Detailed Fate Research Articles

Extensive Cell Movements Accompany Formation of the Otic Placode

During development, the vertebrate inner ear arises from the otic placode, a thickened portion of the ectoderm next to the hindbrain. Here, the first detailed fate maps of this region in the chick embryo are presented. At head process stages, placode precursors are scattered throughout a large region of the embryonic ectoderm, where they intermingle with future neural, neural crest, epidermal, and other placode cells. Within the next few hours, dramatic cell movements shift the future otic placode cells toward the midline and ultimately result in convergence to their final position next to rhombomeres 5–6. Individual cells and small cell groups undergo constant cell rearrangements and appear to sort out from nonotic cells. While the major portion of the otic placode is derived from the nonneural ectoderm, the neural folds also contribute cells to the placode at least until the four-somite stage. Comparison of these fate maps with gene expression patterns at equivalent stages reveals molecular heterogeneity of otic precursor cells in terms of their expression of dlx5, msx1, Six4, and ERNI. Although Pax2 expression coincides with the region where otic precursors are found from stage 8, not all Pax2-positive cells will ultimately contribute to the otic placode.

Fate map of the chicken neural plate at stage 4.

A detailed fate map was obtained for the early chick neural plate (stages 3d/4). Numerous overlapping plug grafts were performed upon New-cultured chick embryos, using fixable carboxyfluorescein diacetate succinimidyl ester to label donor chick tissue. The specimens were harvested 24 hours after grafting and reached in most cases stages 9-11 (early neural tube). The label was detected immunocytochemically in wholemounts, and cross-sections were later obtained. The positions of the graft-derived cells were classified first into sets of purely neural, purely non-neural and mixed grafts. Comparisons between these sets established the neural plate boundary at stages 3d/4. Further analysis categorized graft contributions to anteroposterior and dorsoventral subdivisions of the early neural tube, including data on the floor plate and the eye field. The rostral boundary of the neural plate was contained within the earliest expression domain of the Ganf gene, and the overall shape of the neural plate was contrasted and discussed with regard to the expression patterns of the genes Plato, Sox2, Otx2 and Dlx5 (and others reported in the literature) at stages 3d/4.

Lasers for Discrete Gene Manipulation

The transgenic (Tg) expression of ectopic genes in organisms allows researchers to observe developmental outcomes that deviate from those of the wild-type organism. However, Tg expression in single organs is sometimes desired to determine protein function outside the context of other organs, or when organismic expression is lethal. Halloran et al . use laser-induced gene expression to track development within and surrounding one cell. Tg zebrafish were made containing the green fluorescent protein (GFP) gene cloned under the control of the heat shock protein (Hsp) 70 promoter. Tg fish subjected to heated water exhibited no morphological defects, had normal axonal development, and reproduced successfully, indicating that heat induction of GFP did not have any deleterious developmental effects. Laser-induced heating of individual cells in GFP Tg fish induced GFP production in 24-94% of the cells treated, depending on the cell type. The laser heat did not damage the cells. The authors then created fish containing a fusion of the Hsp70 promoter and semaphorin 3A1, a gene whose product causes repulsion of motor neurons and inhibition of growth cone extension. Laser excitation of one muscle cell led to the inhibition of motor neuron extension at only that muscle cell. Thus, laser-induced gene expression in single cells may give investigators the ability to trace cell lineages and very detailed fate maps in vertebrates. Halloran, M.C., Sato-Maeda, M., Warren, J.T., Su, F., Lele, Z., Krone, P.H., Kuwada, J.Y., and Shoji, W. (2000). Laser-induced gene expression in specific cells of transgenic zebrafish. Development 127 : 1953-1960 [Online Journal]

Modeling Aquatic Mercury Fate in Clear Lake, Calif.

This paper describes the construction and application of a numerical model representing mercury transformations and bioavailability in the aquatic environment of Clear Lake, Calif. The initial application described here provides the basis for more detailed fate and transport modeling of the lake. Results show that total Hg and MeHg concentrations in the lake may be reasonably modeled as functions of sediment total Hg. Calibrated rates of methylation and benthic exchange compare favorably with rates observed in the field and at other lakes. The model suggests the importance of accurately defining a biochemically “available” fraction of total Hg, and demonstrates that even a relatively small amount of available Hg in lake sediments can account for high levels of potentially toxic methylmercury in the water and sediment bed. Indications are that Clear Lake is experiencing a net loss of Hg at a rate of less than 2%/year. At such a remediation rate, mercury concentrations in the lake would meet current water quality criteria in a little over 200 years.

Cell fate in the chick limb bud and relationship to gene expression

We have produced detailed fate maps for mesenchyme and apical ridge of a stage 20 chick wing bud. The fate maps of the mesenchyme show that most of the wing arises from the posterior half of the bud. Subapical mesenchyme gives rise to digits. Cell populations beneath the ridge in the mid apical region fan out into the anterior tip of the handplate, while posterior cell populations extend right along the posterior margin. Subapical mesenchyme of the leg bud behaves similarly. The absence of anterior bending of posterior cell populations has implications when considering models of vertebrate limb evolution. The fatemaps of the apical ridge show that there is also a marked anterior expansion and cells that were in anterior apical ridge later become incorporated into non-ridge ectoderm along the margin of the bud. Mesenchyme and apical ridge do not expand in concert--the apical ridge extends more anteriorly. We used the fatemaps to investigate the relationship between cell lineage and elaboration of Hoxd-13 and Fgf-4 domains. Hoxd-13 and Fgf-4 are initially expressed posteriorly until about the mid-point of the early wing bud in mesenchyme and apical ridge respectively. Later in development, the genes come to be expressed throughout most of the handplate and apical ridge respectively. We found that at the proximal edge of the Hoxd-13 domain, cell populations stopped expressing the gene as development proceeded and found no evidence that the changes in extent of the domains were due to initiation of gene expression in anterior cells. Instead the changes in extent of expression fit with the fate maps and can be attributed to expansion and fanning out of cell populations initially expressing the genes.

A fate map of the vegetal plate of the sea urchin (Lytechinus variegatus) mesenchyme blastula.

Previous lineage tracing experiments have shown that the vegetal blastomers of cleavage stage embryos give rise to all the mesoderm and endoderm of the sea urchin larva. In these studies, vegetal blastomers were labeled no later than the sixth cleavage division (60-64 cell stage). In an earlier study we showed that single cells in the vegetal plate of the blastula stage Lytechinus variegatus embryo could be labeled in situ with the fluorescent, lipophilic dye, DiI(C18), and that cells labeled in the central region of the vegetal plate of the mesenchyme blastula primarily gave rise to homogeneous clones consisting of a single secondary mesenchyme cell (SMC) type (Ruffins and Ettensohn (1993) Dev. Biol. 160, 285-288). Our clonal labeling showed that a detailed fate map could be generated using the DiI(C18) labeling technique. Such a fate map could provide information about the spatial relationships between the precursors of specific mesodermal and endodermal cell types and information concerning the movements of these cells during gastrulation and later embryogenesis. We have used this method to construct the first detailed fate map of the vegetal plate of the sea urchin embryo. Ours is a latitudinal map; mapping from the plate center, where the mesodermal precursors reside, through the region which contains the endodermal precursors and across the ectodermal boundary. We found that the precursors of certain SMC types are segregated in the mesenchyme blastula stage vegetal plate and that prospective germ layers reside within specific boundaries. To determine whether the vegetal plate is radially symmetrical with respect to mesodermal cell fates, single blastomeres of four cell stage embryos were injected with lysyl-rhodamine dextran (LRD). The resulting ectodermal labeling patterns were classified and correlated with the SMC types labeled. This analysis indicates that the dorsal and ventral blastomers do not contribute equally to SMC derivatives in L. variegatus.

Transparent things: cell fates and cell movements during early embryogenesis of zebrafish.

Development of an animal embryo involves the coordination of cell divisions, a variety of inductive interactions and extensive cellular rearrangements. One of the biggest challenges in developmental biology is to explain the relationships between these processes and the mechanisms that regulate them. Teleost embryos provide an ideal subject for the study of these issues. Their optical lucidity combined with modern techniques for the marking and observation of individual living cells allow high resolution investigations of specific morphogenetic movements and the construction of detailed fate maps. In this review we describe the patterns of cell divisions, cellular movements and other morphogenetic events during zebrafish early development and discuss how these events relate to the formation of restricted lineages.

Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres

A detailed fate map of all the progeny derived from each of the blastomeres of the 4- and 8-cell stage South African clawed frog (Xenopus laevis) embryo is presented. Each "identified" blastomere that results from stereotypic cleavages has a characteristic set of progeny that distinguishes it from the other blastomeres of the embryo. The 4-cell dorsal (D) blastomere is the major progenitor of the stomodeum, cement gland, retina, notochord, head somite, pharynx and liver. The 4-cell ventral (V) blastomere is the major progenitor of the trunk and fin epidermis, ventral somite, nephrotome, lateral plate mesoderm and proctodeum. The other organs are derived from both blastomeres. At the next cell division, the animal hemisphere daughters of both blastomeres (D1 and V1, respectively) become the major progenitors for head ectodermal and mesodermal structures, and the vegetal hemisphere daughters become the major progenitors for trunk mesodermal (D2) or trunk endodermal (V2) structures. Semiquantitative lineage diagrams, using data from this and from previous studies demonstrate that as cleavage proceeds from the 2- to the 32-cell stage, the progenitors for particular organs or for specific regions of organs segregate into defined regions of the blastula. To determine whether this segregation is related to the position of the blastomere or to its geneological lineage, we compared the fates of radial 8-cell blastomeres to those of stereotypic 8-cell blastomeres. Radial blastomeres have fates nearly equivalent to the sum of the two 16-cell blastomeres that occupy the same position in the embryo, demonstrating that fate depends upon blastomere position rather than lineage.

Mathematical models for evaluation of fate of chemicals in the environment.

For assessing the potential hazard of the chemicals to human and other organisms, it is required to evaluate ambient concentrations of the chemicals in air, water, and soil media. Mathematical models have been developed and used for estimating the fate and concentrations of chemicals released into the environment. The principal approaches of the models are based on the mass balance equations expressed in terms of ; ·load into the environment, ·equilibrium and rate constants for transport, transfer, and transformation processes, ·environmental conditions. They are classified into three types of modelling approaches ; ·partition equilibrium model, ·simplified fate simulation model, ·detailed fate simulation model. It may be possible to estimate the amount of the chemicals exposed to human and environmental organisms, based on the model outputs, combined with environmental monitoring data.

Fates of the blastomeres of the 32-cell-stage Xenopus embryo

A detailed fate map of all of the progeny derived from each of the blastomeres of the 32-cell-stage South African clawed frog embryo ( Xenopus laevis), which were selected for stereotypic cleavages, is presented. Individual blastomeres were injected with horseradish peroxidase and all of their descendants in the late tailbud embryo (stages 32 to 34) were identified after histochemical processing of serial tissue sections and whole-mount preparations. The progeny of each blastomere were distributed characteristically, both in phenotype and location. Most organs were populated largely by the descendants of particular sets of blastomeres, the progeny of each often being restricted to defined spatial addresses. Thus, the descendants of any one blastomere were distinct and predictable when embryos were preselected for stereotypic cleavages. However, variations among embryos were common and the frequencies with which one may expect organs to contain progeny from any particular blastomere are reported. The differences in the fates of the 16-cell-stage blastomeres and their 32-cell-stage daughter blastomeres are outlined and can be grouped into three general categories. The two daughter cells may give rise to equal numbers of cells in a particular organ, one daughter cell may give rise to many more of the cells in an organ derived from the mother blastomere, or one daughter cell may give rise to all of the progeny in an organ derived from the mother blastomere. Thus, cell fates are segregated during cleavage stages in both symmetric and asymmetric manners, and the lineages exhibit a diversification mode ( G. S. Stent, 1985, Philos. Trans R. Soc. London Ser. B 312, 3–19) of cell division.

The Honeybee Blastoderm Fate Map

SummaryThe distribution of male and female tissue in 25 cuticular structures was recorded for 1555 adult honeybee gynandromorphs derived from 8 queens. Using the gynandromorph data, distances between structures on the blastoderm were calculated with a FORTRAN programme. The distances thus obtained were used in a BASIC programme to construct a detailed fate map showing the location of 24 structures. The distances between structures on the map fitted the gynandromorphic data well. The fate map was consistent with previously published maps for the honeybee, Drosophila and Habrobracon juglandis. Distances between structures on the blastoderm were not influenced by either the queen producing the gynandromorphs or the frequency of gynandromorphs in a queen's progeny. The median ocellus appeared to be a complex, interacting focus mapping at the site of the lateral ocellus with male bilaterally domineering.

Clonal organization of the central nervous system of the frog. II. Clones stemming from individual blastomeres of the 32- and 64-cell stages

Horseradish peroxidase injected into individual blastomeres of 32- and 64-cell embryos of Xenopus laevis was identified in cells of the central nervous system (CNS) at larval stages 31 to 39. The CNS received contributions from 24 blastomeres of the 32-cell stage and 38 blastomeres of the 64-cell stage. The region of CNS in which all of the labeled descendants of a single blastomere were dispersed is called a clonal domain. Mingling of labeled and unlabeled cells always occurred in a clonal domain, but boundaries were seen between such a labeled region and completely unlabeled regions. These boundaries occurred at various places in the CNS but were most frequently seen in the transverse plane at the level of the isthmus between mesencephalon and rhombencephalon and in the horizontal plane between dorsal and ventral regions of the CNS. When the maternal clonal domain was partitioned between the descendants of the daughter cells, the partitioning occurred only at one or both of those boundaries. The constant relationship between the position of each of the initially labeled blastomeres and the final spatial distribution of the labeled descendants in the CNS provided a detailed fate map of the main regions of the CNS in the 32- and 64-cell embryo.

Pattern Formation in Asymmetrical and Symmetrical Imaginal Discs ofDrosophila melanogaster

The polar coordinate model for pattern regulation in epimorphic fields (French et al ., 1976) predicts that bilaterally symmetrical fields will show different kinds of regulative behavior depending on the direction of the cut. These predictions have been tested using the male genital disc of Drosophila melanogaster . First, a detailed fate map was established by examining the fate of disc fragments subjected to immediate metamorphosis in host larvae. Then the regulative abilities of various fragments were examined by culturing them for seven days in adult abdomens, before transfer to larvae for metamorphosis. When the disc was bisected by a vertical cut (parallel to the line of symmetry) then fragments smaller than half of the disc underwent duplication with some simultaneous regeneration, while fragments larger than half of the disc underwent regeneration. If the disc was bisected by a bilaterally symmetrical cut across the line of symmetry, wound healing resulted in the confrontation of cells from similar positions on the right and left sides of the fragment, and no regulation occurred. With the exception of regeneration occurring during duplication of small lateral fragments, these results are as predicted by the polar coordinate model.

Discussion; the fate of antigen.

I should like to discuss an approach to the problem of immunological tolerance, which is closely akin to Burnet & Fenner’s (1949) ‘self-marker’ hypothesis, although its emphasis is less on the question of how antibodies are produced than on the detailed fate of potentially antigenic molecules when they leave the plasma and lymph and are catabolized. Dr Cinader’s figures certainly suggest that in tolerant rabbits heterologous albumin is eliminated from the plasma at rates which are substantially identical with those of homologous albumin. This is true also of heterologous plasma proteins in normal rabbits during the period before antibodies appear. Furthermore, we have found that in normal rats, in which albumin and globulin are eliminated at markedly different rates (Campbell, Cuthbertson, Matthews & McFarlane 1956), heterologous albumin behaves much like rat albumin and heterologous γ -globulin like rat γ -globulin for a week or more. It is a reasonable working hypothesis that foreign materials in the circulation are in the first place removed from the tissue fluids by the same mechanisms as deal with native materials possessing similar physico-chemical properties (e.g. molecular weight, surface charge). We are largely ignorant of what these mechanisms might be, except for the part played by reticulo-endothelial cells in removing particulate matter and denatured or aggregated proteins. However, the use by Coons, Leduc & Kaplan (1951) of fluorescent antibody to locate antigenic material in histological sections has shown that apparently undenatured proteins (e.g. bovine albumin, human γ -globulin) introduced into the plasma are detectable in immunologically active form within a wide variety of cells, such as renal tubule cells, liver parenchyma or vascular endothelium, in addition to cells of the reticuloendothelial system proper. There are possibly at least two processes going on concurrently: first, a non-selective ingestion of their surrounding fluid (with all that it contains) by cells which are widely distributed in the body, and are not necessarily confined to the reticulo-endothelial system. The mechanism may well be one of vacuolar ingestion (‘pinocytosis’), for which there is some circumstantial evidence, but which also presents difficulties. This would be one mechanism responsible for normal plasma protein catabolism. Secondly, there may be a selective removal of particulate or aggregated material, especially when negatively charged, by cells of the reticulo-endothelial system, notably by liver Küpffer cells.