Comparative gene expression analysis and fate mapping studies suggest an early segregation of cardiogenic lineages in Xenopus laevis

Retrospective clonal analysis in mice suggested that the vertebrate heart develops from two sources of cells called first and second lineages, respectively. Cells of the first lineage enter the linear heart tube and initiate terminal differentiation earlier than cells of the second lineage. It is thought that both heart lineages arise from a common progenitor cell population prior to the cardiac crescent stage (E7.5 of mouse development). The timing of segregation of different lineages as well as the molecular mechanisms underlying this process is not yet known. Furthermore, gene expression data for those lineages are very limited. Here we provide the first comparative study of cardiac marker gene expression during Xenopus laevis embryogenesis complemented by single cell RT-PCR analysis. In addition we provide fate mapping data of cardiac progenitor cells at different stages of development. Our analysis indicates an early segregation of cardiac lineages and a fairly complex heterogeneity of gene expression in the cardiac progenitor cells. Furthermore, this study sets a reference for all further studies analyzing cardiac development in X. laevis.     

Interspecific cell markers and lineage in mammals

Study of cell lineage in the mammalian embryo has relied heavily on the use of chimeras to follow the fate of genetically marked cells in later development. Such studies have often been limited by the types of genetic markers available; there are very few markers that allow analysis of the spatial distribution of individual cells at all stages of development. We have developed a marker system that is based on the identification of cells of Mus musculus origin in M. musculus-M. caroli chimeras by in situ DNA-DNA hybridization using a cloned probe to M. musculus satellite DNA. This provides the first ubiquitous in situ cell marker system for mammalian chimeras. We have recently refined the system by the use of biotin-labelled probes and detection of hybridization by streptavidin-peroxidase binding. This increases both the speed and the resolution of the assay. We have used the marker for cell lineage analysis in both embryonic and adult chimeras and results from analysis of the derivatives of early cell lineages in later development and study of coherent growth versus cell mixing in the postimplantation embryo are presented. The importance of understanding embryonic cell lineages as a prelude to molecular studies is emphasized.     

L-MBP is expressed in epithelial cells of mouse small intestine

The mannan-binding proteins (L-MBP and S-MBP, also denoted MBL-C and MBL-A), mainly produced in liver and existing in liver and serum, play important roles in the innate immunity against a variety of pathogens. Total RNA from mouse tissues were screened for MBP mRNA by RT-PCR. In addition to liver, S-MBP mRNA was detected in lung, kidney, and testis, and L-MBP mRNA was detected in kidney, thymus, and small intestine. Quantitative RT-PCR revealed that the small intestine is a predominant site of extrahepatic expression of L-MBP. Western blotting with polyclonal Abs against rat L-MBP demonstrated this protein in Triton X-100 extracts of the small intestine obtained from mice that had undergone systemic perfusion. Immunohistochemical staining with an mAb against mouse L-MBP and in situ hybridization revealed that L-MBP is selectively expressed in some villous epithelial cells of the small intestine. These findings suggest that L-MBP plays a role in mucosal innate immunity.     

A stop-EGFP transgenic mouse to detect clonal cell lineages generated by mutation

The investigation of cell lineages and clonal organization in tissues is facilitated by techniques that allow labelling of clonal cell lineages. Here, we describe a novel transgenic mouse that allows clonal cell lineages to be traced in virtually any tissue. A green fluorescent cell lineage is generated by a random mutation at an enhanced green fluorescent protein gene that carries a premature stop codon, ensuring clonality. The transgenic system allows efficient detection of mutations and stem-cell fate mapping in the epidermis using live mice, as well as in the kidney and liver post-mortem. Cell lineages that descended from single epidermal stem cells were found to be capable of generating three adjacent corneocytes using the system, providing evidence for horizontal migration of epidermal cells between epidermal proliferative units (EPUs), in contrast to the classical EPU model. The transgenic mouse system is expected to provide a novel tool for stem-cell lineage studies.     

SV40 T antigen induced chromosomal changes reflect a process that is both clastogenic and aneuploidogenic and is ongoing throughout neoplastic progression of human fibroblasts.

In human fibroblasts, the expression of SV40 large T antigen is known to cause a variety of chromosomal aberrations and especially dicentric chromosomes. In some cases, the later aberrations have been reported to be reversible telomeric associations. We report here aberration and chromosome number studies of twenty-nine T antigen positive lineages, studied from their initiation by transfection of T antigen sequences into human diploid fibroblasts, until crisis or immortalization occurred or, in some cases until the lines became tumorigenic in nude mice. The data show that T antigen consistently produced chromosomal instability of both number and structure by an active process that began before transformation indicators were positive and continued throughout neoplastic progression. The most frequently observed aberrations were dicentric chromosomes, which were shown to be true dicentrics by examination by in situ hybridization with telomeric sequences. These data are consistent with the hypothesis that T antigen causes human fibroblasts to become neoplastically transformed by successive rounds of chromosomal mutation and lineage evolution.     

Intercellular Interactions,Position, and Polarity in Establishing Blastocyst Cell Lineages and Embryonic Axes

The formation of the three lineages of the mouse blastocyst provides a powerful model system to study interactions among cell behavior, cell signaling, and lineage development. Hippo signaling differences between the inner and outer cells of the early cleavage stages, combined with establishment of a stably polarized outer epithelium, lead to the establishment of the inner cell mass and the trophectoderm, whereas FGF signaling differences among the individual cells of the ICM lead to gradual separation and segregation of the epiblast and primitive endoderm lineages. Events in the late blastocyst lead to the formation of a special subset of cells from the primitive endoderm that are key sources for the signals that establish the subsequent body axis. The slow pace of mouse early development, the ability to culture embryos over this time period, the increasing availability of live cell imaging tools, and the ability to modify gene expression at will are providing increasing insights into the cell biology of early cell fate decisions.     

Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse.

The proto-oncogene int-2 has been implicated in the formation of mouse mammary-tumour-virus-induced mammary tumours. Analysis of the predicted coding sequence indicates that int-2 is a member of the fibroblast growth factor family. Previous studies using Northern blot analysis suggested that normal expression of int-2 may be confined to extra-embryonic endoderm lineages of embryonic stages of mouse development. We have used in situ hybridization and Northern blot analysis to examine directly int-2 expression in embryo stem cells and in the developing embryo from early gastrulation to midsomite stages. Complex patterns of accumulation of int-2 RNA were observed in embryonic and extra-embryonic tissues. The data suggest multiple roles for int-2 in development which may include migration of early mesoderm cells and induction of the otocyst.     

Localization of actin messenger RNA during early ascidian development

The spatial distribution of RNA sequences during early development of the ascidian, Styela plicata, was determined by in situ hybridization with poly(U) and cloned DNA probes. Styela eggs and embryos contain three colored cytoplasmic regions of specific morphogenetic fates, the ectoplasm, endoplasm, and myoplasm. These cytoplasmic regions participate in ooplasmic segregation after fertilization and are distributed to different cell lineages during early embryogenesis. n situ hybridization with poly(U) suggests that poly(A)+RNA is unevenly distributed in eggs and embryos, with about 45% in the ectoplasm, 50% in the endoplasm, and only 5% in the myoplasm. In situ hybridization with a histone DNA probe showed that histone RNA sequences were not localized in eggs or embryos and distributed between the three cytoplasmic regions according to their volumes. In situ hybridization with an actin DNA probe showed actin RNA was localized in the myoplasm and ectoplasm of eggs and embryos with about 45% present in the myoplasm, 40% in the ectoplasm, and only 15% in the endoplasm. These results suggest that a large proportion of the egg actin mRNA is localized in the myoplasm, participates in ooplasmic segregation after fertilization, and is differentially distributed to the mesodermal cell lineages during embryogenesis. Analysis of the translation products of egg mRNA suggests that the localized mRNA codes for a cytoplasmic actin isoform.     

Identification of a putative intestinal stem cell and early lineage marker; musashi-1

There are few reliable markers for adult stem cells and none for those of the intestinal epithelium. Previously, indirect experimental approaches have predicted stem cell position and numbers. The Musashi-1 (Msi-1) gene encodes an RNA binding protein associated with asymmetric divisions in neural progenitor cells. Two-day-old, adult, and 4.5 h, 1-, 2-, 4- and 12-day post-irradiation samples of BDF1 mouse small intestine, together with some samples of mouse colon were stained with a rat monoclonal antibody to Musashi-1 (14 H-1). Min ( + / - ) mice with small intestinal adenomas of varying sizes were also analysed. Samples of human small and large bowel were also studied but the antibody staining was weak. Musashi-1 expression was observed using immunohistochemistry in neonatal, adult, and regenerating crypts with a staining pattern consistent with the predicted number and distribution of early lineage cells including the functional stem cells in these situations. Early dysplastic crypts and adenomas were also strongly Musashi-1 positive. In situ hybridization studies showed similar expression patterns for the Musashi mRNA and real-time quantitative RT-PCR showed dramatically more Msi-1 mRNA expression in Min tumours compared with adjacent normal tissue. These observations suggest that Musashi-1 is a marker of stem and early lineage progenitor cells in murine intestinal tissue.     

Balancing on the crest – Evidence for disruption of the enteric ganglia via inappropriate lineage segregation and consequences for gastrointestinal function

Normal enteric nervous system (ENS) development relies on numerous factors, including appropriate migration, proliferation, differentiation, and maturation of neural crest (NC) derivatives. Incomplete rostral to caudal migration of enteric neural crest-derived progenitors (ENPs) down the gut is at least partially responsible for the absence of enteric ganglia that is a hallmark feature of Hirschsprung disease (HSCR). The thought that ganglia proximal to aganglionosis are normal has guided surgical procedures for HSCR patients. However, chronic gastrointestinal dysfunction suffered by a subset of patients after surgery as well as studies in HSCR mouse models suggest that aberrant NC segregation and differentiation may be occurring in ganglionated regions of the intestine. Studies in mouse models that possess enteric ganglia throughout the length of the intestine (non-HSCR) have also found that certain genetic alterations affect neural crest lineage balance and interestingly many of these mutants also have functional gastrointestinal (GI) defects. It is possible that many GI disorders can be explained in part by imbalances in NC-derived lineages. Here we review studies evaluating ENS defects in HSCR and non-HSCR mouse models, concluding with clinical implications while highlighting areas requiring further study.     

Proenkephalin A is expressed in mesodermal lineages during organogenesis.

Proenkephalin A (PEA) encodes several neuropeptides with an opioid activity, as well as other peptides with as yet unknown functions. As an initial step toward finding possible roles for PEA gene products in non-neuronal tissues, we have determined sites of PEA expression during mouse embryonic development, employing in situ hybridization. We report here the unexpected observation that in addition to its abundance in brain, PEA RNA is expressed in non-differentiated mesodermal cells of diverse lineages in the process of their development into several adult tissues and organs; it drops to undetectable levels upon terminal differentiation of these tissues. In a particular example of differentiating mesoderm, the developing kidney, the transient expression of PEA mRNA and of its encoded peptide Met-enkephalin was demonstrated by both in situ and Northern blot hybridizations, as well as by a radioimmunoassay. These findings suggest a novel role for PEA-derived peptide(s) in mesoderm growth or differentiation during organogenesis.     

The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.     

Detection of casein fragments in an invertebrate and in a vertebrate using in situ hybridization

Using a non-radioactive in situ hybridization procedure it has been demonstrated that both invertebrates such as the mollusc Mytilus galloprovincialis and lower vertebrates such as the fish Cyprinus carpio express bovine alphaS1- and kappa-casein homologous mRNAs. In particular, positive results were found in molluscan immunocytes, and in cells located in different fish tissues: intestine, endocrine pancreas and kidney. These findings suggest that the casein genes are highly conserved throughout evolution.     

Rab17, a novel small GTPase, is specific for epithelial cells and is induced during cell polarization

The rab subfamily of small GTPases has been demonstrated to play an important role in the regulation of membrane traffic in eukaryotic cells. Compared with nonpolarized cells, epithelial cells have distinct apical and basolateral transport pathways which need to be separately regulated. This raises the question whether epithelial cells require specific rab proteins. However, all rab proteins identified so far were found to be equally expressed in polarized and nonpolarized cells. Here we report the identification of rab17, the first epithelial cell- specific small GTPase. Northern blot analysis on various mouse organs, revealed that the rab17 mRNA is present in kidney, liver, and intestine but not in organs lacking epithelial cells nor in fibroblasts. To determine whether rab17 is specific for epithelial cells we studied its expression in the developing kidney. We found that rab17 is absent from the mesenchymal precursors but is induced upon their differentiation into epithelial cells. In situ hybridization studies on the embryonic kidney and intestine revealed that rab17 is restricted to epithelial cells. By immunofluorescence and immunoelectron microscopy on kidney sections, rab17 was localized to the basolateral plasma membrane and to apical tubules. Rab proteins associated with two distinct compartments have been found to regulate transport between them. Therefore, our data suggest that rab17 might be involved in transcellular transport.     

Epithelial lineages of the small intestine have unique patterns of GATA expression

The ability of the GATA family of factors to interact with numerous other factors, co-factors, and repressors suggests that they may play key roles in tissues and cells where they are expressed. Adult mouse small intestine has been shown to express GATA-4, GATA-5, and GATA-6, where they have been implicated in the activation of a number of intestinal genes. Determination of which GATA factor(s) are involved in a specific function in tissues expressing multiple family members has proven difficult. The immunohistochemical analysis presented here demonstrate that within the mouse small intestine GATA-4/-5/-6 are found to be uniquely distributed among the various differentiated lineages of the intestinal epithelium. Among differentiated cells GATA-4 is found only in the villous enterocytes. GATA-5 is absent from enterocytes, but was found in the remaining lineages: goblet, Paneth and enteroendocrine. Additionally, high levels of GATA-6 are found in only one of these differentiated cell types, the enteroendocrine lineage. The observed distribution suggests that the GATA factors may have distinct roles in lineage allocation, lineage maintenance, and/or terminal differentiation events in small intestine.     

Expression of an Nkx3.1-CRE gene using ROSA26 reporter mice

The genetic locus of Nkx3.1, an early murine marker of sclerotome and prostate development, was disrupted by a knock in of CRE recombinase via homologous recombination in embryonic stem cells. Cell fate mapping revealed previously unidentified cell lineages expanded from Nkx3.1-expressing cell populations and recapitulated reported Nkx3.1 expression patterns. In lineage trace experiments of E18.5 Nkx3.1-CRE; R26R embryos novel staining was observed in areas of the lungs, portions of the duodenum, and vertebral elements of the skeleton. beta-galactosidase activity measured in Nkx3.1-CRE; R26R and Nkx3.2-CRE; R26R embryos was observed in overlapping regions of the sclerotome but no apparent change in Nkx3.1 expression was seen in the Nkx3.2 mutants by in situ hybridization.