Cell Lineage Analysis Research Articles

Analysis of cell lineage relationships in taste buds.

Taste buds are a heterogeneous population of cells exhibiting diverse morphological and biochemical characteristics. Because taste buds arise from multiple progenitors, the different types of taste cells may represent distinct lineages. The present study was undertaken to determine the following: (1) how many progenitors contribute to a taste bud, and (2) whether the specific subpopulation of serotonin-immunoreactive (IR) taste cells are related by lineage to a restricted set of progenitor cells. These questions were addressed using cell lineage analysis of taste buds from H253 X-inactivation mosaic mice. After random X-inactivation of the lacZ transgene, the tongue of hemizygous female mice displays discrete patches of epithelial cells, which are either beta-galactosidase (beta-gal) positive or beta-gal negative. By analyzing the proportion of the two differently stained cell populations in taste buds located at the boundary between positive and negative epithelial patches, we can determine the minimum number of progenitors that may contribute to the formation of a taste bud. The presence of taste buds containing only 6-12% labeled cells indicates that at least eight progenitors contribute to an average taste bud of 55 cells, assuming progenitors contribute equally to the cell population. Cell lineage analysis of serotonin-IR taste cells in such mixed taste buds suggests that this subpopulation likely arises from only one to two progenitors and often is related by lineage. Thus, at least some of the cell types in a taste bud represent distinct lineages of cells and are not merely phenotypic stages as a cell progresses from a young to a mature state.

In vivo Cell Lineage Analysis During Chemical Hepatocarcinogenesis in Rats Using Retroviral-Mediated Gene Transfer: Evidence for Dedifferentiation of Mature Hepatocytes

Feeding adult rats with a diet containing 2-acetylaminofluorene (2-AAF) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Although oval cells may be facultative liver stem cells, the actual relationship between oval cells and liver cancer has not been clearly established in vivo. Our goal was to label hepatic cells in vivo using retroviral vectors and follow their fate during the early steps of chemically induced hepatocarcinogenesis. Oval cell proliferation was induced by continuous feeding with a carcinogenic diet containing 2-AAF. We used two different strategies to genetically label hepatic cells: (a) labeling of proliferating cells in rats fed 2-AAF by injecting recombinant retroviral vectors containing the β-galactosidase gene either in a peripheral vein or in the common bile duct at the peak of oval cell proliferation and (b) prelabeling of hepatocytes by intravenously injecting recombinant vectors 1 day after partial hepatectomy and 1 week before subsequent administration of 2-AAF. Using the first strategy, transgene expression occurred in both oval cells and hepatocytes. Using the second strategy, we could selectively label, and hence study the fate of, differentiated hepatocytes. In the latter case, we observed clusters of β-galactosidase–positive hepatocytes, some of them also expressing preneoplastic markers such as gamma-glutamyl transpeptidase as well as the placental form of glutathione-S-transferase. These results demonstrate that preneoplastic foci can originate from mature hepatocytes and are consistent with the hypothesis that dedifferentiation of mature hepatocytes may occur during the course of carcinogenic regimen.

Analysis of Cell Lineage Relationships in Taste Buds

Taste buds are a heterogeneous population of cells exhibiting diverse morphological and biochemical characteristics. Because taste buds arise from multiple progenitors, the different types of taste cells may represent distinct lineages. The present study was undertaken to determine the following: (1) how many progenitors contribute to a taste bud, and (2) whether the specific subpopulation of serotonin-immunoreactive (IR) taste cells are related by lineage to a restricted set of progenitor cells. These questions were addressed using cell lineage analysis of taste buds from H253 X-inactivation mosaic mice. After random X-inactivation of the <i>lacZ</i>transgene, the tongue of hemizygous female mice displays discrete patches of epithelial cells, which are either β-galactosidase (β-gal) positive or β-gal negative. By analyzing the proportion of the two differently stained cell populations in taste buds located at the boundary between positive and negative epithelial patches, we can determine the minimum number of progenitors that may contribute to the formation of a taste bud. The presence of taste buds containing only 6–12% labeled cells indicates that at least eight progenitors contribute to an average taste bud of 55 cells, assuming progenitors contribute equally to the cell population. Cell lineage analysis of serotonin-IR taste cells in such mixed taste buds suggests that this subpopulation likely arises from only one to two progenitors and often is related by lineage. Thus, at least some of the cell types in a taste bud represent distinct lineages of cells and are not merely phenotypic stages as a cell progresses from a young to a mature state.

A new moss genetics: targeted mutagenesis in Physcomitrella patens.

The potential of moss as a model system to study plant biology is associated with their relatively simple developmental pattern that nevertheless resembles the basic organization of the body plan of land plants, the direct access to cell-lineage analysis, their similar responses to plant growth factors and environmental stimuli as those observed in other land plants, and the dominance of the gametophyte in the life cycle that facilitates genetic approaches. Transformation studies in the moss Physcomitrella patens have revealed a totally unique feature for plants, i.e., that foreign DNA sequences integrate in the genome preferentially at targeted locations by homologous recombination, enabling for the first time in plants the application of the powerful molecular genetic approaches used routinely in bacteria, yeast, and since 1989, the mouse embryonic stem cells. This article reviews our current knowledge of Physcomitrella patens transformation and its unique suitability for functional genomic studies.

Modeling stem cell development by retrospective analysis of gene expression profiles in single progenitor-derived colonies.

The process of development of various cell types is often based on a linear or deterministic paradigm. This is true, for example, for osteoblast development, a process that occurs through the differentiation of a subset of primitive fibroblast progenitors called colony-forming unit-osteoblasts (CFU-Os). CFU-O differentiation has been subdivided into three stages: proliferation, extracellular matrix development and maturation, and mineralization, with characteristic changes in gene expression at each stage. Few analyses have asked whether CFU-O differentiation, or indeed stem cell differentiation in general, may follow more complex and nondeterministic paths, a possibility that may underlie the substantial number of discrepancies in published reports of progenitor cell developmental sequences. We analyzed 99 single colonies of osteoblast stem/primitive progenitor cells cultured under identical conditions. The colonies were analyzed by global amplification poly(A) polymerase chain reaction to determine which of nine genes had been expressed. We used the expression profiles to develop a statistically rigorous map of the cell fate decisions that occur during osteoprogenitor differentiation and show that different developmental routes can be taken to achieve the same end point phenotype. These routes appear to involve both developmental "dead ends" (leading to the expression of genes not correlated with osteoblast-associated genes or the mature osteoblast phenotype) and developmental flexibility (the existence of multiple gene expression routes to the same developmental end point). Our results provide new insight into the biology of primitive progenitor cell differentiation and introduce a powerful new quantitative method for stem cell lineage analysis that should be applicable to a wide variety of stem cell systems.

Ubiquitous expression of goat cyclin T1 in transgenic mice.

Ubiquitous gene expression has a variety of applications in transgenesis that include, for example cell lineage analyses in chimeras and gain-of-function related to xenotransplantations. Although several promoters have already been used to these aims, they often do not reliably or reproducibly target gene expression in mice. We have recently reported the site-independent expression of a bacterial artificial chromosome (BAC)-derived goat alpha-lactalbumin transgene in the mammary gland of mice and the subsequent localisation within the insert of this BAC of the cyclin T1 locus. This ubiquitously expressed gene encodes for a protein that acts as a co-factor for the HIV nuclear transcriptional activator. In the present paper, we report that the goat BAC transgene, which encompasses around 30 kb of the cyclin T1 promoter, also confers ubiquitous expression of this gene in the six transgenic mouse lines analysed. These results suggest that the cyclin T1 promoter could be a useful alternative to target ubiquitous gene expression in transgenics.

In vivo cell lineage analysis in cyproterone acetate–treated rat liver using genetic labeling of hepatocytes

Genetic labeling using recombinant retroviruses is a powerful strategy for the study of cell lineage in the liver. However, this type of vector is only able to infect dividing cells. The synthetic steroid cyproterone acetate (CPA) is mitogenic and carcinogenic in the adult rat liver. In this study, we used retroviral vectors carrying the nuclear targeted β-galactosidase gene to selectively label and follow the fate of hepatocytes dividing on administration of CPA. Labeled cells as well as those in mitosis were preferentially located around the portal tract, whereas apoptotic bodies were predominant in the pericentral area. Labeled hepatocytes did not disappear after apoptosis, suggesting a preferential elimination of nontransduced cells. The presence of labeled binucleated hepatocytes showed the persistence of a binucleation process. Finally, we performed long-term analysis of labeled cells in transgenic animals tolerant for β-galactosidase and treated with 2-acetylaminofluorene (2-AAF) to promote the growth of CPA-initiated hepatocytes. The presence of β-galactosidase–positive hepatocyte clones showed that hepatocytes divided during treatment with 2-AAF. Only 3% of β-galactosidase clones were positive for the placental form of glutathione S-transferase (GSTp), indicating the absence of a preferential appearance of preneoplastic foci in the population of β-galactosidase–labeled hepatocytes. In conclusion, our results show that the mitogenic and tumor-initiating activities of CPA are directed toward different hepatocyte populations. (HEPATOLOGY .)

Pancreatic cell lineage analyses in mice.

Considerable knowledge of the ontogeny of the endocrine pancreas has been gained in recent years, mainly through the use of two complementary genetic approaches in transgenic mice: gene inactivation or overexpression (to assess gene function) and genetic labeling of precursor cells (to determine cell lineages). In recent years, in vivo Cre/loxP-based direct cell tracing experiments show that (i) all pancreatic cells differentiate from pdx1-expressing precursors, (ii) p48 is involved in the exocrine and endocrine pancreatic lineages, (iii) islet endocrine cells derive from ngn3-expressing progenitor cells, and (iv) insulin cells do not derive from glucagon- expressing progenitors. Lineage analyses allow the identification of progenitor cells from which mature cell types differentiate. Once identified, such progenitors can be labeled and isolated, and their differentiation and gene expression profiles studied in vitro. Understanding pancreatic cell lineages is highly relevant for future cell replacement therapies in diabetic patients, helping to define the identity of putative (endodermal) pancreatic stem cells.

Pathways and Fate of Migratory Cells During Late Tooth Organogenesis

Tissue recombination experiments and cell lineage analyses of the developing neural crest have documented the role and central pathways of migratory cells during early craniofacial development. In the present study, regional pathways of cells during late peripheral morphogenesis were investigated using the crown stage tooth organ as a model. Homing targets during tooth integument formation were analyzed to understand the fate of migratory cells involved in late tooth organogenesis and the developmental origin of periodontal tissues. After surgical removal of the oral mucosa, the oral aspect of the dental follicle of lower first mouse molar teeth was labeled using a fluorescent contact dye. Following sacrifice after 0, 2, 4, and 6 days, labeled cells were detected in the dental follicle, in the alveolar bone, and in the periodontal ligament adjacent to the molar root. The distribution of labeled tissues was reconstructed three-dimensionally via confocal microscopy. Using a tooth molar organ culture system, labeled cells within the dental follicle were documented traveling in the apical direction. Our results indicated that cell migration during tooth organogenesis was following specific pathways and that cells within the circumference of the dental follicle were migrating in the apical direction. We speculate that migratory cells passing through the dental follicle connective tissue may contribute to the formation of the periodontium. The present documentation visualizes pathways, role, and dynamics of extensive cell movements during late tooth organogenesis.

Deuterostome evolution: early development in the enteropneust hemichordate, Ptychodera flava.

Molecular and morphological comparisons indicate that the Echinodermata and Hemichordata represent closely related sister-phyla within the Deuterostomia. Much less is known about the development of the hemichordates compared to other deuterostomes. For the first time, cell lineage analyses have been carried out for an indirect-developing representative of the enteropneust hemichordates, Ptychodera flava. Single blastomeres were iontophoretically labeled with Dil at the 2- through 16-cell stages, and their fates followed through development to the tornaria larval stage. The early cleavage pattern of P. flava is similar to that of the direct-developing hemichordate, Saccoglossus kowalevskii, as well as that displayed by indirect-developing echinoids. The 16-celled embryo contains eight animal "mesomeres," four slightly larger "macromeres," and four somewhat smaller vegetal "micromeres." The first cleavage plane was not found to bear one specific relationship relative to the larval dorsoventral axis. Although individual blastomeres generate discrete clones of cells, the appearance and exact locations of these clones are variable with respect to the embryonic dorsoventral and bilateral axes. The eight animal mesomeres generate anterior (animal) ectoderm of the larva, which includes the apical organ; however, contributions to the apical organ were found to be variable as only a subset of the animal blastomeres end up contributing to its formation and this varies from embryo to embryo. The macromeres generate posterior larval ectoderm, and the vegetal micromeres form all the internal, endomesodermal tissues. These blastomere contributions are similar to those found during development of the only other hemichordate studied, the direct-developing enteropneust, S. kowalevskii. Finally, isolated blastomeres prepared at either the two- or the four-cell stage are capable of forming normal-appearing, miniature tornaria larvae. These findings indicate that the fates of these cells and embryonic dorsoventral axial properties are not committed at these early stages of development. Comparisons with the developmental programs of other deuterostome phyla allow one to speculate on the conservation of some key developmental events/mechanisms and propose basal character states shared by the ancestor of echinoderms and hemichordates.

The formation of syncytia within the visceral musculature of the Drosophila midgut is dependent on duf, sns and mbc.

The visceral musculature of the Drosophila midgut consists of an inner layer of circular and an outer layer of longitudinal muscles. Here, we show that the circular muscles are organised as binucleate syncytia that persist through metamorphosis. At stage 11, prior to the onset of the fusion processes, we detected two classes of myoblasts within the visceral trunk mesoderm. One class expresses the founder-cell marker rP298-LacZ in a one- to two-cells-wide strip along the ventralmost part of the visceral mesoderm, whereas the adjacent two to three cell rows are characterised by the expression of Sticks-and-stones (SNS). During the process of cell fusion at stage 12 SNS expression decreases within the newly formed syncytia that spread out dorsally over the midgut. At both margins of the visceral band several cells remain unfused and continue to express SNS. Additional rP298-LacZ-expressing cells arise from the posterior tip of the mesoderm, migrate anteriorly and eventually fuse with the remaining SNS-expressing cells, generating the longitudinal muscles. Thus, although previous studies proposed a separate primordium for the longitudinal musculature located at the posteriormost part of the mesoderm anlage, our cell lineage analyses as well as our morphological observations reveal that a second population of cells originates from the trunk mesoderm. Mutations of genes that are involved in somatic myoblast fusion, such as sns, dumbfounded (duf) or myoblast city (mbc), also cause severe defects within the visceral musculature. The circular muscles are highly unorganised while the longitudinal muscles are almost absent. Thus the fusion process seems to be essential for a proper visceral myogenesis. Our results provide strong evidence that the founder-cell hypothesis also applies to visceral myogenesis, employing the same genetic components as are used in the somatic myoblast fusion processes.

Multiple Inductive Signals Are Involved in the Development of the Ctenophore Mnemiopsis leidyi

Ctenophores possess eight longitudinally arrayed rows of comb plate cilia. Previous intracellular cell lineage analysis has shown that these comb rows are derived from two embryonic lineages, both daughters of the four e1 micromeres (e11 and e12) and a single daughter of the four m1 micromeres (the m12 micromeres). Although isolated e1 micromeres will spontaneously generate comb plates, cell deletion experiments have shown that no comb plates appear during embryogenesis following the removal of e1 descendents. Thus, the m1 lineage requires the inductive interaction of the e1 lineage to contribute to comb plate formation. Here we show that, although m12 cells are normally the only m1 derivatives to contribute to comb plate formation, m11 cells are capable of generating comb plates in the absence m12 cells. The reason that m11 cells do not normally make comb rows may be attributable either to their more remote location relative to critical signaling centers (e.g., e1 descendants) or to inhibitory signals that may be provided by other nearby cells such as sister cells m12. In addition, we show that the signals provided by the e1 lineage are not sufficient for m1-derived comb plate formation. Signals provided by endomesodermal progeny of either the E or the M lineages (the 3E or 2M macromeres) are also required.

Trunk Lateral Cell-Specific Genes of the Ascidian Halocynthia roretzi

Abstract Cell lineage analysis of the ascidian Halocynthia roretzi demonstrated that a pair of cells situated on the right and left sides of 64-cell stage embryos, termed the A7.6 cells, give rise to trunk lateral cells (TLCs) of the tadpole larva and that after metamorphosis TLCs give rise to various mesodermal tissues of the adult, in particular all of the blood cells or coelomic cells. Here we report the isolation and characterization of cDNA clones for two TLC-specific genes HrTLC1 and HrTLC2. HrTLC1 encodes for a novel protein while HrTLC2 encodes for a polypeptide with RNA recognition motifs. Zygotic expression of HrTLC1 and HrTLC2 begins at the neural plate stage and transcripts of both these genes are restricted to TLCs.

A method for the generation of conditional gene repair mutations in mice

Conditional gene repair mutations in the mouse can assist in cell lineage analyses and provide a valuable complement to conditional gene inactivation strategies. We present a method for the generation of conditional gene repair mutations that employs a loxP-flanked (floxed) selectable marker and transcriptional/translational stop cassette (neostop) located within the first intron of a target gene. In the absence of Cre recombinase, expression of the targeted allele is suppressed generating a null allele, while in the presence of Cre, excision of neostop restores expression to wild-type levels. To test this strategy, we have generated a conditional gene repair allele of the mouse Huntington's disease gene homolog (HDH:). Insertion of neostop within the HDH: intron 1 generated a null allele and mice homozygous for this allele resembled nullizygous HDH: mutants and died after embryonic day 8.5. In the presence of a cre transgene expressed ubiquitously early in development, excision of neostop restored HDH: expression and rescued the early embryonic lethality. A simple modification of this strategy that permits the generation of conventional gene knockout, conditional gene knockout and conditional gene repair alleles using one targeting construct is discussed.

Cell Lineage Analysis during Liver Development Using the spfash-Heterozygous Mouse

Biliary epithelial cells differentiate from periportal hepatoblasts during fetal mouse liver development. It remains to be determined whether each hepatoblast is equivalent for differentiation into hepatocytes and biliary epithelial cells in normal liver development. To resolve this question, the mosaic pattern of ornithine transcarbamylase (OTC) expression was analyzed in the hepatoblast population of spfash (sparse-fur with abnormal skin and hair)-heterozygous fetal mouse livers, in which random inactivation of either the X chromosome carrying the spfash gene (causing OTC deficiency) or its wild-type gene occurs. Aggregates (patches) of OTC-positive hepatoblasts showed very complex patterns, and their shapes and size distributions were similar in sections from periportal regions and nonperiportal regions of the fetal liver in which bile duct differentiation by periportal hepatoblasts occurred. Average sizes of periportal patches were larger than those of nonperiportal patches because of the presence of more hemopoietic cells in the latter region. The OTC mosaicism in periportal bile duct progenitors and hepatoblast islands of other liver parenchyma was also similar. These results suggest that the growth patterns of hepatoblasts are similar in both periportal and nonperiportal regions. Isolated three-dimensional patches comprising hepatoblasts giving rise to only biliary epithelial cells or hepatoblasts giving rise to both hepatocytes and biliary epithelial cells were observed in periportal regions. In nonperiportal regions, patches consisting of hepatoblasts differentiating into hepatocytes were also seen. Thus, it is likely that there are three lineages for the developmental fates of hepatoblasts: hepatoblasts giving rise to only biliary epithelial cells, hepatoblasts giving rise to only hepatocytes, and hepatoblasts giving rise to both of them.

Stomatal development and patterning in Arabidopsis leaves

The functional unit for gas exchange between plants and the atmosphere is the stomatal complex, an epidermal structure composed of two guard cells, which delimit a stomatal pore, and their subsidiary cells. In the present work, we define the basic structural unit formed in Arabidopsis thaliana during leaf development, the anisocytic stomatal complex. We perform a cell lineage analysis by transposon excision founding that at least a small percentage of stomatal complexes are unequivocally non‐clonal. We also describe the three‐dimensional pattern of stomata in the Arabidopsis leaf. In the epidermal plane, subsidiary cells of most stomatal complexes contact the subsidiary cells of immediately adjacent complexes. This minimal distance between stomatal complexes allows each stoma to be circled by a full complement of subsidiary cells, with which guard cells can exchange water and ions in order to open or to close the pore. In the radial plane, stomata (and their precursors, the meristemoids) are located at the junctions of several mesophyll cells. This meristemoid patterning may be a consequence of signals that operate along the radial axis of the leaf, which establish meristemoid differentiation precisely at these places. Since stomatal development is basipetal, these radially propagated signals may be transmitted in the axial direction, thus guiding stomatal development through the basal end of the leaf.

Gene transfer into chicken embryos by retrovirus vectors

While chickens have many properties that are advantageous for embryological studies, their genetic analysis has been restricted. However, by using retrovirus vector systems in combination with classical techniques of experimental developmental biology, it has recently become possible to analyze the function of genes involved in the development of this organism. Avian retrovirus vectors are unique in that they can be divided into two categories: replication-competent and replication-defective (replication-incompetent). By choosing the vectors correctly, there are many experimental applications of these vectors such as induction of constitutive (or regulated) gene expression in a restricted region of tissues, organs and embryos; cell lineage analysis; and formation of concentration gradients of morphogens in micromass cultures. In this paper, several retrovirus vectors available for the chicken will be introduced and their applications in developmental biology will be reviewed.

Review: Hepatic Oval Cells: "Knocking on Forbidden Doors"

Hepatic oval “stem” cells were first described in 1956 by E. Farber (1), and are considered to be a cell capable of developing into hepatocytes or bile duct epithelial cells. Other investigators suggest that they are the progeny of a hepatic stem cell. Numerous in vivo and in vitro studies have documented a central role of oval cells in liver biology and carcinogenesis (2–4). In addition to the effects seen in the liver, oval cells (or cells very similar to oval cells) have been implicated in the architecture of the regenerating pancreas after injury (5–6) and they may be involved in the regeneration of other organs as well. While there is a precise purpose for this cell population in the liver, the mechanism(s) by which oval cells activate, proliferate and differentiate are poorly understood. Despite forty years of research and 300 publications on hepatic oval “stem” cells their ontogeny remains a mystery. “Oval cell proliferation” describes the occurrence of nonparenchymal cells detected in large numbers at early stages of carcinogenesis. The population is heterogeneous and contains cells that differ in their differentiation state and potential in their lineage commitment. “Oval cell compartment” is also used to describe the cells invading the parenchyma after the administration of certain carcinogens (7–8). Farber (1991) argued that although oval cells might be the progenitors of hepatocelluar carcinomas (HCC) induced by 3 9 methyl-4 9 -dimethyl-amino-azo-benzene (DMAB), this conclusion can not be generalized to other models of hepatocarcinogenesis (3). It is his view that cell lineage analyses cannot be achieved in a culture system(s) and that the markers used in studies of hepatocarcinogenesis can be considered reliable lineage markers for work done in vivo (3). Farber also reported that in each oval cell compartment model the oval cells will express different isoenzyme profiles, however, certain hepatic alpha-fetoprotien (AFP) and biliary cytokeratin-19 (CK-19) markers will always be expressed (3). In the DMAB model, the single most important conclusion provided by this data is that hepatocytes could be generated from nonparenchymal progenitor cells (3,9). This model establishes the key principle that some type of progenitor cell exists even if it turns out that precursor cell differentiation occurs only infrequently. Evarts et al. (1987 and 1989) show that the oval cell compartment is activated extensively in a 2-Acetylaminofluorene/two thirds partial-hepatecomy (2-AAF/PHx) model. This model is a variation of the SoltFarber protocol (2,10) and has become the industry standard. Figure 1 represents a timeline for this type of protocol. Under these conditions hepatocyte proliferation is heavily suppressed, and it was possible to label oval cells with [3H]-thymidine. This procedure allowed them the ability to follow the fate of the labeled cells. Four to five days after [3H]-thymidine injection, the label was found in basophilic hepatocytes expressing AFP and albumin (2). The authors concluded that there was a precursor-product relationship between the labeled cells of the oval cell compartment and the basophilic hepatocytes. Further studies, presented by the same investigators , show that proliferation of the oval cells and hepatocyte differentiation at early stages of carcinogenesis are associated with the activation of the stellate or Ito cells, which may reg-

New approach to cell lineage analysis in mammals using the Cre-loxP system.

The Cre-loxP site-specific recombination system was used for cell lineage analysis in mammals. We constructed an expression plasmid, pCETZ-17, which consists of cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a portion of the rabbit beta-globin gene, loxP-flanked DNA sequence (containing enhanced green fluorescent protein (EGFP) cDNA), and lacZ gene encoding E. coli beta-galactosidase (beta-gal). When circular pCETZ-17 plasmid DNA was microinjected into the pronuclei of fertilized eggs and these eggs were allowed to develop to two-cell stage, 62.8% (59/94) of the two-cell embryos exhibited distinct fluorescence in one or both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X-Gal, a substrate for beta-gal. When both circular plasmids, pCETZ-17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co-injected into fertilized eggs, almost all (87.0%, 47/54) embryos exhibited low or no fluorescence, but 51.9% (27/52) exhibited positive staining for beta-gal activity. This indicates that transient expression of the Cre recombinase gene removed the loxP-flanked DNA sequence in pCETZ-17 and then caused expression of the downstream lacZ sequence. We next microinjected pCETZ-17 into the pronuclei of fertilized eggs, cultured these injected eggs for 1 day, and collected only two-cell embryos expressing EGFP in both blastomeres. One blastomere of the EGFP-expressing two-cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 1 day up to four-cell stage. When the developing four-cell embryos were subjected to staining with X-Gal, cell lineage-related staining pattern for beta-gal activity was observed in most (77.8%, 7/9) embryos. These findings were further confirmed using two-cell embryos derived from a transgenic mouse line carrying CETZ-17 transgene. Thus, our system, which is based on transient expression of the Cre recombinase gene directly introduced into nuclei of embryonic cells by microinjection, is a powerful means for cell lineage analysis in mammals.

Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis.

Neural crest cells are multipotential stem cells that contribute extensively to vertebrate development and give rise to various cell and tissue types. Determination of the fate of mammalian neural crest has been inhibited by the lack of appropriate markers. Here, we make use of a two-component genetic system for indelibly marking the progeny of the cranial neural crest during tooth and mandible development. In the first mouse line, Cre recombinase is expressed under the control of the Wnt1 promoter as a transgene. Significantly, Wnt1 transgene expression is limited to the migrating neural crest cells that are derived from the dorsal CNS. The second mouse line, the ROSA26 conditional reporter (R26R), serves as a substrate for the Cre-mediated recombination. Using this two-component genetic system, we have systematically followed the migration and differentiation of the cranial neural crest (CNC) cells from E9.5 to 6 weeks after birth. Our results demonstrate, for the first time, that CNC cells contribute to the formation of condensed dental mesenchyme, dental papilla, odontoblasts, dentine matrix, pulp, cementum, periodontal ligaments, chondrocytes in Meckel's cartilage, mandible, the articulating disc of temporomandibular joint and branchial arch nerve ganglia. More importantly, there is a dynamic distribution of CNC- and non-CNC-derived cells during tooth and mandibular morphogenesis. These results are a first step towards a comprehensive understanding of neural crest cell migration and differentiation during mammalian craniofacial development. Furthermore, this transgenic model also provides a new tool for cell lineage analysis and genetic manipulation of neural-crest-derived components in normal and abnormal embryogenesis.