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Implications of the mitotic selective chromatid segregationphenomenon for vertebrate development

Authors: Armakolas A1, Koutsilieris M1, Klar AJS2

1Laboratory of Experimental Physiology, Athens Medical School Mikras Asias 75,Athens 115 27, Greece; 2Gene Regulation and Chromosome BiologyLaboratory, 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 lateralitydevelopment, developmental mechanism

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

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Abstract

Asymmetric cell division is a fundamental process of cell biology. It is required togenerate cellular diversity and tissue renewal during development. During thisdevelopmentally regulated process, mitosis yields two non-equivalent daughtercells that arise from the same parental cell. Despite inheriting identical geneticmaterial, daughter cells often exhibit properties different from one another. Toexplain this mechanism of cellular differentiation, several hypotheses have beenadvanced, mostly involving studies of invertebrate model systems and, to a lesserextent, of mammals. Here we review the evidence obtained from diversebiological systems to highlight how different mechanisms interplay to accomplishcellular differentiation. We focus primarily on studies relevant to a relativelyrecently proposed model that builds on the replication history of specific Watsonversus Crick DNA strands of a chromosome. It proposes generation of non-equivalent sister chromatids by epigenetic means followed by selectivedistribution of thus differentiated chromatids between daughter cells. We presentimplications of this chromosome modification/inheritance model for mice visceralorgans body axis laterality development.

Introduction

Every multi-cellular organism consists of a variety of different cell types that giverise to different tissues and organs. The development in most organismsstarts from a single cell, which after multiple cell divisions gives rise to

different tissues, with each tissue containing cells of specific type(s). Thesignificance of asymmetric cell division for the development of multi-cellularorganisms, including vertebrates, is widely recognized. For example, astem cell often generates one daughter cell that is committed todifferentiation, while the other daughter cell maintains stem-cellcharacteristics to generate cellular diversity of each organism. Four majorhypotheses have been proposed for explaining the phenomenon ofasymmetric cell division. These can be broadly separated into two majorcategories: those that suggest that asymmetry is imposed by the cellsexposure to extra-cellular environment, and others postulate that theinformation for cellular differentiation is found internally in the cell causingasymmetric cell division of the progenitor cell.

Prominent among the first type, the morphogen-gradient model suggests thattwo initially identical daughter cells become different because theyencounter different microenvironments that induce and repress differentsets of genes in a cell. A special case of this hypothesis concerns differentstem cell niches. In this case, precise cellular location influences the stemcells division by integrating signals emanating from adjoining cells in thenitch to induce daughter cells to differ from one another. Alternatively, thefate of each daughter cell may be determined by cell intrinsic factors. Inthis case, either the cytoplasmic cell-fate determinants are segregated intoonly 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 mechanismsoperate in different tissues.

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The morphogen-gradient model

Morphogens are believed to be secreted cell-signalling molecules that helporganize a field of surrounding cells into different cell-differentiated patterns.According to a popular hypothesis, morphogens form a gradient of concentrationemanating from a localized source, and thereby determine the arrangement andfate of responding cells according to the different concentrations of themorphogen perceived by them. The idea of the morphogen gradient is intimatelyassociated with the concept of positional information in a field. A cell is believedto read its position in a concentration gradient and it develops its fateaccordingly. It is believed that a single event, the emission of a factor(morphogen) from a source, can lead to the formation of many different celltypes to accomplish their correct spatial positioning in the body of the organism(21). While most of the evidence supporting this hypothesis came from studiesofDrosophila, mammalian models for limb patterning have also been designed totest the hypothesis (34, 89, 92). Indeed, defining the possible role of morphogengradients in vertebrate systems has been a focus of increasing interest for manydecades. Although this has been a prominent hypothesis proposed for

development, by no means has it been accepted because many major questionsremain to be answered. It is necessary to determine the morphogensbiochemical identity, how the concentration gradient is formed, how the absoluteconcentration of the gradient is maintained, and what are the factors that createand maintain it for different precise levels of concentration in a developmentalfield. Furthermore, it is believed that a number of different morphogens affectcells differently. So, what is the mechanism of differential response of cells todifferent threshold concentrations of morphogens? How do receptors on cellsurface transduce this information to the nucleus to create the appropriate geneexpression 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 theprocess of asymmetric cell division, at least in studies with mammals. In a relatedissue, host tissue-specific signals have been implicated in the biology ofmetastatic cancer (63). Also, development of refractoriness to hormones and tochemotherapy in clinical responses has been demonstrated at metastatic sites(91). Although the cellular signalling phenomenon is well established in theaforementioned cases, it is not clear whether the morphogen-gradient model canreadily explain general development as well as the origin and proliferation ofcancer.

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 recentlyfrom studies of the female germ line in Drosophila flies. In this prominent modelfor stem cell biology, one daughter cell (the stem cell) self-renews at eachdivision, while the other cell stops to proliferate after a few cell divisions and itsprogeny differentiates. The cells asymmetry is regulated by extracellular signalsemanating from the surrounding ovarian stem cell niche. Two biochemicalpathways are believed to be involved in the process of asymmetric cell division(100, 53, 54). Specifically, the germline stem-cell niche signaling requires twosignalling pathways: the TGF and an unknown pathway that is defined by the YBand PIWI proteins. The effect of those two pathways is to repress the expressionof the bag of marbles (bam), a gene that is necessary and sufficient forpromoting stem cell differentiation (12, 86). The niche function is additionallyassisted by the Hedgehog signaling pathway, and it also requires niche celladhesion 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 stemcell spindle to the niche cells. This attachment is performed by a spectrin-richstructurethe spectrosomeand a cytoplasmic dynein-mediated mechanism (1,58). After division, the daughter cell that loses the niche contact starts thedifferentiation process. A recent study (64) implicates microRNAs, operatingthrough 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 differentiationdecisions are made at the single cell level, whereby the fate of sister cells isspecified to differ because of an asymmetric cell division that requires cell-cellcontact.

Recently it was also demonstrated that the Drosophila male germ line stem cellsact in a similar way. In this case, the interaction of stem cells with the somaticniche cells (hub cells) promotes the asymmetric cell division. As a stem celldivides, one pole of its mitotic spindle is anchored to the niche cells, ensuring theasymmetric division by allowing only one of the two daughter cells to maintaincontact with the niche cells. Thus, only the attached cell retains the stem-cell

fate. The attachment complex in this case contains cadherin, catenin, and theadenomatous polyposis coli 2 (APC2) protein. Moreover, maintenance of germline stem cells is achieved by the hub cells secretion of an unpaired ligand of theJAK-STAT pathway (53, 54, 100).

Although the role of niches in the asymmetric division in mammalian stem cellshas not been as clearly defined, there is some evidence that such niches mayexist there and they may function in ways similar to those employed by flies. Afew in vitro studies exist, where surrounding niche cells may be required for theasymmetric cell divisions in mouse embryonic basal epidermal cells, embryonicneuroepithelial cells, and hematopoietic progenitor cells. Interestingly, theswitching of proliferating cells to a differentiated type involves a change in

orientation of cleavage plane from perpendicular to parallel in reference to theplane of the apical lamina (45). Furthermore, the hematopoietic progenitor cellsare capable of both symmetric (proliferation) and asymmetric (differentiation)divisions in cultures supported by stromal cells (13, 23, 48, 99). The nichesprovide transientsignals for stem cell division to support ongoing tissueregeneration. The balance between proliferation-inhibiting and proliferation-promotingsignals is the key to homeostatic regulation of stem cell maintenanceversus tissue regeneration. Loss of the niche can lead to lossof stem cells, thusindicating the reliance of stem cells on nichesignals. Interestingly, aberrations ofstem cell controls lead to cancerous growth. Recent evidence indicates theinvolvement of niches for cancer development. Cancer stem cells may arise froma mutation leading to self-sufficient cell proliferation, orthey may also involve

deregulation or alteration of the niche by

dominant proliferation-promotingsignals. Furthermore, the molecularmachinery used by normal stem cells forhoming on to or mobilizingfrom the niche may be used by cancer stem cells forinvasionand metastasis, as well as to create a diversion of cancer celles responseto 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, especiallyDrosophila and the earthworm C. elegans. Evidence is obtained by

implicating proteins that exhibit alterations in cells known to proceedasymmetric cell division, such as embryonic and adult stem cells,Drosophila sensory organ precursor (SOP) cells, and neural precursor cells

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Despite the continuously increasing number of proteins that are suggested toparticipate in asymmetric cell division in diverse systems, it is not clear howthose proteins are non-equivalently distributed between sister cells and howprecisely their presence or absence causes daughter cells todevelopmentally differ from one another. It is clear that sister cellsasymmetry 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 cellularpolarity, other mechanisms might also operate to promote differential generegulation of daughter cells. Recent studies of such a mechanism promotingselective chromatid segregation in mitosis as newly discovered cell biologyphenomenon are reviewed below.

Sister chromatids are inherently non-equivalent both by DNA strandsequence and by their replication history

Another model suggested that asymmetric cell division might be promoted bydifferentiation of sister chromatids by epigenetic means, whereby

developmentally important gene(s) are allowed to express in only one specificmember of the pair of sister chromatids from one or a specific set ofchromosomes, followed by selective segregation of thus differentiated sisterchromatids to daughter cells. DNA replication is a fundamental process used byall living organisms. It is used to duplicate chromosomes, and that forms thebasis ofbiological inheritance of genetic material. The two DNA strands of thechromosome carry genetic information complementary to one another, and bothstrands serve as a template for the synthesis of the other strand duringchromosome duplication. Barring mitotic recombination, normally, the templatestrands continuity is preserved in its entirety, and the new strand is assembledon it (61). The resulting double-stranded DNA replicas are equivalent to oneanother because proofreading and error-checking mechanisms operate to ensure

near-perfect fidelity. Thus, each chromosome replication event produces twopaired daughter chromosome copies that in the G2 phase of the cell cycle areconventionally referred to as sister chromatids. One chromatid contains a DNAreplication template (older) strand, designated as the Watson (W) strand; andthe 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 Cstrand and the younger W strand (designated WC chromatid) (40). Thus,because of differences in the replication history of DNA strands, the sisterchromatids are formally non-equivalent with one another even though they carryidentical base sequences. One chromatid copy from each chromosome isdelivered to each daughter cell during cell division. It is generally believed that

sister chromatids derived from a homologous pair of chromosomes aresegregated randomly to daughter cells, but recent studies have found cases ofselective chromatid segregation. Moreover, it is suggested that such a biasedchromatid segregation phenomenon might constitute the mechanism ofasymmetric cell division in eukaryotes (39).

The fission yeasts chromosome II sister chromatids differ in competence

to switch cell type

The evidence for producing differentiated sister chromatids only came fromstudies of mating-type inter-conversion of fission yeast cells. In this single-celledorganism, a haploid cell exists in one of the two mating cell types. A remarkablefeature of this system is that cell type changes spontaneously and very efficientlyin mitosis by chromosomally heritable but reversible gene replacement, a processcalled mating-type switching. There, a non-switchable cell undergoes two

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consecutive asymmetric cell divisions to produce one switch-in-four relatedgranddaughter cells (Figure 1). The mating cell-type is determined by one of thetwo, 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 geneduring its replication in only one of the two sister chromatids, and this eventinitiates the precise pattern of switching observed in cell pedigrees.

Consequently, based on possessing the older W versus older C strand, only thespecific chromatid acquires the imprint. Inheritance of thus differentiatedchromatids produces a stem cell-like asymmetric cell division pattern such thatone of the daughter cells remains non-switchable, like the parental cell, while theother daughter, which inherits the imprinted chromosome, becomes switchable.The switchable daughter undergoes a second asymmetric cell division such thatthe chromatid inheriting the imprinted strand acquires a double-strandedchromosomal break during chromosome replication. This site-specific breakdirects the DNA recombination gene conversion event that replaces the residentmat1 allele with information of the opposite allele. The information to replacemat1 is copied from the transcriptionally inactive library copies, mat2Pandmat3Mloci located near mat1 (16, 38, 41).

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

switching in fission yeast cell pedigrees. The Mu cell always produces one Mudaughter, 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 cells differentiation is simply dictated by the inheritance of

specific mat1 DNA strands.

It is clear that the developmental program in both asymmetric divisions is strictlyadvanced by the act of DNA replication itself. In particular, DNA replication

produces non-equivalent sister chromatids, and their differentiation is strictlybased on their inheritance of Watson versus Crick, older versus younger, andimprinted versus non-imprinted DNA strands. In addition, stable epigenetic states

Mu

MuMs

MsPu MuMs

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

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of gene expression through heterochromatin assembly at the mat2/3 region inthe chromosome are faithfully replicated, along with chromosomal DNA (27).Remarkably, such epigenetic states are inherited both in mitosis and meiosis asconventional Mendelian factors. Thus, in contrast to the prokaryotic paradigmfor maintaining the state of gene expression/repression requiring the continuedpresence of the inducer/repressor, perpetuating the specific epigenetic state in

eukaryotes does not require the continued presence of a factor that initiallycaused a change in the state of gene expression. Most remarkably, the specificepigenetic states are maintained by cis-acting gene controls. The concepts ofsister chromatid differentiation and inheritance of mitotically stable epigeneticstates discovered in the yeast model system are likely to help us explain cellulardifferentiation in multi-cellular organisms. At present, only fission yeastdifferentiation has been shown to employ this mechanism. Perhaps this isbecause such studies at the single cell level are not possible with other systems,or alternatively, such a mechanism only operates in fission yeast. Given thesimplicity of the mechanism tied to DNA replication, in principle, it might operatewidely in biology.

Biased chromatid segregation of the entire genome?

As stated above, sister chromatid copies of a chromosome, with respect to thoseof its homolog, are assumed to be segregated randomly to daughter cells ofdiploid organisms. If, however, sister chromatids are made functionally non-equivalent, as presented above only in the case of fission yeast, evolution of amechanism for selective segregation of sister chromatids can be envisioned toexploit chromatid differences in biology. In mammals, encouraging resultssuggest that non-random segregation of DNA strands, and therefore ofchromatids, might occur in cells undergoing asymmetric cell division. Suchevidence has been mostly obtained by pulse-chase, bromodeoxyuridine ortritium, base-labelling experiments in the mouse muscle fibre stem cells,

epithelial cells of the small intestine, mammary gland, and neuronal stem cells. Inthese special cases, it is suggested that selective retention of the older/templateDNA strands by stem cells of all chromosomes occurs and that all the newlysynthesized strands are selectively delivered to the differentiating cell in thefollowing stem cell division (23, 35, 45, 46, 48, 60, 70-72, 78, 79). However, ithas not been possible to definitively ascertain whether all chromosomes indeedundergo asymmetric segregation, because biased segregation of only a set ofchromosomes can explain the findings. Such is the Cairns (9) immortal strandhypothesis for segregating older template DNA strands to asymmetrically dividingself-renewing stem cells. This hypothesis was proposed as a mechanism toprotect stem cells from inheriting DNA replication errors so as to avoid futurecancer 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 cellimprinting to accomplish mating-type differentiation of sister cells in yeast (Figure1). Similarly, in principle, differential gene regulation of developmentallyimportant gene(s) in eukaryotes may be accomplished by epigenetic moietiesinstalled in the Watson versus Crick, older versus newer, strand-specific fashionin cells at specific stages of development. This may occur by differentialheterochromatin assembly and/or by altering cytosine base methylation of therelevant gene to repress or to activate transcription by disrupting the generepressing epigenetic controls. Thereby, cell differentiation might occur bycontrolling gene(s) expression by epigenetic means. Supporting the epigeneticnotion, the white-opaque transition of cell type in the pathogenic yeast Candidaalbicans can be experimentally induced by treating cells with trichostatin A, a

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histone deacetylase inhibitor (43). In this fashion, non-equivalent sisterchromatids of one or a specific set of chromosomes might be produced byepigenetic means at specific cell divisions in development to effect cellulardifferentiation.

In the case of haploid and unicellular organism of fission yeast presented above,

biased chromatid segregation would not have any obvious biologicalconsequence. For the chromatid asymmetry to become a crucial part of amechanism for cellular differentiation in diploid and multi-cellular organisms, aprocess for selective chromatid segregation is required. This proposal exploits thesequence 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 inmitosis (39). The model postulated evolution of a selective chromatidsegregation mechanism, whereby one daughter cell inherits both WCchromosomes, one copy from each homolog, and the other daughter cell therebyinherits both WC copies from the progenitor cell (Figure 2). By specifying onlythe 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:CCdenotes non-random segregation of template DNA strands to a specific daughtercell, whereby the WW daughter cell inherits both WC copies and the CC daughtercell inherits both WC replicas. This model specifically recognizes which one of theDNA strands of a chomatid is W, which one is C, which one is older, and whichone was synthesized in the last replication cycle.

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

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

W C W C

Mitosis

Or

W W : C CW C : W C

Chr. 7 pair in G1

Chromatid number:

WC

W C W C

WC

W C W C W C W CWC WC WC WC

DNA Replication

Segregationpattern

1 2 3 4

1 3 2 4 1 4 3 2

Daughter cell pairs

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

G2

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the double helix configuration are presented as straight lines. The W and C strands are

defined by their specific 53 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:WCsegregation mode, as both inherit WC plus WC chromosomes.

The challenging problem has how been to experimentally test the segregationpattern of a specific chromosome during cell division. The key insight thatchallenged the commonly held belief came from an attempt to explain an unusualresult 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 Southernanalysis, by performing methylation-sensitive restriction enzyme digestion of theSnrpn gene located in the middle of Chr. 7; the Mepigenetic allele is methylated

in the maternal homolog and unmethylated in the paternal P allele byconventional parent-of-origin imprinting (2, 55). The result of experimentallyinduced recombination with the site-specific Cre/loxp system in mouse embryonicstem cells (ES) (55) was subsequently interpreted to suggest the existence of aselective chromatid segregation phenomenon (40), as explained below.

One remarkable result noted in a study was that in all 432 Chr. 7 recombinantsanalyzed, each produced an M/Mand P/Ppair of homozygous progeny cells(Figure 3). Normally, G2 recombination events between non-sister chromatids areresolved to generate a mixture of homozygous and heterozygous products formarkers located distal to the crossover point. Authors explained the onlyhomozygous products result by invoking the terms of so-called X and Z

segregation (55), previously coined only to describe the distal markers status ofrecombinants in studies with Drosophila. The recombinant chromatids segregateaway from each other in the X segregation, and this results in homozygosis(Figure 3). Alternatively, it is called the Z segregation if both recombinedchromatids co-segregate to the same daughter cell and thereby maintainheterozygosis. A similar result of obtaining preferentially homozygousrecombinants in Drosophila, by employing an altogether different FLP/FRTrecombination system, was previously explained by invoking the X segregationterminology (6, 25, 69). According to the prevailing explanation for both therecombination systemsCre/loxp in mouse and FLP/FRT in Drosophilait wassuggested that somehow the recombination systems themselves causerecombinant chromatids to segregate always from each other, resulting in the Xsegregation pattern (6, 25, 28, 55). This explanation postulated a meiotic-reduction-division 1-like process in which sister chromatids followingrecombination remain attached in regions distal to the crossover point andtherefore segregate together to one pole of the mitotic spindle. Such a model isunlikely, as it requires chromatid segregation to occur through chromosomeregions different from their centromeres. Also, this constraint is unlikely to beimposed by two different site-specific recombination systems, that, too, in twodifferent organisms where recombination was induced by systems that are notindigenous to cells of either species. Moreover, such constraints imposed by therecombination process in mouse cells should have resulted in a similarly biasedsegregation in other cell lineages where, instead, an unbiased segregation patternwas 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 chromatidsto progeny cells normally occurs irrespective of the involvement of mitotic

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recombination. It was proposed that the Chr. 7 selective chromatid segregationnormally occurs through centromeric segregation in ES cells (2, 40), andconsequently, recombined chromatids always segregate away from each other toresult in markers homozygosis (Figure 3). For clarity, this kind of distribution wasnamed as the WW:CC segregation pattern by referring to the older DNA strandsspecifically at the centromere of each chromatid/chromosome (Figures 2 and 3).

By changing growth conditions, the cells containing this recombination systemwere changed to several other cell types. Only the WW:CC segregation wasobserved in endodermal cells (Table 1). In contrast, all the recombinant culturesexamined for each class of pancreatic, mesodermal, and cardiomyocyte cellsexhibited P/Mheterozygosis in about one-third of recombinants, indicating theirless biased or near random segregation pattern (2).

Figure 3: The recombination model employed to define the chromatidsegregation 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. ThePand Mallelic constitution is determined with Southern

analysis. To obtain the result of all recombinants becoming homozygous forPand 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

WC), and it must be followed by selective distribution of centromere copies, asindicated in the drawing. Therefore, all M/MandP/Phomozygous recombinants,

G2 recombination segregation modes

2WC

M

P

P

1WC

W2C

W1C

M

P

M

M

M

P

P

Segregation pattern: WW:CC = X WC:WC = ZDistalmarkers: Homozygous Heterozygous

1W

1W

2W

2C

1C

2C

Mitosis

M2W

P

1C

Or

M1W

1C

P2W2C

loxP

loxP

G1 recombination

M

1W1C

P

2W2C

loxP

Daughter cells chromosomes

Key: loxp, the site-specific recombination cassette inserted in both homologs near thecentromere (oval circle); XX crossover, G2 phase recombination of indicated non-sisterchromatids; Pand M, chromosomal markers located in the middle of the chromosome; all othersymbols are defined in Figure 1.

Chr. 7 pair in G1

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conventionally termed to have originated from X segregation, are interpreted to reflect

the WW:CC segregation process, and allP/Mrecombinants, 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 eitherpattern is found in different cells of a culture.

Another unanticipated result was obtained with neuroectoderm cells, whichshowed yet another kind of segregation mode. In this case, all 160 recombinantsanalysed maintained the P/Mconstitution, a result consistent with the biasedWC:WC (i.e., G2-Z) segregation pattern and/or recombination in them occurs inG1 (Figure 3). Therefore, it was suggested that regulation of chromatiddistribution to progeny cells occurs in ES cells, irrespective of mitoticrecombination occurring in them (40). Indeed, the analysis of recombinantsconstitutes a procedure to discern the segregation mode of a specificchromosome in mitoses. To obtain the homozygous (i.e., WW:CC = X

segregation) result, it was proposed that only specific non-sister chromatids (WCwith WC) must have been allowed to participate in recombination in the G2phase, followed by selective segregation of chromatid sequences through thecentromere, and that recombination in the G1 phase must not have occurred,because such events would produce P/Mheterozygous recombinants (Figure 3).Perhaps the biased chromatid segregation process itself restricts which pair of thenon-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 cellsand suggest that the pattern is not invariant, as it changes with the cell type invery interesting ways (2, 55). Furthermore, this process seems to bechromosome-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 bedetermined whether the ES/endoderm and the neuroectoderm results differbecause of cell-type regulated distinction between chromatids that are permittedto recombine or due to differences in the mode of chromatid segregation. Assomatic general recombination normally occurs rarely (46), it is difficult toimagine that a chromatid choice mechanism only dedicated for controllingexperimentally induced recombination has evolved in mouse cells. Therefore, weenvision that likely a chromosome-specific non-random segregation processinherently operates in some mouse cell lineages, that such a process indirectlyinfluences the choice of recombining chromatids, and that this process might alsodisallow 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. Theyexhibit a unidirectional minus-end movement on microtubules, and are classifiedas either cytoplasmic or axonemal. Cytoplasmic dyneins have been implicated invesicular transport, nuclear migration, and spindle orientation, whereas axonemaldyneins produce the motor forces that cause the sliding of adjacent microtubulesin the axoneme leading to cilia and flagella movement. Dyneins function as largemulti-subunit complexes containing up to three heavy chains, which include one

with the force-producing motor domain, and several intermediate and lightchains. Dyneins are considered to be key players in the intrinsic cell division

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process in Drosophila (93). It has been suggested that since dyneins areanchored at the cell cortex, as well as at the microtubules, a pulling force may begenerated leading to specific spindle positioning in the cell. Evidence for the LRD(protein) function on asymmetric cell division came by conditional depletion andmild RNAi inactivation studies of several dynein proteins, such as the dyneinheavy-chain gene (dhc-1), the Lissencephaly protein1 (LIS-1) and the dynein-

associated component road-block (DYRB-1) in C. elegans (65). Since all thedynein mutants exhibited a marked reduction of spindle pulling forces, dyneinsare required for proper spindle positioning in cells ofC. elegans embryos (14, 65,67). Furthermore, cytoplasmic dynein comprises one of the components toinduce the attachment of one pole of the stem cell spindle to the niche cells,thereby promoting asymmetric cell division in Drosophila female germ line stemcell mitoses (10, 29). Although dynein seem to be the common factor involved inasymmetric cell division in diverse systems, how precisely dynein proteinpromotes asymmetric cell division remains unknown.

The left-right dynein (lrd) is an axonemal dynein heavy chain-encoding gene. Anextensive expression analysis revealed that, in addition to symmetric expression

in the embryonic node, apparently transient asymmetric expression oflrdoccursin the head-fold region of 0- to 5-somite stage mouse embryos. At later stages,lrdis 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 visceralorgans left-right (LR) development (17, 18, 24, 56). In addition, expression wasseen in non-ciliated cells (5), in several ciliated cell types in newborn mice, theepithelial lining of the nasal cavity, and in the ependymal lining of the thirdventricle of the brain (97).

A spontaneous missense mutation in lrdcauses randomization of LR lateralitysuch that one-half of mice develop with mirror-imaged visceral organs, ascompared with wild-type mice (47). The LRD proteins function in visceral

laterality specification in mice was confirmed by generating a targeted deletion ofthe ATP binding domain of the lrdgene. Like the missense mutation, the deletionmutant similarly produced LR axis randomization (83). Similarly, homozygotes ofdifferent subunits of dynein exhibit visceral organs randomization in humans (1).The placement of gut and stomach is also under the LR axis control. Thissuggests that lrdexpression in the developing gut may be involved in LRspecification. Although the gut tube begins to close at E8.5 in mice, the gut doesnot initiate handed asymmetric coiling until after mid-gestation. Expression oflrdin the gut endoderm is only found up to the E9.5 stage, suggesting that lrdmaynot directly function in the lateralization of the gut, but rather it acts through anindirect mechanism, as speculated for heart looping. In the heart case, thetransient 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 thepericardiac region. Taking this into account, it has been speculated that thetransient asymmetric domain of LRD expression functions in the transfer of left-right patterning information from the midline node to influence the rightwardlooping of the heart tube. Expression oflrdin E10.512.5 embryos was alsofound in the floor plate of the neural tube, the midline structure that has beenimplicated in LR specification (83, 84, 85).The SSIS model was first proposed to explain the LR randomization phenotype ofthe iv(situs inversus) mutant mice. By the model, the ivgene, later discoveredto encode LRD (85), mediates WW:CC segregation (Figure 2) to produceasymmetric cell division in the embryo when initially the visceral laterality isspecified (39). According to the molecular data discussed above from studies ofDrosophila, mice and in humans, it is possible that LRD is involved in asymmetriccell division. Therefore, the hypothesized role of dynein according to the SSIS

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model for LR axis determination was investigated by determining its role in theselective Chr. 7 segregation process, even though it is not known whether thischromosome concerns axis specification. Very encouraging results were obtained(3): First, there is a perfect correlation between lrdmRNA presence/absence andthe Chr. 7 segregation mode in all the six cell lineages that were examined (Table1). Specifically, lrdgene is expressed in cultures that follow selective segregation

patterns, but not in those that follow an unbiased pattern (3). Second, after lrdmRNA inactivation, by RNAi technology, each of the ES, endoderm, andneuroectoderm cell lines disrupted their selective segregation mode (Table 1).Third, neuroectoderm cells after LRD depletion produced M/Mheterozygousrecombinants; they must have been generated by recombination in G2 (Figure3). Thus, neuroectoderm cells must normally follow the WC:WC segregationmode to produce P/Mheterozygous recombinants where recombined chromatidsare always delivered to the same daughter cell. As both the WW:CC and WC:WCselective patterns are found in different lineages, we conclude that therecombination process itself does not dictate generation of either the X or the Zsegregation outcome (6, 25, 28, 55), rather selective segregation processinherently operates in specific cell lineages (2, 3, 42); LRD likely functions

directly in the selective Chr. 7 segregation mechanism. These results support theexistence of the selective strand segregation feature of the SSIS model proposedfor LR axis determination in vertebrates (39, 40).

QuickTime and adecompressor

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 bycell type, as indicated. ON means expressed; OFF means silent.

The mechanism of how the LR symmetry of the embryo is initially broken remainsdevelopmental biologys key unanswered question (59, 73, 87, 88, 89). Defining

the molecular function of the lrdgene in axis development is required to explainthe fascinating LR axis randomization phenotype of the mutant mice. The mostpopular model for the LRD protein function postulates its role in the motility ofmonocilia developed on nodal cells of the mouse embryo, called the nodal flowhypothesis (66). Notably, the mutant mice develop immotile cilia. According tothe nodal flow hypothesis, the cilia-generated leftward embryonic fluid flowestablishes an asymmetric gradient of a morphogen across the embryo,constituting a mechanism for LR patterning (8). However, it remains acontroversial model when applied to the LR axis determination. The work inmouse cells supports the theory that the dynein motor protein plays acytoplasmic role in LR patterning and that it does not involve extra-cellular ciliamotion (73).

The SSIS model was proposed as an alternative model for explainingdevelopment of LR asymmetry through asymmetric cell division during

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development. Thus, this model is based on cell lineage and it is designed toexploit the inherent base sequence differences in the W and C strands, theirreplication history (98), and the epigenetic events that might be installed at thetime of chromosome replication during development (16, 37, 76). The result ofrandom chromatid segregation in LRD knockdown cells is consistent with therandom LR visceral phenotype of the lrd-/lrd-mutant mice. Also, the findings that

only one-half of heterozygous Chr. 11 three independent translocation carriersdevelop schizophrenia and bipolar brain psychiatric diseases have been argued tosupport the SSIS model for human brain LR laterality development by postulatingrandom strand/chromatid segregation occurring owing to rearrangements (40).The same logic might explain the Kartageners 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 modelis too vague to be experimentally tested. Discovery of extra-cellular signalsthat promote cellular differentiation through cellcell contact, such as thosein germ line nitches ofDrosophila, do not necessarily evidence themorphogen-gradient model designed for explaining patterning in muchlarger areas of the embryo. Apparently, a very interesting new avenue forresearch has opened up to accomplish cellular differentiation throughasymmetric cell division by regulating chromosome distribution. Studies ofthe Cre/loxp and FLP/FRT site-specific recombination systems quoted abovewere conducted only to develop tools for chromosomal manipulation. Wepropose that this approach also identifies the mode of sister chromatidsegregation in mitosis. Liu et al. (55) conducted their study only with Chr. 7and 11. Fortuitously, Chr. 7 exhibited two types of selective segregationpatterns, which we showed to be LRD-dependent, and which changes with

the cell type in most interesting ways (2, 3). The sole function known forLRD is for axis determination. These results raise the tantalizing possibilitythat Chr. 7 might specify LR axis determination in mice, in accord with theSSIS model. If so, one genetic test of this suggestion is that micegenetically engineered to contain an inversion of both Chr. 7 centromeresshould develop situs inversus in all animals. Also, some of the cell intrinsicfactors implicated in promoting asymmetric cell division might function bydictating cellular polarity required for selective chromatid distribution. Forexample, the cell-fate determining Numb protein is located in the mothercentrosome (44), a structure found at the pole of the mitotic spindle. Also,the basal body of monocilia originates from one of the two centrosomes ofthe spindle. This new cell biological phenomenon of selective chromatid

segregation may explain development in mammals and the origin of brainLR laterality developmental disorders of psychoses in humans. In futurestudies more direct approaches should be designed to further investigatethe mechanism of the DNA strand segregation process and to scrutinize itsproposed role in development. It has been speculated that the sistercentromeres might be differentiated during replication by epigenetic meansto identify them for biased segregation (52). In sum, we hypothesize thatsuch a mechanism may be crucial in evolution of form, for cellulardifferentiation and development, and for maintaining the integrity of parent-of-originspecific imprints in somatic lineages by discouraging mitoticrecombination perhaps through chromosome compartmentalization in thenucleus.

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AcknowledgmentsResearch in the Laboratory of Experimental Physiology is supported by theGeneral Secretariat of Research and Development, Ministry of Development,Greek Government, and by the Research Academy of the National andKapodistrian University of Athens. The Intramural Research Program, Center forCancer Research, National Cancer Institute at Frederick, National Institutes of

Health, USA, supports research in the Klar laboratory.

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