Analysis of mouse eye development with chimeras and mosaics

Analysis of experimental mouse chimeras (chimaeras) and mosaics provides a means of investigating patterning and differentiation within the developing mammalian eye. Chimeric and mosaic mice carry two or more genetically distinct cell populations and extend the repertoire of analytical tools available to the geneticist. Here we review the impact these techniques have had on our understanding of eye organogenesis. Chimeras and mosaics are routinely used to investigate cell lineages, patterns of growth and gene function, and provide a means to clear analytical hurdles that otherwise limit standard genetic approaches. In particular, chimeras are used to investigate the roles of genes in tissues that do not develop in conventional mutant or knock-out mice, to test whether genes act cell autonomously or non-autonomously in different tissues and to dissect tissue-tissue interactions in less tractable, complex systems. Chimeras, in which cells of different genetic composition are mixed at a fine-scale cellular level, may provide qualitatively different data from mosaic mice with conditional knockouts. The uses of chimeras, Cre-loxP mosaics and in vitro tissue recombination for study of ocular organogenesis are compared. Wider use of mosaics and chimeras should provide further insights into eye development.     

Growth and development of the mouse retinal pigment epithelium. II. Cell patterning in experimental chimaeras and mosaics

The retinal pigment epithelium (PE), with pigmentation as a cell-autonomous marker, was analyzed in three types of mice: congenic pigmented----albino chimaeras, X-inactivation mosaics (Cattanach's translocation), and mosaics homozygous for the pink-eyed unstable mutation, which contain rare fully pigmented cells. In 10 chimaeric and 34 X-inactivation eyes, the proportionate mix in the right and left eyes of an individual animal was similar, the mix was approximately constant in all parts of a given eye, average patch size was larger toward the periphery of the PE, and peripheral patches tended to be elongated in the radial dimension. In all 44 whole mounts from pink-eyed unstable mutants, patches of 1-12 pigmented cells, each representing a single clone, were scattered throughout the PE; they tended to be larger with increasing distance from the optic nerve head. The collective data are consistent with significant cell mixing prior to specification of the two eye fields, during early organ-forming stages, and during later development of the PE. The tendency of peripheral patches to orient radially reflects the edge-biased pattern of cell proliferation in the PE. Cell mixing appears to be more prominent posteriorly in the PE sheet; growth proceeds anteriorly for more generations.     

Interspecies chimeras of mammals

Chimaeras between closely related and distant mammalian species were obtained during the last decade using the aggregation and injection methods. In the chimaeras between the closely related species Mus musculus--M. caroli, the cell clones of both parental species are equally represented; such chimaeras are fertile and their development does not differ from that of intraspecific chimaeras. But in the chimaeras between distant species mouse--rat or sheep--goat a selective predominance of cells with one genotype over those with another was noted. This leads to the total elimination of cell populations with one genotype, rat in particular in the mouse--rat chimaeras, or, possibly, to the sterility of the sheep--goat chimaeras.     

The theory of clonal mixing during growth

The component clones of an embryo may mingle as they grow. The pattern which results from clonal mixing can be seen in adult genetic mosaics. This paper relates the final type and degree of mixing to the random diffusive mobility of individual cells during growth. It is shown that mixing is slight if d ? a, and extensive if d ? a, where a is the average distance between neighbouring cells, and d is the average distance through which a cell diffuses in the course of one cell cycle.     

Influence of cell fate mechanisms upon retinal mosaic formation: a modelling study

Many types of retinal neurone are arranged in a spatially regular manner so that the visual scene is uniformly sampled. Several mechanisms are thought to be involved in the development of regular cellular positioning. One early-acting mechanism is the lateral inhibition of neighbouring cells from acquiring the same fate, mediated by Delta-Notch signalling. We have used computer modelling to test whether lateral inhibition might transform an initial population of undifferentiated cells into more regular populations of two types of differentiated cells. Initial undifferentiated cells were positioned randomly, subject only to a minimal distance constraint. Each undifferentiated cell then acquired either primary or secondary fate using one of several lateral inhibition mechanisms. Mosaic regularity was assessed using the regularity index and the packing factor. We found that for irregular undifferentiated mosaics, the arrangement of resulting primary (but not secondary) fate cells was more regular than in the initial undifferentiated population. However, for regular undifferentiated mosaics, no further increases in the regularity of the primary fate mosaics were observed. We have used this model to test the specific hypothesis that on- and off-centre retinal ganglion cells emerge from an initial, irregular undifferentiated population of ganglion cells. Lateral inhibition can subdivide an initially irregular population into two types of cell that are mildly regular. However, lateral inhibition alone is insufficient to produce mosaics of the same regularity as observed experimentally. Likewise, and in contrast to earlier reports, cell death alone is insufficient to match the regularity of experimental mosaics. We conclude that lateral inhibition can transform irregular distributions into regular mosaics, upon which subsequent processes (such as lateral cell movement or cell death) can further refine mosaic regularity.     

Evidence for selection against tetraploid cells in tetraploid<-->diploid mouse chimaeras before the late blastocyst stage

Tetraploid (4n) cells do not contribute equally to all tissues of midgestation mouse chimaeras and mosaics. Our previous studies of early blastocysts showed that 4n cells are preferentially allocated to the mural trophectoderm of the early blastocyst and this may contribute to the restricted distribution pattern seen at later stages. In this study of later-stage blastocysts we found evidence for selection against 4n cells. The contribution of 4n cells to 4n<-->2n chimaeric blastocysts decreased between E3.5 and E4.5 days, whereas the composition of 2n<-->2n controls changed little over this period. These results suggest that, prior to implantation, blastocysts have already lost some tetraploid cells from their embryonic and extra-embryonic lineages due to a combination of preferential allocation of 4n cells to the mural trophectoderm and selection against 4n cells throughout the embryo.     

A Modeling Study on How Cell Division Affects Properties of Epithelial Tissues Under Isotropic Growth

Cell proliferation affects both cellular geometry and topology in a growing tissue, and hence rules for cell division are key to understanding multicellular development. Epithelial cell layers have for long times been used to investigate how cell proliferation leads to tissue-scale properties, including organism-independent distributions of cell areas and number of neighbors. We use a cell-based two-dimensional tissue growth model including mechanics to investigate how different cell division rules result in different statistical properties of the cells at the tissue level. We focus on isotropic growth and division rules suggested for plant cells, and compare the models with data from the Arabidopsis shoot. We find that several division rules can lead to the correct distribution of number of neighbors, as seen in recent studies. In addition we find that when also geometrical properties are taken into account other constraints on the cell division rules result. We find that division rules acting in favor of equally sized and symmetrically shaped daughter cells can best describe the statistical tissue properties.     

A cellular lineage analysis of the chick limb bud

The chick limb bud has been used as a model system for studying pattern formation and tissue development for more than 50 years. However, the lineal relationships among the different cell types and the migrational boundaries of individual cells within the limb mesenchyme have not been explored. We have used a retroviral lineage analysis system to track the fate of single limb bud mesenchymal cells at different times in early limb development. We find that progenitor cells labeled at stage 19-22 can give rise to multiple cell types including clones containing cells of all five of the major lateral plate mesoderm-derived tissues (cartilage, perichondrium, tendon, muscle connective tissue, and dermis). There is a bias, however, such that clones are more likely to contain the cell types of spatially adjacent tissues such as cartilage/perichondrium and tendon/muscle connective tissue. It has been recently proposed that distinct proximodistal segments are established early in limb development; however our analysis suggests that there is not a strict barrier to cellular migration along the proximodistal axis in the early stage 19-22 limb buds. Finally, our data indicate the presence of a dorsal/ventral boundary established by stage 16 that is inhibitory to cellular mixing. This boundary is demarcated by the expression of the LIM-homeodomain factor lmx1b.     

Spatial interaction models: from human geography to plant-herhivore interactions

The spatial pattern of defoliation by mammalian herbivores across vegetation mosaics bas been frequently discussed, but rarely spatially quantified. Here we considered the role of plant-herbivore interactions in determining the spatial distribution of shrub defoliation by a large mammalian herbivore across a grass-shrub mosaic.
We investigated the spatial pattern of heather defoliation by sheep in heather-grass mosaics. Heather-grass mosaics are two-phased vegetation mosaics, in which a spatially localized plant community (grass) fulfils nutritional needs, whilst a spatially extensive plant community (heather) meets energy requirements but is nutritionally marginal.
We used a spatial analysis method, originating from human geography, to show that heather defoliation was not spread across the mosaic homogeneously, but that the spatial pattern was determined by geometric characteristics of the mosaic, grazing intensity, and the contrast between preferred and less preferred communities.
The spatial analysis method proved to be a powerful tool to describe the spatial pattern of shrub defoliation. Applications of the method are explored and the implications of the spatial distribution of shrub defoliation are discussed.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. II. Effect of the steel locus on spermatogenesis

Mutant mice of Sl/Sld genotype are deficient in melanocytes, erythrocytes, mast cells and germ cells. Deficiency of melanocytes, erythrocytes and mast cells is not attributable to an intrinsic defect in their precursor cells but to a defect in the tissue environment that is necessary for migration, proliferation and/or differentiation. We investigated the mechanism of germ cell deficiency in male Sl/Sld mice by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent white stripes were obtained. Two of four such chimaeras were fertile and the phenotypes of resulting progenies showed that some Sl/Sld germ cells had differentiated into functioning sperms in the testis of the chimaeras. In cross sections of the testes of chimaeras, both differentiated and nondifferentiated tubules were observed. However, the proportions of type A spermatogonia to Sertoli cells in both types of tubules were comparable to the values observed in differentiated tubules of normal +/+ mice. We reconstructed the whole length of four tubules from serial sections. Differentiated and nondifferentiated segments alternated in a single tubule. The shortest differentiated segment contained about 180 Sertoli cells and the shortest nondifferentiated segment about 150 Sertoli cells. These results suggest that Sertoli cells of either Sl/Sld or +/+ genotype make discrete patches and that differentiation of type A spermatogonia does not occur in patches of Sl/Sld Sertoli cells.     

Difference in the retinal cone mosaic pattern between zebrafish and medaka: cell-rearrangement model

In fish retina, four kinds of photoreceptor cells (or cones) are two-dimensionally arranged in a very regular manner, forming cone mosaics. Mosaic pattern differs between species--two typical patterns are "row mosaic" and "square mosaic", exemplified by the cone mosaics in zebrafish and in medaka, respectively. In this paper, we study a cell-rearrangement model. Cells with pre-fixed fate exchange their locations between nearest neighbors and form regular mosaic patterns spontaneously, if the adhesive force between nearest neighbors and between next-nearest neighbors depend on their cell types in an appropriate manner. The same model can produce both row and square mosaic patterns. However, if the cell-cell interaction is restricted to nearest neighbors only, the square mosaic (medaka pattern) cannot be generated, showing the importance of interaction between next-nearest neighbors. In determining whether row mosaic (zebrafish pattern) or square mosaic (medaka pattern) is to be formed, two shape factors are very important, which control the way adhesions in different geometric relations are combined. We also developed theoretical analysis of the parameter ranges for the row mosaic and the square mosaic to have higher total adhesion than alternative spatial patterns.     

Specific detection of Salmonella typhimurium proteins synthesized intracellularly.

Studies of the proteins Salmonella typhimurium synthesizes under conditions designed to more closely approximate the in vivo environment, i.e., in cell and tissue culture, are not easily interpreted because they have involved chemical inhibition of host cell protein synthesis during infection. The method which we have developed allows specific labeling of bacterial proteins without interfering with host cell metabolic activities by using a labeled lysine precursor which mammalian cells cannot utilize. We have resolved the labeled proteins using two-dimensional electrophoresis and autofluorography. We were able to detect 57 proteins synthesized by S. typhimurium during growth within a human intestinal epithelial cell line. Of the 57 proteins detected, 34 appear to be unique to the intracellular environment, i.e., they are not seen during growth of the bacteria in tissue culture medium alone. Current (and future) efforts are directed at organizing the 34 proteins into known stress response groups, determining the cellular locations of the proteins (outer or inner membrane, etc.), and comparing the pattern of proteins synthesized within an intestinal epithelial cell to the pattern synthesized during growth within other tissues.     

A multiscale model of complex endothelial cell dynamics in early angiogenesis

We introduce a hybrid two-dimensional multiscale model of angiogenesis, the process by which endothelial cells (ECs) migrate from a pre-existing vascular bed in response to local environmental cues and cell-cell interactions, to create a new vascular network. Recent experimental studies have highlighted a central role of cell rearrangements in the formation of angiogenic networks. Our model accounts for this phenomenon via the heterogeneous response of ECs to their microenvironment. These cell rearrangements, in turn, dynamically remodel the local environment. The model reproduces characteristic features of angiogenic sprouting that include branching, chemotactic sensitivity, the brush border effect, and cell mixing. These properties, rather than being hardwired into the model, emerge naturally from the gene expression patterns of individual cells. After calibrating and validating our model against experimental data, we use it to predict how the structure of the vascular network changes as the baseline gene expression levels of the VEGF-Delta-Notch pathway, and the composition of the extracellular environment, vary. In order to investigate the impact of cell rearrangements on the vascular network structure, we introduce the mixing measure, a scalar metric that quantifies cell mixing as the vascular network grows. We calculate the mixing measure for the simulated vascular networks generated by ECs of different lineages (wild type cells and mutant cells with impaired expression of a specific receptor). Our results show that the time evolution of the mixing measure is directly correlated to the generic features of the vascular branching pattern, thus, supporting the hypothesis that cell rearrangements play an essential role in sprouting angiogenesis. Furthermore, we predict that lower cell rearrangement leads to an imbalance between branching and sprout elongation. Since the computation of this statistic requires only individual cell trajectories, it can be computed for networks generated in biological experiments, making it a potential biomarker for pathological angiogenesis.     

The effects of cell size and ploidy on cell allocation in mouse chimaeric blastocysts

In a previous study of mouse tetraploid<-->diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in post-implantation stage tetraploid<-->diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid<-->diploid chimaeric blastocysts.     

Pattern formation of the cone mosaic in the zebrafish retina: a cell rearrangement model

In fish retinas, cone photoreceptor cells are arranged in two-dimensional regular patterns, called cone mosaics. In the zebrafish retina, four subtypes of cone cells, which are maximally sensitive to different wavelengths of light, appear in quasi-periodic patterns. The pattern formation mechanism is unknown. Here, I develop a mathematical model to examine whether cell adhesion can explain the formation of the zebrafish mosaic. I assume that the movement of differentiated cells is responsible for generating the pattern, and that the movement rate is modified by cell adhesion. The pattern is formed if the magnitudes of cell adhesion between cell types are chosen appropriately. I determine the conditions of cell adhesion for generating the pattern. I also compare this cell rearrangement model with a previously studied model in which the pattern is formed by transitions of cell fate. The condition for obtaining the focal pattern is looser in the cell rearrangement model than in the fate transition model.     

BIO-LGCA: A cellular automaton modelling class for analysing collective cell migration

Collective dynamics in multicellular systems such as biological organs and tissues plays a key role in biological development, regeneration, and pathological conditions. Collective tissue dynamics—understood as population behaviour arising from the interplay of the constituting discrete cells—can be studied with on- and off-lattice agent-based models. However, classical on-lattice agent-based models, also known as cellular automata, fail to replicate key aspects of collective migration, which is a central instance of collective behaviour in multicellular systems. To overcome drawbacks of classical on-lattice models, we introduce an on-lattice, agent-based modelling class for collective cell migration, which we call biological lattice-gas cellular automaton (BIO-LGCA). The BIO-LGCA is characterised by synchronous time updates, and the explicit consideration of individual cell velocities. While rules in classical cellular automata are typically chosen ad hoc, rules for cell-cell and cell-environment interactions in the BIO-LGCA can also be derived from experimental cell migration data or biophysical laws for individual cell migration. We introduce elementary BIO-LGCA models of fundamental cell interactions, which may be combined in a modular fashion to model complex multicellular phenomena. We exemplify the mathematical mean-field analysis of specific BIO-LGCA models, which allows to explain collective behaviour. The first example predicts the formation of clusters in adhesively interacting cells. The second example is based on a novel BIO-LGCA combining adhesive interactions and alignment. For this model, our analysis clarifies the nature of the recently discovered invasion plasticity of breast cancer cells in heterogeneous environments.     

Morphogenesis and formation of a pattern in hydra. I. Distribution of morphogenetic potential

True multicellularity is characterized by complex interactions between individual cells of the organism as well as by organization of cell masses into spatially and functionally determined structures promoting the exchange of information. Morphogenetic processes--genetically programmed generation of structures--always correlate with determination and maintenance of a pattern, i.e. a system of spatial relationships between them. Hydroid polyps provide a wide variety of approaches to study morphogenesis and patterning. Being comparatively simply organized, these animals have nevertheless certain developed mechanisms underlaying such processes as regeneration of missing structures, recovery of normal pattern after dissociation of polyps into single cells, tissue transdifferentiation in non-complementary chimaeras. An important feature of regeneration of hydroid polyps is its independence of the nerve net elements; the basis for regeneration is rather stored in epithelial cells and in their interactions. Phenomenological data, provided in the XVIII-XX centuries, allowed to propose several theoretical models of pattern regulation in hydra. The main goal of this paper is to review contemporary models of morphogenesis and patterning in the hydroid polyps.     

Cell Growth and Size Homeostasis in Silico

Cell growth in size is a complex process coordinated by intrinsic and environmental signals. In a research work performed by a different group, size distributions of an exponentially growing population of mammalian cells were used to infer cell-growth rate in size. The results suggested that cell growth was neither linear nor exponential, but subject to size-dependent regulation. To explain the observed growth pattern, we built a mathematical model in which growth rate was regulated by the relative amount of mRNA and ribosomes in a cell. Under the growth model and a stochastic division rule, we simulated the evolution of a population of cells. Both the sampled growth rate and size distribution from this in silico population agreed well with experimental data. To explore the model space, alternative growth models and division rules were studied. This work may serve as a starting point to understand the mechanisms behind cell growth and size regulation using predictive models.     

Cell Growth and Size Homeostasis in Silico

Cell growth in size is a complex process coordinated by intrinsic and environmental signals. In a research work performed by a different group, size distributions of an exponentially growing population of mammalian cells were used to infer cell-growth rate in size. The results suggested that cell growth was neither linear nor exponential, but subject to size-dependent regulation. To explain the observed growth pattern, we built a mathematical model in which growth rate was regulated by the relative amount of mRNA and ribosomes in a cell. Under the growth model and a stochastic division rule, we simulated the evolution of a population of cells. Both the sampled growth rate and size distribution from this in silico population agreed well with experimental data. To explore the model space, alternative growth models and division rules were studied. This work may serve as a starting point to understand the mechanisms behind cell growth and size regulation using predictive models.