Implications of the mitotic selective chromatid segregation

phenomenon for vertebrate development

Authors: Armakolas A1, Koutsilieris M1, Klar AJS2

1
Laboratory of Experimental Physiology, Athens Medical School Mikras Asias 75,
Athens 115 27, Greece; 2Gene Regulation and Chromosome Biology
Laboratory, Building 539, P. O. Box B, NCI-Frederick, Frederick, MD 21702-
1201, USA.

Corresponding authors: 1 thanosarmakolas@yahoo.com and 2 Klar@ncifcrf.gov

Running title: Selective chromatid segregation

Key words for use in the reviewing process: asymmetric cell division;
selective chromatid segregation; mouse cell mitosis, visceral laterality
development, developmental mechanism

Words: 7329 (excluding 104 references), 3 figures, 1 table.

1
Abstract

Asymmetric cell division is a fundamental process of cell biology. It is required to

generate cellular diversity and tissue renewal during development. During this
developmentally regulated process, mitosis yields two non-equivalent daughter
cells that arise from the same parental cell. Despite inheriting identical genetic
material, daughter cells often exhibit properties different from one another. To
explain this mechanism of cellular differentiation, several hypotheses have been
advanced, mostly involving studies of invertebrate model systems and, to a lesser
extent, of mammals. Here we review the evidence obtained from diverse
biological systems to highlight how different mechanisms interplay to accomplish
cellular differentiation. We focus primarily on studies relevant to a relatively
recently proposed model that builds on the replication history of specific “Watson”
versus “Crick” DNA strands of a chromosome. It proposes generation of non-
equivalent sister chromatids by epigenetic means followed by selective
distribution of thus differentiated chromatids between daughter cells. We present
implications of this chromosome modification/inheritance model for mice visceral
organs’ body axis laterality development.

Introduction

Every multi-cellular organism consists of a variety of different cell types that give
rise to different tissues and organs. The development in most organisms
starts from a single cell, which after multiple cell divisions gives rise to
different tissues, with each tissue containing cells of specific type(s). The
significance of asymmetric cell division for the development of multi-cellular
organisms, including vertebrates, is widely recognized. For example, a
stem cell often generates one daughter cell that is committed to
differentiation, while the other daughter cell maintains stem-cell
characteristics to generate cellular diversity of each organism. Four major
hypotheses have been proposed for explaining the phenomenon of
asymmetric cell division. These can be broadly separated into two major
categories: those that suggest that asymmetry is imposed by the cells’
exposure to extra-cellular environment, and others postulate that the
information for cellular differentiation is found internally in the cell causing
asymmetric cell division of the progenitor cell.

Prominent among the first type, the morphogen-gradient model suggests that
two initially identical daughter cells become different because they
encounter different microenvironments that induce and repress different
sets of genes in a cell. A special case of this hypothesis concerns different
stem cell niches. In this case, precise cellular location influences the stem
cell’s division by integrating signals emanating from adjoining cells in the
nitch to induce daughter cells to differ from one another. Alternatively, the
fate of each daughter cell may be determined by cell intrinsic factors. In
this case, either the cytoplasmic cell-fate determinants are segregated into
only one of the two daughter cells during mitosis, or alternatively, non-
random chromatid segregation to daughter cells confers non-
equivalency on them. Perhaps different combinations of these mechanisms
operate in different tissues.

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The morphogen-gradient model

Morphogens are believed to be secreted cell-signalling molecules that help

organize a field of surrounding cells into different cell-differentiated patterns.
According to a popular hypothesis, morphogens form a gradient of concentration
emanating from a localized source, and thereby determine the arrangement and
fate of responding cells according to the different concentrations of the
morphogen perceived by them. The idea of the morphogen gradient is intimately
associated with the concept of positional information in a field. A cell is believed
to read its position in a concentration gradient and it develops its fate
accordingly. It is believed that a single event, the emission of a factor
(morphogen) from a source, can lead to the formation of many different cell
types to accomplish their correct spatial positioning in the body of the organism
(21). While most of the evidence supporting this hypothesis came from studies
of Drosophila, mammalian models for limb patterning have also been designed to
test the hypothesis (34, 89, 92). Indeed, defining the possible role of morphogen
gradients in vertebrate systems has been a focus of increasing interest for many
decades. Although this has been a prominent hypothesis proposed for
development, by no means has it been accepted because many major questions
remain to be answered. It is necessary to determine the morphogen’s
biochemical identity, how the concentration gradient is formed, how the absolute
concentration of the gradient is maintained, and what are the factors that create
and maintain it for different precise levels of concentration in a developmental
field. Furthermore, it is believed that a number of different morphogens affect
cells differently. So, what is the mechanism of differential response of cells to
different threshold concentrations of morphogens? How do receptors on cell
surface transduce this information to the nucleus to create the appropriate gene
expression response to accomplish cellular differentiation?

Although much data has been published about the role of the morphogen
gradient in embryonic development, there is no direct evidence that it affects the
process of asymmetric cell division, at least in studies with mammals. In a related
issue, host tissue-specific signals have been implicated in the biology of
metastatic cancer (63). Also, development of refractoriness to hormones and to
chemotherapy in clinical responses has been demonstrated at metastatic sites
(91). Although the cellular signalling phenomenon is well established in the
aforementioned cases, it is not clear whether the morphogen-gradient model can
readily explain general development as well as the origin and proliferation of
cancer.

Stem cell niches promote asymmetric cell division

Although the morphogen model has long been a prominent model under study,
the major evidence that might be construed as supporting it came only recently
from studies of the female germ line in Drosophila flies. In this prominent model
for stem cell biology, one daughter cell (the stem cell) self-renews at each
division, while the other cell stops to proliferate after a few cell divisions and its
progeny differentiates. The cell’s asymmetry is regulated by extracellular signals
emanating from the surrounding ovarian stem cell niche. Two biochemical
pathways are believed to be involved in the process of asymmetric cell division
(100, 53, 54). Specifically, the germline stem-cell niche signaling requires two
signalling pathways: the TGF and an unknown pathway that is defined by the YB
and PIWI proteins. The effect of those two pathways is to repress the expression
of the “bag of marbles” (bam), a gene that is necessary and sufficient for
promoting stem cell differentiation (12, 86). The niche function is additionally
assisted by the Hedgehog signaling pathway, and it also requires niche cell
adhesion mediated by epithelial cadherin molecules (36, 80). Notably, the

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asymmetric cell division is promoted by the attachment of one pole of the stem
cell spindle to the niche cells. This attachment is performed by a spectrin-rich
structure—the spectrosome—and a cytoplasmic dynein-mediated mechanism (1,
58). After division, the daughter cell that loses the niche contact starts the
differentiation process. A recent study (64) implicates microRNAs, operating
through chromatin organization, in the choice mechanism for stem cell versus
differentiated lineages development. The most alluring feature of this system,
likely to be useful for differentiation in general, is that robust differentiation
decisions are made at the single cell level, whereby the fate of sister cells is
specified to differ because of an asymmetric cell division that requires cell-cell
contact.

Recently it was also demonstrated that the Drosophila male germ line stem cells
act in a similar way. In this case, the interaction of stem cells with the somatic
niche cells (hub cells) promotes the asymmetric cell division. As a stem cell
divides, one pole of its mitotic spindle is anchored to the niche cells, ensuring the
asymmetric division by allowing only one of the two daughter cells to maintain
contact with the niche cells. Thus, only the attached cell retains the stem-cell
fate. The attachment complex in this case contains cadherin, catenin, and the
adenomatous polyposis coli 2 (APC2) protein. Moreover, maintenance of germ
line stem cells is achieved by the hub cells’ secretion of an unpaired ligand of the
JAK-STAT pathway (53, 54, 100).

Although the role of niches in the asymmetric division in mammalian stem cells
has not been as clearly defined, there is some evidence that such niches may
exist there and they may function in ways similar to those employed by flies. A
few in vitro studies exist, where surrounding niche cells may be required for the
asymmetric cell divisions in mouse embryonic basal epidermal cells, embryonic
neuroepithelial cells, and hematopoietic progenitor cells. Interestingly, the
switching of proliferating cells to a differentiated type involves a change in
orientation of cleavage plane from perpendicular to parallel in reference to the
plane of the apical lamina (45). Furthermore, the hematopoietic progenitor cells
are capable of both symmetric (proliferation) and asymmetric (differentiation)
divisions in cultures supported by stromal cells (13, 23, 48, 99). The niches
provide transient signals for stem cell division to support ongoing tissue
regeneration. The balance between proliferation-inhibiting and proliferation-
promoting signals is the key to homeostatic regulation of stem cell maintenance
versus tissue regeneration. Loss of the niche can lead to loss of stem cells, thus
indicating the reliance of stem cells on niche signals. Interestingly, aberrations of
stem cell controls lead to cancerous growth. Recent evidence indicates the
involvement of niches for cancer development. Cancer stem cells may arise from
a mutation leading to self-sufficient cell proliferation, or they may also involve
deregulation or alteration of the niche by dominant proliferation-promoting
signals. Furthermore, the molecular machinery used by normal stem cells for
homing on to or mobilizing from the niche may be used by cancer stem cells for
invasion and metastasis, as well as to create a diversion of cancer celles’ response
to chemotherapies (82).

Asymmetric division by cell intrinsic factors

Most of the insight into the process of cell-intrinsic asymmetric divisions in multi-
cellular organisms has come from invertebrate model systems, especially
Drosophila and the earthworm C. elegans. Evidence is obtained by
implicating proteins that exhibit alterations in cells known to proceed
asymmetric cell division, such as embryonic and adult stem cells,
Drosophila sensory organ precursor (SOP) cells, and neural precursor cells

4
 (4, 7, 49, 50, 57, 75, 95). According to one model, asymmetric cell division
constitutes a four-step process that leads to the generation of two non-
equivalent daughter cells. It has been suggested that initially the symmetry
within a parental cell is interrupted by the presence of a certain signal that
initiates the sequel of the asymmetric cell division. This signal has been
assumed to be a factor from the surroundings or from neighbouring cells.
During the second step, the parental cell becomes polarized and that
usually involves the polarization of the actomyocin network. It was found in
C. elegans that the evolutionary conserved PAR proteins and associated
components are key players in the process of polarization (22, 62). There,
the process starts shortly after fertilization. An association of PAR-3, PAR-6,
and atypical protein kinase C forms a complex (33, 90). This complex
retracts towards the anterior, along with the contractile actomyosin
network, where PAR2 and PAR1 are localized in the non-contractile regions
of the cytoskeleton (15). It has also been found that the retention of the
anterior and posterior cortical domains as distinct entities is due to the
presence of PAR5 and due to reciprocal interactions of PAR proteins with
actomyocin; these interactions are necessary for proper actomyocin
movement (26, 30, 31, 62, 77). Maintenance of polarity is achieved by a
number of proteins associated with the PARs, such as CDC-37 and CDC-42
(11). In third step, the cell fate determinants are localized towards specific
regions of the polarized cell. Finally, during step four the mitotic spindle
forms. The spindle is positioned non-randomly in the parental cell to allow
non-equivalent distribution of cell-fate determinants between daughter
cells. Cell fate determinants are considered to be the mRNA and/or
cytoplasmic proteins. Due to asymmetric cell division, the two daughter
cells end up with different mRNA/protein content. In addition, in some
cases this leads to difference in the size of daughter cells, such as those of
the surface neuroectoderm and SOP cells in Drosophila. In the former case,
each neuroblast undergoes an asymmetric cell division to produce a larger
apical neuroblast and a smaller basal ganglion mother cell. The asymmetric
cell division in this case has been attributed to the presence of the prospero
transcription factor in the basal ganglion mother cell (20, 32, 81, 96). In
the latter case, asymmetric SOP division yields two different precursor cells,
where each of the posterior pIIa cell gives rise to a socket and a hair cell,
and the pIIb anterior cell gives rise to a neuron and a sheath cell. In both
the well-studied cases mentioned above, the major cell-fate determinant is
believed to be the Numb protein (51, 75).

Influenced by the above-mentioned studies of invertebrates, the Numb protein is

also suggested to promote asymmetric cell division in vertebrates, such as
in larval neuroblasts and SOP cell divisions. It is present throughout the
cytoplasm of progenitor cells (102). Numb has two homologs in mouse, the
Numb and the Numblike (104, 103, 101). The combined absence of these
two proteins leads to the depletion of progenitor cells without affecting the
production of neurons. This suggests that both homologs are playing roles
in segregating cell-fate determinants (68). It was also demonstrated that
both proteins are required for the maintenance of cadherin-based adhesion
and for establishing polarity of neural progenitor cells; both processes are
believed to be associated with the asymmetric cell division (74). Numblike
protein instead is localized in the cytoplasm, and it does not show the
asymmetric segregation observed with Numb; therefore, its role in
asymmetric cell division remains unexplained. The results from mRNA
knockdown in different types of cells have been inconsistent. Despite that,
it has been assumed that Numb and Numblike factors are important for
promoting asymmetric cell divisions in the vertebrate cerebral cortex tissue.

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Despite the continuously increasing number of proteins that are suggested to
participate in asymmetric cell division in diverse systems, it is not clear how
those proteins are non-equivalently distributed between sister cells and how
precisely their presence or absence causes daughter cells to
developmentally differ from one another. It is clear that sister cells
asymmetry results from their differential expression of developmentally
important genes. This logic suggests that, in addition to the above-
discussed cell intrinsic mechanisms required for establishing cellular
polarity, other mechanisms might also operate to promote differential gene
regulation of daughter cells. Recent studies of such a mechanism promoting
selective chromatid segregation in mitosis as newly discovered cell biology
phenomenon are reviewed below.

Sister chromatids are inherently non-equivalent both by DNA strand

sequence and by their replication history

Another model suggested that asymmetric cell division might be promoted by

differentiation of sister chromatids by epigenetic means, whereby
developmentally important gene(s) are allowed to express in only one specific
member of the pair of sister chromatids from one or a specific set of
chromosomes, followed by selective segregation of thus differentiated sister
chromatids to daughter cells. DNA replication is a fundamental process used by
all living organisms. It is used to duplicate chromosomes, and that forms the
basis of biological inheritance of genetic material. The two DNA strands of the
chromosome carry genetic information complementary to one another, and both
strands serve as a template for the synthesis of the other strand during
chromosome duplication. Barring mitotic recombination, normally, the template
strand’s continuity is preserved in its entirety, and the new strand is assembled
on it (61). The resulting double-stranded DNA replicas are equivalent to one
another because proofreading and error-checking mechanisms operate to ensure
near-perfect fidelity. Thus, each chromosome replication event produces two
paired daughter chromosome copies that in the G2 phase of the cell cycle are
conventionally referred to as sister chromatids. One chromatid contains a DNA
replication template (older) strand, designated as the Watson (W) strand; and
the other a complementary strand synthesized in the previous replication cycle,
the Crick (C’, prime denotes strand synthesized in the previous replication cycle)
strand (Figure 1). Consequently, its sister chromatid consists of the older C
strand and the younger W’ strand (designated W’C chromatid) (40). Thus,
because of differences in the replication history of DNA strands, the sister
chromatids are formally non-equivalent with one another even though they carry
identical base sequences. One chromatid copy from each chromosome is
delivered to each daughter cell during cell division. It is generally believed that
sister chromatids derived from a homologous pair of chromosomes are
segregated randomly to daughter cells, but recent studies have found cases of
selective chromatid segregation. Moreover, it is suggested that such a biased
chromatid segregation phenomenon might constitute the mechanism of
asymmetric cell division in eukaryotes (39).

The fission yeast’s chromosome II sister chromatids differ in competence

to switch cell type

The evidence for producing differentiated sister chromatids only came from
studies of mating-type inter-conversion of fission yeast cells. In this single-celled
organism, a haploid cell exists in one of the two mating cell types. A remarkable
feature of this system is that cell type changes spontaneously and very efficiently
in mitosis by chromosomally heritable but reversible gene replacement, a process
called mating-type switching. There, a non-switchable cell undergoes two

6
consecutive asymmetric cell divisions to produce one switch-in-four related
granddaughter cells (Figure 1). The mating cell-type is determined by one of the
two, P (Plus) or M (Minus), mating-type information residing at the mating-type 1
(mat1) locus. The DNA strand-specific imprint is installed in the mat1 gene
during its replication in only one of the two sister chromatids, and this event
initiates the precise pattern of switching observed in cell pedigrees.
Consequently, based on possessing the older W versus older C strand, only the
specific chromatid acquires the imprint. Inheritance of thus differentiated
chromatids produces a stem cell-like asymmetric cell division pattern such that
one of the daughter cells remains non-switchable, like the parental cell, while the
other daughter, which inherits the imprinted chromosome, becomes switchable.
The switchable daughter undergoes a second asymmetric cell division such that
the chromatid inheriting the imprinted strand acquires a double-stranded
chromosomal break during chromosome replication. This site-specific break
directs the DNA recombination gene conversion event that replaces the resident
mat1 allele with information of the opposite allele. The information to replace
mat1 is copied from the transcriptionally inactive library copies, mat2P and
mat3M loci located near mat1 (16, 38, 41).

Mu

Ms Mu

Ms Pu Ms Mu

Key: M & P, cell’s mating type; u, unswitchable cell;

s, switchable cell

Figure 1: Asymmetric cell divisions confer the indicated pattern of mating-type

switching in fission yeast cell pedigrees. The Mu cell always produces one Mu
daughter, while the other daughter is most often Ms (reviewed in 41). The Ms cell in
turn produces a switched Pu daughter and the other daughter is most often of the Ms
type. Thus, only one in four grandchildren cells of the Mu grandparental cell switches
through two consecutive stem cell-like asymmetric cell divisions. The Pu cell in turn
produces a similar pattern of switching to the M type in subsequent cell divisions.
Most remarkably, the cell’s differentiation is simply dictated by the inheritance of
specific mat1 DNA strands.

It is clear that the developmental program in both asymmetric divisions is strictly

advanced by the act of DNA replication itself. In particular, DNA replication
produces non-equivalent sister chromatids, and their differentiation is strictly
based on their inheritance of Watson versus Crick, older versus younger, and
imprinted versus non-imprinted DNA strands. In addition, stable epigenetic states

7
of gene expression through heterochromatin assembly at the mat2/3 region in
the chromosome are faithfully replicated, along with chromosomal DNA (27).
Remarkably, such epigenetic states are inherited both in mitosis and meiosis as
conventional Mendelian “factors”. Thus, in contrast to the prokaryotic paradigm
for maintaining the state of gene expression/repression requiring the continued
presence of the inducer/repressor, perpetuating the specific epigenetic state in
eukaryotes does not require the continued presence of a factor that initially
caused a change in the state of gene expression. Most remarkably, the specific
epigenetic states are maintained by cis-acting gene controls. The concepts of
sister chromatid differentiation and inheritance of mitotically stable epigenetic
states discovered in the yeast model system are likely to help us explain cellular
differentiation in multi-cellular organisms. At present, only fission yeast
differentiation has been shown to employ this mechanism. Perhaps this is
because such studies at the single cell level are not possible with other systems,
or alternatively, such a mechanism only operates in fission yeast. Given the
simplicity of the mechanism tied to DNA replication, in principle, it might operate
widely in biology.

Biased chromatid segregation of the entire genome?

As stated above, sister chromatid copies of a chromosome, with respect to those

of its homolog, are assumed to be segregated randomly to daughter cells of
diploid organisms. If, however, sister chromatids are made functionally non-
equivalent, as presented above only in the case of fission yeast, evolution of a
mechanism for selective segregation of sister chromatids can be envisioned to
exploit chromatid differences in biology. In mammals, encouraging results
suggest that non-random segregation of DNA strands, and therefore of
chromatids, might occur in cells undergoing asymmetric cell division. Such
evidence has been mostly obtained by pulse-chase, bromodeoxyuridine or
tritium, base-labelling experiments in the mouse muscle fibre stem cells,
epithelial cells of the small intestine, mammary gland, and neuronal stem cells. In
these special cases, it is suggested that selective retention of the older/template
DNA strands by stem cells of all chromosomes occurs and that all the newly
synthesized strands are selectively delivered to the differentiating cell in the
following stem cell division (23, 35, 45, 46, 48, 60, 70-72, 78, 79). However, it
has not been possible to definitively ascertain whether all chromosomes indeed
undergo asymmetric segregation, because biased segregation of only a set of
chromosomes can explain the findings. Such is the Cairns (9) immortal strand
hypothesis for segregating older template DNA strands to asymmetrically dividing
self-renewing stem cells. This hypothesis was proposed as a mechanism to
protect stem cells from inheriting DNA replication errors so as to avoid future
cancer development; it was not proposed to explain cellular differentiation.

Discovery of the selective chromatid segregation phenomenon

Recall the above-discussed case of chromatid differentiation by somatic cell

imprinting to accomplish mating-type differentiation of sister cells in yeast (Figure
1). Similarly, in principle, differential gene regulation of developmentally
important gene(s) in eukaryotes may be accomplished by epigenetic moieties
installed in the Watson versus Crick, older versus newer, strand-specific fashion
in cells at specific stages of development. This may occur by differential
heterochromatin assembly and/or by altering cytosine base methylation of the
relevant gene to repress or to activate transcription by disrupting the gene
repressing epigenetic controls. Thereby, cell differentiation might occur by
controlling gene(s) expression by epigenetic means. Supporting the epigenetic
notion, the white-opaque transition of cell type in the pathogenic yeast Candida
albicans can be experimentally induced by treating cells with trichostatin A, a

8
histone deacetylase inhibitor (43). In this fashion, non-equivalent sister
chromatids of one or a specific set of chromosomes might be produced by
epigenetic means at specific cell divisions in development to effect cellular
differentiation.

In the case of haploid and unicellular organism of fission yeast presented above,
biased chromatid segregation would not have any obvious biological
consequence. For the chromatid asymmetry to become a crucial part of a
mechanism for cellular differentiation in diploid and multi-cellular organisms, a
process for selective chromatid segregation is required. This proposal exploits the
sequence differences between DNA strands to accomplish cellular differentiation.
Such a Somatic Strand-specific Imprinting and selective chromatid Segregation
(SSIS) model was initially proposed to generate non-equivalent daughter cells in
mitosis (39). The model postulated evolution of a selective chromatid
segregation mechanism, whereby one daughter cell inherits both W’C
chromosomes, one copy from each homolog, and the other daughter cell thereby
inherits both WC’ copies from the progenitor cell (Figure 2). By specifying only
the older chromosome strands of chromatids/chromosomes for simplicity, the
term WW:CC was coined, where W reflects WC’ and C reflects CW’ strand-
containing chromatid/chromosome (40). In other words, the term WW:CC
denotes non-random segregation of template DNA strands to a specific daughter
cell, whereby the WW daughter cell inherits both WC’ copies and the CC daughter
cell inherits both W’C replicas. This model specifically recognizes which one of the
DNA strands of a chomatid is W, which one is C, which one is older, and which
one was synthesized in the last replication cycle.

WC WC

Chr. 7 pair in G1

DNA Replication
W C’ W’ C W C’ W’ C

G2
Mitosis
Chromatid number: 1 2 3 4

Or

1 3 2 4 1 4 3 2
W C’ W C’ W’C W’C W C’ W’C W C’ W’C

Segregation W C : W C
pattern W W : C C
Daughter cell pairs

Key: W, template Watson strand in green colour; C, template Crick strand in red; W’ and C’
strands in black are synthesized in the present DNA replication cycle; 1 to 4 numbers indicate
specific chromatids in the parent cell; red and green arrows indicate the colour-matched template strand inherited by a c
.

Figure 2: Two theoretical possibilities of selective chromatid distribution of Chr.

7 in mouse cell mitoses. For clarity, chromosomal DNA strands found normally in

9
the double helix configuration are presented as straight lines. The W and C strands are
defined by their specific 5’–3’ DNA sequence orientation. In the WW:CC designated
pattern, both template (older) W (arbitrarily coloured green) strand-containing
chromatids are segregated to one daughter cell and both older C (red) strand-
containing chromatids are segregated to the other daughter cells to affect an
asymmetric cell division. Equivalent daughter cells are produced in the WC:WC
segregation mode, as both inherit WC’ plus W’C chromosomes.

The challenging problem has how been to experimentally test the segregation
pattern of a specific chromosome during cell division. The key insight that
challenged the commonly held belief came from an attempt to explain an unusual
result obtained by inducing mitotic recombination of mouse chromosome (Chr.) 7
(40). Both the chromatid origin and the distal marker P (for paternal allele) or M
(for maternal allele) in the recombinant lines were detected with Southern
analysis, by performing methylation-sensitive restriction enzyme digestion of the
Snrpn gene located in the middle of Chr. 7; the M epigenetic allele is methylated
in the maternal homolog and unmethylated in the paternal P allele by
conventional parent-of-origin imprinting (2, 55). The result of experimentally
induced recombination with the site-specific Cre/loxp system in mouse embryonic
stem cells (ES) (55) was subsequently interpreted to suggest the existence of a
selective chromatid segregation phenomenon (40), as explained below.

One remarkable result noted in a study was that in all 432 Chr. 7 recombinants
analyzed, each produced an M/M and P/P pair of homozygous progeny cells
(Figure 3). Normally, G2 recombination events between non-sister chromatids are
resolved to generate a mixture of homozygous and heterozygous products for
markers located distal to the crossover point. Authors explained the only
homozygous products result by invoking the terms of so-called X and Z
segregation (55), previously coined only to describe the distal markers status of
recombinants in studies with Drosophila. The recombinant chromatids segregate
away from each other in the X segregation, and this results in homozygosis
(Figure 3). Alternatively, it is called the Z segregation if both recombined
chromatids co-segregate to the same daughter cell and thereby maintain
heterozygosis. A similar result of obtaining preferentially homozygous
recombinants in Drosophila, by employing an altogether different FLP/FRT
recombination system, was previously explained by invoking the X segregation
terminology (6, 25, 69). According to the prevailing explanation for both the
recombination systems—Cre/loxp in mouse and FLP/FRT in Drosophila—it was
suggested that somehow the recombination systems themselves cause
recombinant chromatids to segregate always from each other, resulting in the X
segregation pattern (6, 25, 28, 55). This explanation postulated a meiotic-
reduction-division 1-like process in which sister chromatids following
recombination remain attached in regions distal to the crossover point and
therefore segregate together to one pole of the mitotic spindle. Such a model is
unlikely, as it requires chromatid segregation to occur through chromosome
regions different from their centromeres. Also, this constraint is unlikely to be
imposed by two different site-specific recombination systems, that, too, in two
different organisms where recombination was induced by systems that are not
indigenous to cells of either species. Moreover, such constraints imposed by the
recombination process in mouse cells should have resulted in a similarly biased
segregation in other cell lineages where, instead, an unbiased segregation pattern
was observed (see below).
An alternative model, specifically invoked to explain the result of obtaining only X
segregation in ES cells, suggested that selective distribution of sister chromatids
to progeny cells normally occurs irrespective of the involvement of mitotic

10
recombination. It was proposed that the Chr. 7 selective chromatid segregation
normally occurs through centromeric segregation in ES cells (2, 40), and
consequently, recombined chromatids always segregate away from each other to
result in markers homozygosis (Figure 3). For clarity, this kind of distribution was
named as the WW:CC segregation pattern by referring to the older DNA strands
specifically at the centromere of each chromatid/chromosome (Figures 2 and 3).
By changing growth conditions, the cells containing this recombination system
were changed to several other cell types. Only the WW:CC segregation was
observed in endodermal cells (Table 1). In contrast, all the recombinant cultures
examined for each class of pancreatic, mesodermal, and cardiomyocyte cells
exhibited P/M heterozygosis in about one-third of recombinants, indicating their
less biased or near random segregation pattern (2).

Segregation pattern: WW:CC = X WC:WC = Z

Distal markers: Homozygous Heterozygous

Daughter cells’ chromosomes

M P
M 1W
1W
1C
M C΄ M M
1W M 2W 2W
1C loxP
W΄
2W loxP P 1C P Mitosis
Or
P M
2C 2W 1W
C΄ 1C P P
Chr. 7 pair in G1 W΄ P
2C 2C 2C
G2 recombination segregation modes

1W P
1C
2W loxP M
G1 recombination 2C

Key: loxp, the site-specific recombination cassette inserted in both homologs near the
centromere (oval circle); XX crossover, G2 phase recombination of indicated non-sister
chromatids; P and M, chromosomal markers located in the middle of the chromosome; all other
symbols are defined in Figure 1.

Figure 3: The recombination model employed to define the chromatid

segregation mode (modified from Armakolas and Klar, 2007). The newly
synthesized C’ strand is coloured brown and W’ in blue, and all other notations are
defined in Figure 2. Mitotic crossover at the loxp sites is experimentally induced by
transiently expressing Cre recombinase in cells (55). The crossover event generates
one chromatid with a functional hypoxanthine phosphoribosyl transferase minigene
and those colonies inheriting the marker are selected by growing in an appropriate
selective medium. The P and M allelic constitution is determined with Southern
analysis. To obtain the result of all recombinants becoming homozygous for P and M
alleles, as in ES and endoderm cells (Table 1), recombination must occur not in the
G1 but in the G2 phase, only between specific non-sister chromatids (e.g., WC’ with
W’C), and it must be followed by selective distribution of centromere copies, as
indicated in the drawing. Therefore, all M/M and P/P homozygous recombinants,

11
conventionally termed to have originated from X segregation, are interpreted to reflect
the WW:CC segregation process, and all P/M recombinants, conventionally termed to
have originated from Z segregation, are proposed to result from the WC:WC
segregation process. Therefore, the selective DNA strand segregation phenomenon
comprises the mechanism for generating either only X or only Z segregation in
different cell lineages. A random distribution is thought to have occurred when either
pattern is found in different cells of a culture.

Another unanticipated result was obtained with neuroectoderm cells, which

showed yet another kind of segregation mode. In this case, all 160 recombinants
analysed maintained the P/M constitution, a result consistent with the biased
WC:WC (i.e., G2-Z) segregation pattern and/or recombination in them occurs in
G1 (Figure 3). Therefore, it was suggested that regulation of chromatid
distribution to progeny cells occurs in ES cells, irrespective of mitotic
recombination occurring in them (40). Indeed, the analysis of recombinants
constitutes a procedure to discern the segregation mode of a specific
chromosome in mitoses. To obtain the homozygous (i.e., WW:CC = X
segregation) result, it was proposed that only specific non-sister chromatids (WC’
with W’C) must have been allowed to participate in recombination in the G2
phase, followed by selective segregation of chromatid sequences through the
centromere, and that recombination in the G1 phase must not have occurred,
because such events would produce P/M heterozygous recombinants (Figure 3).
Perhaps the biased chromatid segregation process itself restricts which pair of the
non-sister chromatids is permitted to recombine in the G2 phase, and second,
somehow it also prohibits Chr. 7 homologs recombinational interaction in G1 (2,
3). We therefore advanced the notion of patterned Chr. 7 segregation in ES cells
and suggest that the pattern is not invariant, as it changes with the cell type in
very interesting ways (2, 55). Furthermore, this process seems to be
chromosome-specific because a similar analysis of Chr. 11 produced the usually
expected mixture of homozygous and heterozygous recombinants (55).

If neuroectoderm cells indeed recombine in G2 (see below), it remains to be

determined whether the ES/endoderm and the neuroectoderm results differ
because of cell-type regulated distinction between chromatids that are permitted
to recombine or due to differences in the mode of chromatid segregation. As
somatic general recombination normally occurs rarely (46), it is difficult to
imagine that a chromatid choice mechanism only dedicated for controlling
experimentally induced recombination has evolved in mouse cells. Therefore, we
envision that likely a chromosome-specific non-random segregation process
inherently operates in some mouse cell lineages, that such a process indirectly
influences the choice of recombining chromatids, and that this process might also
disallow recombination in the G1 phase by constraining chromosome interaction,
perhaps through chromosome compartmentalization in the nucleus (94).

Left-Right Dynein protein (LRD) is implicated in the selective chromatid

segregation process

Dyneins are molecular motors that travel on cytoskeleton microtubules. They

exhibit a unidirectional minus-end movement on microtubules, and are classified
as either cytoplasmic or axonemal. Cytoplasmic dyneins have been implicated in
vesicular transport, nuclear migration, and spindle orientation, whereas axonemal
dyneins produce the motor forces that cause the sliding of adjacent microtubules
in the axoneme leading to cilia and flagella movement. Dyneins function as large
multi-subunit complexes containing up to three heavy chains, which include one
with the force-producing motor domain, and several intermediate and light
chains. Dyneins are considered to be key players in the intrinsic cell division

12
process in Drosophila (93). It has been suggested that since dyneins are
anchored at the cell cortex, as well as at the microtubules, a pulling force may be
generated leading to specific spindle positioning in the cell. Evidence for the LRD
(protein) function on asymmetric cell division came by conditional depletion and
mild RNAi inactivation studies of several dynein proteins, such as the dynein
heavy-chain gene (dhc-1), the Lissencephaly protein1 (LIS-1) and the dynein-
associated component road-block (DYRB-1) in C. elegans (65). Since all the
dynein mutants exhibited a marked reduction of spindle pulling forces, dyneins
are required for proper spindle positioning in cells of C. elegans embryos (14, 65,
67). Furthermore, cytoplasmic dynein comprises one of the components to
induce the attachment of one pole of the stem cell spindle to the niche cells,
thereby promoting asymmetric cell division in Drosophila female germ line stem
cell mitoses (10, 29). Although dynein seem to be the common factor involved in
asymmetric cell division in diverse systems, how precisely dynein protein
promotes asymmetric cell division remains unknown.

The left-right dynein (lrd) is an axonemal dynein heavy chain-encoding gene. An

extensive expression analysis revealed that, in addition to symmetric expression
in the embryonic node, apparently transient asymmetric expression of lrd occurs
in the head-fold region of 0- to 5-somite stage mouse embryos. At later stages,
lrd is expressed symmetrically in the floor plate of the neural tube, a midline-
signalling centre, and in a region of the embryo shown to be involved in visceral
organs’ left-right (LR) development (17, 18, 24, 56). In addition, expression was
seen in non-ciliated cells (5), in several ciliated cell types in newborn mice, the
epithelial lining of the nasal cavity, and in the ependymal lining of the third
ventricle of the brain (97).

A spontaneous missense mutation in lrd causes randomization of LR laterality

such that one-half of mice develop with mirror-imaged visceral organs, as
compared with wild-type mice (47). The LRD protein’s function in visceral
laterality specification in mice was confirmed by generating a targeted deletion of
the ATP binding domain of the lrd gene. Like the missense mutation, the deletion
mutant similarly produced LR axis randomization (83). Similarly, homozygotes of
different subunits of dynein exhibit visceral organs’ randomization in humans (1).
The placement of gut and stomach is also under the LR axis control. This
suggests that lrd expression in the developing gut may be involved in LR
specification. Although the gut tube begins to close at E8.5 in mice, the gut does
not initiate handed asymmetric coiling until after mid-gestation. Expression of lrd
in the gut endoderm is only found up to the E9.5 stage, suggesting that lrd may
not directly function in the lateralization of the gut, but rather it acts through an
indirect mechanism, as speculated for heart looping. In the heart case, the
transient asymmetric domain of LRD expression is not localized in the left or right
lateral plate of the mesoderm; instead, it is observed more anterior, dorsal to the
pericardiac region. Taking this into account, it has been speculated that the
transient asymmetric domain of LRD expression functions in the transfer of left-
right patterning information from the midline node to influence the rightward
looping of the heart tube. Expression of lrd in E10.5–12.5 embryos was also
found in the floor plate of the neural tube, the midline structure that has been
implicated in LR specification (83, 84, 85).

The SSIS model was first proposed to explain the LR randomization phenotype of
the iv (situs inversus) mutant mice. By the model, the iv gene, later discovered
to encode LRD (85), mediates WW:CC segregation (Figure 2) to produce
asymmetric cell division in the embryo when initially the visceral laterality is
specified (39). According to the molecular data discussed above from studies of
Drosophila, mice and in humans, it is possible that LRD is involved in asymmetric
cell division. Therefore, the hypothesized role of dynein according to the SSIS

13
model for LR axis determination was investigated by determining its role in the
selective Chr. 7 segregation process, even though it is not known whether this
chromosome concerns axis specification. Very encouraging results were obtained
(3): First, there is a perfect correlation between lrd mRNA presence/absence and
the Chr. 7 segregation mode in all the six cell lineages that were examined (Table
1). Specifically, lrd gene is expressed in cultures that follow selective segregation
patterns, but not in those that follow an unbiased pattern (3). Second, after lrd
mRNA inactivation, by RNAi technology, each of the ES, endoderm, and
neuroectoderm cell lines disrupted their selective segregation mode (Table 1).
Third, neuroectoderm cells after LRD depletion produced M/M heterozygous
recombinants; they must have been generated by recombination in G2 (Figure
3). Thus, neuroectoderm cells must normally follow the WC:WC segregation
mode to produce P/M heterozygous recombinants where recombined chromatids
are always delivered to the same daughter cell. As both the WW:CC and WC:WC
selective patterns are found in different lineages, we conclude that the
recombination process itself does not dictate generation of either the X or the Z
segregation outcome (6, 25, 28, 55), rather selective segregation process
inherently operates in specific cell lineages (2, 3, 42); LRD likely functions
directly in the selective Chr. 7 segregation mechanism. These results support the
existence of the selective strand segregation feature of the SSIS model proposed
for LR axis determination in vertebrates (39, 40).

QuickTime™ and a
decompressor
are needed to see this picture.

Table 1: LRD protein implicated in the selective chromatid segregation in

mouse cell lineages. Expression of the LRD dynein gene is inherently regulated by
cell type, as indicated. ON means expressed; OFF means silent.

The mechanism of how the LR symmetry of the embryo is initially broken remains
developmental biology’s key unanswered question (59, 73, 87, 88, 89). Defining
the molecular function of the lrd gene in axis development is required to explain
the fascinating LR axis randomization phenotype of the mutant mice. The most
popular model for the LRD protein function postulates its role in the motility of
monocilia developed on nodal cells of the mouse embryo, called the “nodal flow”
hypothesis (66). Notably, the mutant mice develop immotile cilia. According to
the nodal flow hypothesis, the cilia-generated leftward embryonic fluid flow
establishes an asymmetric gradient of a “morphogen” across the embryo,
constituting a mechanism for LR patterning (8). However, it remains a
controversial model when applied to the LR axis determination. The work in
mouse cells supports the theory that the dynein motor protein plays a
cytoplasmic role in LR patterning and that it does not involve extra-cellular cilia
motion (73).

The SSIS model was proposed as an alternative model for explaining

development of LR asymmetry through asymmetric cell division during

14
development. Thus, this model is based on cell lineage and it is designed to
exploit the inherent base sequence differences in the W and C strands, their
replication history (98), and the epigenetic events that might be installed at the
time of chromosome replication during development (16, 37, 76). The result of
random chromatid segregation in LRD knockdown cells is consistent with the
random LR visceral phenotype of the lrd-/lrd- mutant mice. Also, the findings that
only one-half of heterozygous Chr. 11 three independent translocation carriers
develop schizophrenia and bipolar brain psychiatric diseases have been argued to
support the SSIS model for human brain LR laterality development by postulating
random strand/chromatid segregation occurring owing to rearrangements (40).
The same logic might explain the Kartagener’s LR axis randomization syndrome,
characterized by immotile cilia due to cytoplasmic dynein defects in humans (1).

Concluding remarks

The commonly invoked morphogen-gradient model for explaining development,

including for situs determination in mice, remains controversial. The model
is too vague to be experimentally tested. Discovery of extra-cellular signals
that promote cellular differentiation through cell–cell contact, such as those
in germ line nitches of Drosophila, do not necessarily evidence the
morphogen-gradient model designed for explaining patterning in much
larger areas of the embryo. Apparently, a very interesting new avenue for
research has opened up to accomplish cellular differentiation through
asymmetric cell division by regulating chromosome distribution. Studies of
the Cre/loxp and FLP/FRT site-specific recombination systems quoted above
were conducted only to develop tools for chromosomal manipulation. We
propose that this approach also identifies the mode of sister chromatid
segregation in mitosis. Liu et al. (55) conducted their study only with Chr. 7
and 11. Fortuitously, Chr. 7 exhibited two types of selective segregation
patterns, which we showed to be LRD-dependent, and which changes with
the cell type in most interesting ways (2, 3). The sole function known for
LRD is for axis determination. These results raise the tantalizing possibility
that Chr. 7 might specify LR axis determination in mice, in accord with the
SSIS model. If so, one genetic test of this suggestion is that mice
genetically engineered to contain an inversion of both Chr. 7 centromeres
should develop situs inversus in all animals. Also, some of the cell intrinsic
factors implicated in promoting asymmetric cell division might function by
dictating cellular polarity required for selective chromatid distribution. For
example, the cell-fate determining Numb protein is located in the ‘mother’
centrosome (44), a structure found at the pole of the mitotic spindle. Also,
the basal body of monocilia originates from one of the two centrosomes of
the spindle. This new cell biological phenomenon of selective chromatid
segregation may explain development in mammals and the origin of brain
LR laterality developmental disorders of psychoses in humans. In future
studies more direct approaches should be designed to further investigate
the mechanism of the DNA strand segregation process and to scrutinize its
proposed role in development. It has been speculated that the sister
centromeres might be differentiated during replication by epigenetic means
to identify them for biased segregation (52). In sum, we hypothesize that
such a mechanism may be crucial in evolution of form, for cellular
differentiation and development, and for maintaining the integrity of parent-
of-origin–specific imprints in somatic lineages by discouraging mitotic
recombination perhaps through chromosome compartmentalization in the
nucleus.

15
Acknowledgments
Research in the Laboratory of Experimental Physiology is supported by the
General Secretariat of Research and Development, Ministry of Development,
Greek Government, and by the Research Academy of the National and
Kapodistrian University of Athens. The Intramural Research Program, Center for
Cancer Research, National Cancer Institute at Frederick, National Institutes of
Health, USA, supports research in the Klar laboratory.

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