allantois. The allantois is surrounded by the amnion, which is a thin, tough membrane filled with amniotic fluid. This fluid serves as a shock absorber during pregnancy, lessening the impact of maternal motion on the developing embryo. In addition to forming the placenta, the chorion also forms an outer membrane around the amnion, adding an additional level of protection. The anatomy of these structures is shown in Figure 3.5.

Figure 3.5. Anatomy of Pregnancy

Amniocentesis is the process of aspirating amniotic fluid by inserting a thin needle into the amniotic sac. The amniotic fluid contains fetal cells that can be examined for chromosomal abnormalities as well as sex determination. Amniocentesis is recommended for pregnant women over 35 if earlier screening tests (blood tests and ultrasound) indicate a high chance of chromosomal abnormalities in the fetus. Women in this age group have a higher rate of meiotic nondisjunction, which can result in genetic aberrations such as Down syndrome.

Once the cell mass implants, it can begin further developmental processes such as gastrulation, the generation of three distinct cell layers. The early developmental processes up to this point are shown in Figure 3.6. Much of our knowledge of development comes from the study of other organisms, which have varying degrees of similarity to human development. In sea urchins, gastrulation begins with a small invagination in the blastula. Cells continue moving toward the invagination, resulting in elimination of the blastocoel. To visualize this, imagine inflating a balloon and then poking one of the sides with your finger. If you kept pushing, eventually the rubber from one side of the balloon would come into contact with the other side. If the two membranes could merge, as occurs in development, this would create a tube through the middle of the balloon. In living things, the result of this process is called a gastrula. The membrane invagination into the blastocoel is called the archenteron, which later develops into the gut. The opening of the archenteron is called the blastopore. In deuterostomes, such as humans, the blastopore develops into the anus. In protostomes, it develops into the mouth.

Figure 3.6. Early Stages of Embryonic Development

How can we remember the blastopore’s fate in protostomes vs. deuterostomes? Think about how parents talk to toddlers&mdashdeuterostome starts with deu, which looks like duo, meaning two. Thus, deuterostomes develop the anus&mdashthe orifice associated with “number two”&mdashfrom the blastopore. Protostomes must start at the other end (the mouth).

Primary Germ Layers

Eventually, some cells will also migrate into what remains of the blastocoel. This establishes three layers of cells called primary germ layers.

The outermost layer is called the ectoderm and gives rise to the integument, including the epidermis, hair, nails, and the epithelia of the nose, mouth, and lower anal canal. The lens of the eye, nervous system (including adrenal medulla), and inner ear are also derived from ectoderm.

The middle layer is called the mesoderm and develops into several different systems including the musculoskeletal, circulatory, and most of the excretory systems. Mesoderm also gives rise to the gonads as well as the muscular and connective tissue layers of the digestive and respiratory systems and the adrenal cortex.

The innermost layer is called the endoderm and forms the epithelial linings of the digestive and respiratory tracts, including the lungs. The pancreas, thyroid, bladder, and distal urinary tracts, as well as parts of the liver, are derived from endoderm.

·&emspEctoderm&mdash“attracto”derm (things that attract us to others, such as cosmetic features and “smarts”)

·&emspMesoderm&mdash“means”oderm (the means of getting around as an organism, such as bones and muscle the means of getting around in the body, such as the circulatory system the means of getting around, such as the gonads)

·&emspEndoderm&mdashlinings of “endernal” organs (the digestive and respiratory tract, and accessory organs attached to these systems)

MCAT EXPERTISE

The MCAT likes to test on the dual embryonic origin of the adrenal glands. The adrenal cortex is derived from the mesoderm, but the adrenal medulla is derived from the ectoderm (because the adrenal medulla contains some nervous tissue).

Differentiation

So how is it that cells with the same genes are able to develop into such distinctly different cell types with highly specialized functions? Primarily, it is by selective transcription of the genome. In other words, only the genes needed for that particular cell type are transcribed. Thus, in pancreatic islet cells, the genes to produce specific hormones (insulin, glucagon, or somatostatin) are turned on, while these same genes are turned off in other cell types. Selective transcription is often related to the concept of induction, which is the ability of one group of cells to influence the fate of other nearby cells. This process is mediated by chemical substances called inducers, which diffuse from the organizing cells to the responsive cells. These chemicals are responsible for processes such as the guidance of neuronal axons. This process also ensures proximity of different cell types that work together within an organ.

Once the three germ layers are formed, neurulation, or development of the nervous system, can begin. Remember that the nervous system is derived from the ectoderm. How, then, do cells originating on the surface of the embryo (ectoderm) end up inside the final organism? First, a rod of mesodermal cells known as the notochord forms along the long axis of the organism like a primitive spine (in fact, small remnants of notochord persist in the intervertebral discs between vertebrae). The notochord induces a group of overlying ectodermal cells to slide inward to form neural folds, which surround a neural groove, as shown in Figure 3.7. The neural folds grow toward one another until they fuse into a neural tube, which gives rise to the central nervous system. At the tip of each neural fold are neural crest cells. These cells migrate outward to form the peripheral nervous system (including the sensory ganglia, autonomic ganglia, adrenal medulla, and Schwann cells) as well as specific cell types in other tissues (such as calcitonin-producing cells of the thyroid, melanocytes in the skin, and others). Finally, ectodermal cells will migrate over the neural tube and crests to cover the rudimentary nervous system.

Figure 3.7. Formation of the Neural Tube

Failure of the neural tube to close results in either spina bifida (in which some or all of the spinal cord may be exposed to the outside world) or anencephaly (in which the brain fails to develop and the skull is left open). The severity of spina bifida’s effects range from no significant distress to death, whereas anencephaly is universally fatal. Women who wish to conceive are encouraged to take folate (folic acid) to prevent this complication it is recommended that all women of childbearing age supplement their diets with folate in advance of getting pregnant because neurulation often occurs before pregnancy is detected.

PROBLEMS IN EARLY DEVELOPMENT

Early development is a highly sensitive time during the development of a human being. During this stage, as the germ layers are forming and then organogenesis (the production of organs) begins, teratogens may have far-reaching and highly detrimental effects. Teratogens are substances that interfere with development, causing defects or even death of the developing embryo. However, each teratogen will not have the same effect on every embryo or fetus. It is believed that the genetics of the individual embryo influences the effects of the teratogen. In addition to genetics, the route of exposure, length of exposure, rate of placental transmission of the teratogen, and the exact identity of the teratogen will also affect the overall outcome. Some common teratogens include alcohol, prescription drugs, viruses, bacteria, and environmental chemicals including polycyclic aromatic hydrocarbons.

In addition to teratogens, development can also be influenced by maternal health. Certain conditions may cause changes in the overall physiology of the mother, resulting in overexposure or underexposure of the embryo or fetus to certain chemicals. For example, diabetic mothers with hyperglycemia (high blood glucose) can have poor birth outcomes. Overexposure to sugar in utero can lead to a fetus that is too large to be delivered and who suffers from hypoglycemia soon after birth (due to synthesizing very high levels of insulin to compensate). Maternal folic acid deficiency may prevent complete closure of the neural tube, resulting in spina bifida or anencephaly, in which parts of the nervous system are exposed to the outside world or covered with a thin membrane. However, like teratogens, maternal health issues can have variable effects on the developing fetus. Spina bifida may be so severe as to result in profound disability, or may be completely asymptomatic and only detected by a tuft of hair overlying the area. Overall, trends and associations can certainly be found between various environmental conditions and genes during development however, it is somewhat unpredictable and highly variable.

MCAT Concept Check 3.1:

Before you move on, assess your understanding of the material with these questions.

1. What is the difference between determinate and indeterminate cleavage?

2. From zygote to gastrula, what are the various stages of development?

3. During which stage of development does implantation occur?

4. What are the primary germ layers, and what organs are formed from each?

## Chapter One - Regulation of the Embryonic Cell Cycle During Mammalian Preimplantation Development

The preimplantation development stage of mammalian embryogenesis consists of a series of highly conserved, regulated, and predictable cell divisions. This process is essential to allow the rapid expansion and differentiation of a single-cell zygote into a multicellular blastocyst containing cells of multiple developmental lineages.

This period of development, also known as the germinal stage, encompasses several important developmental transitions, which are accompanied by dramatic changes in cell cycle profiles and dynamics. These changes are driven primarily by differences in the establishment and enforcement of cell cycle checkpoints, which must be bypassed to facilitate the completion of essential cell cycle events.

Much of the current knowledge in this area has been amassed through the study of knockout models in mice. These mouse models are powerful experimental tools, which have allowed us to dissect the relative dependence of the early embryonic cell cycles on various aspects of the cell cycle machinery and highlight the extent of functional redundancy between members of the same gene family.

This chapter will explore the ways in which the cell cycle machinery, their accessory proteins, and their stimuli operate during mammalian preimplantation using mouse models as a reference and how this allows for the usually well-defined stages of the cell cycle to be shaped and transformed during this unique and critical stage of development.

## Developmental Biology. 6th edition.

Cleavage in most frog and salamander embryos is radially symmetrical and holoblastic, just like echinoderm cleavage. The amphibian egg, however, contains much more yolk. This yolk, which is concentrated in the vegetal hemisphere, is an impediment to cleavage. Thus, the first division begins at the animal pole and slowly extends down into the vegetal region (Figure 10.1 see also Figures 2.2D and 8.4). In the axolotl salamander, the cleavage furrow extends through the animal hemisphere at a rate close to 1 mm per minute. The cleavage furrow bisects the gray crescent and then slows down to a mere 0.02𠄰.03 mm per minute as it approaches the vegetal pole (Hara 1977).

#### Figure 10.1

Cleavage of a frog egg. Cleavage furrows, designated by Roman numerals, are numbered in order of appearance. (A, B) Because the vegetal yolk impedes cleavage, the second division begins in the animal region of the egg before the first division has divided (more. )

Figure 10.2A is a scanning electron micrograph showing the first cleavage in a frog egg. One can see the difference in the furrow between the animal and the vegetal hemispheres. Figure 10.2B shows that while the first cleavage furrow is still cleaving the yolky cytoplasm of the vegetal hemisphere, the second cleavage has already started near the animal pole. This cleavage is at right angles to the first one and is also meridional. The third cleavage, as expected, is equatorial. However, because of the vegetally placed yolk, this cleavage furrow in amphibian eggs is not actually at the equator, but is displaced toward the animal pole. It divides the frog embryo into four small animal blastomeres (micromeres) and four large blastomeres (macromeres) in the vegetal region. This unequal holoblastic cleavage establishes two major embryonic regions: a rapidly dividing region of micromeres near the animal pole and a more slowly dividing vegetal macromere area (Figure 10.2C Figure 2.2E). As cleavage progresses, the animal region becomes packed with numerous small cells, while the vegetal region contains only a relatively small number of large, yolk-laden macromeres.

#### Figure 10.2

Scanning electron micrographs of the cleavage of a frog egg. (A) First cleavage. (B) Second cleavage (4 cells). (C) Fourth cleavage (16 cells), showing the size discrepancy between the animal and vegetal cells after the third division. (A from Beams and (more. )

An amphibian embryo containing 16 to 64 cells is commonly called a morula (plural: morulae from the Latin, “mulberry,” whose shape it vaguely resembles). At the 128-cell stage, the blastocoel becomes apparent, and the embryo is considered a blastula. Actually, the formation of the blastocoel has been traced back to the very first cleavage furrow. Kalt (1971) demonstrated that in the frog Xenopus laevis, the first cleavage furrow widens in the animal hemisphere to create a small intercellular cavity that is sealed off from the outside by tight intercellular junctions (Figure 10.3). This cavity expands during subsequent cleavages to become the blastocoel.

#### Figure 10.3

Formation of the blastocoel in a frog egg. (A) First cleavage furrow, showing a small cleft, which later develops into the blastocoel. (B) 8-cell embryo showing a small blastocoel (arrow) at the junction of the three cleavagefurrows. (From Kalt 1971 (more. )

The blastocoel probably serves two major functions in frog embryos: (1) it permits cell migration during gastrulation, and (2) it prevents the cells beneath it from interacting prematurely with the cells above it. When Nieuwkoop (1973) took embryonic newt cells from the roof of the blastocoel, in the animal hemisphere, and placed them next to the yolky vegetal cells from the base of the blastocoel, these animal cells differentiated into mesodermal tissue instead of ectoderm. Because mesodermal tissue is normally formed from those animal cells that are adjacent to the vegetal endoderm precursors, it seems plausible that the vegetal cells influence adjacent cells to differentiate into mesodermal tissues. Thus, the blastocoel appears to prevent the contact of the vegetal cells destined to become endoderm with those cells fated to give rise to the skin and nerves.

While these cells are dividing, numerous cell adhesion molecules keep the blastomeres together. One of the most important of these molecules is EP-cadherin. The mRNA for this protein is supplied in the oocyte cytoplasm. If this message is destroyed (by injecting antisense oligonucleotides complementary to this mRNA into the oocyte), the EP-cadherin is not made, and the adhesion between the blastomeres is dramatically reduced (Heasman et al. 1994a,b), resulting in the obliteration of the blastocoel (Figure 10.4).

#### Figure 10.4

Depletion of EP-cadherin mRNA in the Xenopus oocyte results in the loss of adhesion between blastomeres and the obliteration of the blastocoel. (A) control embryo (B) EP-cadherin-depleted embryo. (From Heasman et al. 1994b photographs courtesy of J. (more. )

## 8 DISCUSSION

We preface this discussion with the understanding that SCNT has made incredible gains toward understanding totipotency and epigenesis of the early embryo SCNT products are widely called clones and are not considered synthetic although it's relevance to empower SESs remains high. The scope of this review is on cultured cells that are practically and technically distant from real embryonic origins.

With the emerging SESs, we see an abundance of opportunity and a fertile research environment to empower and inform. Recent paradigm shifts in related technologies (cells, cultures, materials, omics, bioinformatics, etc.) have set the stage to seed the current wave of SESs that have near-simultaneously emerged despite a variety of inputs, to generate a broad spectrum of embryo-like outputs. From here, these SESs spur the conversations and discoveries that we need to have to grow our human dimension. From cell potencies, cell-cell interactions, molecular pathways, patterning, implantation, and post-implantation development, a window to the unknown has manifested. At last, we embark on employing SESs to excite our interests to the origins of life and provide two SES examples below.

### 8.1 Correcting human differentiation

Differentiating human PSCs in vitro drives attractive applications such as regenerative medicine and drug development. However, most current differentiated cells are far less functional than the true in vivo equivalent. Human SESs focusing on post-implantation development may be crucial to understand correct differentiation paths to improve in vitro differentiation protocols from PSCs and the quality of the differentiated cells. For instance, the human gastruloid revealed that canonical WNT pathway stimulation by Chiron(CHIR) was important in initiating gastruloid formation and spatial-temporal gene expression despite the fact that neither WNT3A nor BMP4 could do so (Moris et al., 2020 ). Importantly, BMP4 is crucial in PSC patterning SESs (Warmflash et al., 2014 ). These results suggest that an unknown molecular determinant exists in the first steps of differentiation. We believe that further investigation with human SESs will greatly improve upon established PSC technologies and applications.

### 8.2 Symmetry and polarity dynamics

The first clear cell symmetry break occurs in the morula stage as the TE and ICM lineages appear. This event has drawn many developmental biologists’ attention for investigations at the molecular level (e.g., Maitre et al., 2016 Rossant & Tam, 2009 )). Currently, two models explain the molecular mechanism of this specification, although they are not mutually exclusive. One model is the positional model, and the other is the polarity model (Toyooka, 2020 ). Both models conclude that the Hippo-Yap axis segregates the two lineages (Nishioka et al., 2009 ). Curiously, we observed the same regulation in the Yap localization in mouse iBLC-PCs (Kime et al., 2019 ) as mouse embryos have (Nishioka et al., 2009 ). Employing SES with Yap-reporter and genetic engineering may shed new light on the first symmetry break event in embryogenesis.

### 8.3 Closing remarks - research paradigms in culture

Researchers have constantly faced the limitations of cell systems and animal models to find conserved and differentiating aspects of life systems. Our mentor Bruce Conklin once said that “human is the new model system” and pointed convincingly at the ongoing discovery of critical differences that separate our species from animal models differences that weigh heavily on medical relevance tethered to public funding. Basic researchers and animal models have driven the abundance of research put forth, often at the behest of human relevance, but times have changed, and the paradigm is shifting. Human cells are becoming easier to work with. Human cell-based SESs may pave the way to realistic data to improve our development, implantation, pregnancy, the body plan, and regenerative medicine. Conversely, animal models still skirt ethics concerns and provide the most substantive evidence in the form of living animals. The interplay may have shifted such that we may now start a study with human cells and later validate in animal models. Unlike most human biology, the relevance here is human embryogenesis and the ethics carry far more power than the facts. We may need human relevance more than ever, yet the taboo of an ideal human SES model will be long lasting. We anticipate that important designs that limit cell potencies or confine output may enable some aspect of human SESs to find broad acceptance.