Growth factors, signaling pathways, and the regulation of proliferation and differentiation in BC3H1 muscle cells. I. A pertussis toxin-sensitive pathway is involved.

Cells of the nonfusing muscle cell line BC3H1 stop proliferating and express a family of muscle-specific proteins when the FBS concentration is reduced from 20 to 0.5% (Munson, R., K.L. Caldwell, and L. Glaser. 1982. J. Cell Biol. 92:350-356). Several growth factors have been shown to block differentiation in this cell line. To begin to investigate the potential role of G proteins in signal transducing pathways from these receptors, we have examined the effects of cholera toxin (CT) and pertussis toxin (PT) on proliferation and differentiation in BC3H1 cells. PT specifically ADP ribosylates a protein with an apparent molecular mass of 40 kD in BC3H1 cell membranes, whereas CT specifically ADP ribosylates three proteins of 35-43 kD. When added to exponentially growing cells in 20% FBS, CT and PT inhibited [3H]thymidine incorporation by up to 75% in a dose-dependent fashion. We found the synthesis of creatine kinase (CK) and skeletal muscle myosin light chain was reversibly induced in cells in 20% FBS treated with PT, but no increased synthesis was seen in cells treated with CT or in control cells; Northern analysis indicated this induction was at the level of mRNA. In cells shifted to 0.5% FBS, CT inhibited the normally induced synthesis of CK whereas PT potentiated it by approximately 50%. Forskolin also inhibited growth in 20% FBS and differentiation in 0.5% FBS medium in a dose-dependent fashion. both forskolin and CT elevated cAMP levels compared with control or PT-treated cells, suggesting that CT is blocking proliferation and differentiation by elevating cAMP levels. These results establish that a PT-sensitive pathway is involved in regulating proliferation and differentiation in BC3H1 cells, and we postulate that PT functions by ADP ribosylating a G protein that transduces signals from growth factor receptors in these cells.

New insights into segmentation and patterning during vertebrate somitogenesis.

Understanding how muscles and the skeleton develop is essential to understanding how distinct form arises in different vertebrate organisms. The meristic somites give rise to the serially-repeated precursors of the axial skeleton and musculature. Recent studies of somitogenesis have shed light upon the mechanisms of segmentation in vertebrates and the relationship between segmentation and patterning. Significant advances have also been made in connecting signalling molecules that possess somite patterning activity with downstream effector genes.

Fibroblast growth factors in mammalian development.

Polypeptide growth factors are secreted signalling molecules that function as intercellular communicators. Detailed analyses of the expression and function of members of the fibroblast growth factor (FGF) family and their recepotors have demonstrated that the FGF signalling pathways play essential roles in regulating cellular proliferation, differentiation and tissue patterning during vertebrate embryogenesis. Recent studies on the molecular basis of human dysmorphic syndromes have revealed that aberrant FGF signalling during limb and skeletal development can lead to pathogenesis.

Heads or tails: Wnts and anterior-posterior patterning.

Cell-cell communication is critical during embryogenesis for organizing the vertebrate body plan. Members of the Wnt family of secreted signaling molecules possess axis-inducing and posteriorizing activity when overexpressed. Wnt signals are modulated extracellularly by a diverse group of secreted Wnt antagonists and cofactors. Recent work has revealed that inhibition of posteriorly localized Wnt signaling by anteriorly localized Wnt antagonists is critical for inducing the anterior structures, forebrain and heart, from neural ectoderm and mesoderm, respectively. This review centers on the role that Wnts and Wnt antagonists play in the patterning of the vertebrate anterior-posterior axis.

tek, a novel tyrosine kinase gene located on mouse chromosome 4, is expressed in endothelial cells and their presumptive precursors.

A search for protein tyrosine kinases expressed during murine cardiogenesis resulted in the isolation of a novel tyrosine kinase, designated tek, which maps to mouse chromosome 4 between the brown and pmv-23 loci. The deduced amino acid sequence of tek predicts that it encodes a putative receptor tyrosine kinase that contains a 21 amino acid kinase insert and which is most closely related in its catalytic domains to FGFR1 and the product of the ret proto-oncogene. In situ hybridization analysis of adult tissues, as well as sectioned and whole-mount embryos, showed that tek is specifically expressed in the endocardium, the leptomeninges and the endothelial lining of the vasculature from the earliest stages of their development. Moreover, examination of the morphology of tek-expressing cells, and staging of tek expression relative to that of the endothelial cell marker von Willebrand factor, revealed that tek is expressed prior to von Willebrand factor and appears to mark the embryonic progenitors of mature endothelial cells. tek encodes a novel putative receptor tyrosine kinase that may be critically involved in the determination and/or maintenance of cells of the endothelial lineage.

Segmentation of whole cells and cell nuclei from 3-D optical microscope images using dynamic programming.

Communications between cells in large part drive tissue development and function, as well as disease-related processes such as tumorigenesis. Understanding the mechanistic bases of these processes necessitates quantifying specific molecules in adjacent cells or cell nuclei of intact tissue. However, a major restriction on such analyses is the lack of an efficient method that correctly segments each object (cell or nucleus) from 3-D images of an intact tissue specimen. We report a highly reliable and accurate semi-automatic algorithmic method for segmenting fluorescence-labeled cells or nuclei from 3-D tissue images. Segmentation begins with semi-automatic, 2-D object delineation in a user-selected plane, using dynamic programming (DP) to locate the border with an accumulated intensity per unit length greater that any other possible border around the same object. Then the two surfaces of the object in planes above and below the selected plane are found using an algorithm that combines DP and combinatorial searching. Following segmentation, any perceived errors can be interactively corrected. Segmentation accuracy is not significantly affected by intermittent labeling of object surfaces, diffuse surfaces, or spurious signals away from surfaces. The unique strength of the segmentation method was demonstrated on a variety of biological tissue samples where all cells, including irregularly shaped cells, were accurately segmented based on visual inspection.

fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation.

Experiments in amphibians have implicated fibroblast growth factors (FGFs) in the generation and patterning of mesoderm during embryogenesis. We have mutated the gene for fibroblast growth factor receptor 1 (fgfr-1) in the mouse to genetically dissect the role of FGF signaling during development. In the absence of fgfr-1 signaling, embryos displayed early growth defects; however, they remained capable of gastrulating and generating mesoderm. The nascent mesoderm of fgfr-1 homozygous mutant embryos differentiated into diverse mesodermal subtypes, but mesodermal patterning was aberrant. Somites were never generated and axial mesoderm was greatly expanded at the expense of paraxial mesoderm. These results suggest that FGFR-1 transduces signals that specify mesodermal cell fates and regional patterning of the mesoderm during gastrulation.

Expression of the fibroblast growth factor receptor FGFR-1/flg during gastrulation and segmentation in the mouse embryo.

Recent evidence from studies in both amphibians and mammals suggest that the fibroblast growth factor (FGF) family of signaling molecules and their receptors may play regulatory roles during early embryogenesis. We have used both standard and whole-mount in situ hybridization techniques to analyze the temporal and spatial expression patterns of the murine fibroblast growth factor receptor-1 (FGFR-1) in order to help define the role of FGFs in the processes of gastrulation and segmentation. FGFR-1 transcripts were detected in the primitive ectoderm of the egg cylinder embryo but not in the primitive endoderm or ectoplacental cone. During gastrulation, FGFR-1 mRNA were expressed at high levels in the migrating embryonic mesoderm of the mid-streak-stage embryo. Late-streak-stage embryos displayed strong expression in both the embryonic ectoderm and mesoderm. Within the ectodermal lineage, FGFR-1 mRNA later became localized to the neural ectoderm during its formation and continued to be expressed at high levels throughout neural development. In the mesodermal lineage, FGFR-1 transcripts became concentrated in the posterior medial mesoderm of the embryo as it condensed to form paraxial mesoderm. The most striking expression patterns were observed before and during segmentation where FGFR-1 was strongly expressed in the presomitic mesoderm and the rostral half of the newly formed somites. The patterns of expression are consistent with a role for FGFR-1 in posterior mesoderm formation. FGFR-1 may also play significant roles in the formation of neural ectoderm and the early events that establish compartments within the developing somites.

flk-1, an flt-related receptor tyrosine kinase is an early marker for endothelial cell precursors.

We have used RT-PCR to screen pluripotent murine embryonic stem cells to identify receptor tyrosine kinases (RTKs) potentially involved in the determination or differentiation of cell lineages during early mouse development. Fourteen different tyrosine kinase sequences were identified. The expression patterns of four RTKs have been examined and all are expressed in the mouse embryo during, or shortly after, gastrulation. We report here the detailed expression pattern of one such RTK, the flt-related gene flk-1. In situ hybridization analysis of the late primitive streak stage embryo revealed that flk-1 was expressed in the proximal-lateral embryonic mesoderm; tissue fated to become heart. By headfold stages, staining was confined to the endocardial cells of the heart primordia as well as to the blood islands of the visceral yolk sac and the developing allantois. Patchy, speckled staining was detected in the endothelium of all the major embryonic and extraembryonic blood vessels as they formed. During early organogenesis, expression was detected in the blood vessels of highly vascularized tissues such as the brain, liver, lungs and placenta. Since flk-1 was expressed in early mesodermal cells prior to any morphological evidence for endothelial cell differentiation (vasculogenesis), as well as in cells that form blood vessels from preexisting ones (angiogenesis), it appears to be a very early marker of endothelial cell precursors. We have previously reported that another novel RTK, designated tek, was expressed in differentiating endothelial cells. We show here that flk-1 transcripts are expressed one full embryonic day earlier than the first tek transcripts. The expression of these two RTKs appear to correlate with the specification and early differentiation of the endothelial cell lineage respectively, and therefore may play important roles in the establishment of this lineage.

Expression analysis of a Notch homologue in the mouse embryo.

The Drosophila Notch gene has been shown to be involved in the determination of fate in a number of different cell types. Similarly, Notch homologues in Caenorhabditis elegans are involved in cell decision-making steps. It is of interest to determine if a mammalian Notch homologue plays a role in cell fate determination. We have isolated cDNA from a mouse Notch gene using low-stringency hybridization with probes derived from the Xenopus Notch gene. Sequence analysis reveals that this gene possesses EGF repeats, Notch/lin-12 repeats, and CDC-10/SWI-6 repeats, characteristic of other Notch homologues. Northern analysis revealed that the transcript size was roughly 10 kb as has been found for the other Notch genes. We have studied the expression pattern of the gene by both conventional and whole mount in situ hybridization. Expression patterns were consistent with mouse Notch having a determinative role in the formation of mesoderm, somites, and the nervous system.

Chimeric analysis of fibroblast growth factor receptor-1 (Fgfr1) function: a role for FGFR1 in morphogenetic movement through the primitive streak.

Fibroblast growth factor (FGF) signaling has been implicated in the patterning of mesoderm and neural lineages during early vertebrate development. In the mouse, FGF receptor-1 (FGFR1) is expressed in an appropriate spatial and temporal manner to be orchestrating these functions. Mouse embryos homozygous for a mutated Fgfr1 allele (fgfr1(delta tmk)) die early in development, show abnormal growth and aberrant mesodermal patterning. We have performed a chimeric analysis to further study FGFR1 function in the morphogenesis and patterning of the mesodermal germ layer at gastrulation. At E9.5, fgfr1(delta tmk)/fgfr1(delta tmk) cells showed a marked deficiency in their ability to contribute to the extra-embryonic, cephalic, heart, axial and paraxial mesoderm, and to the endoderm of chimeric embryos. Analysis at earlier stages of development revealed that fgfr1(delta tmk)/fgfr1(delta tmk) cells accumulated within the primitive streak of chimeric embryos, and consequently failed to populate the anterior mesoderm and endodermal lineages at their inception. We suggest that the primary defect associated with the fgfr1(delta tmk) mutation is a deficiency in the ability of epiblast cells to traverse the primitive streak. fgfr1(delta tmk)/fgfr1(delta tmk) cells that accumulated within the primitive streak of chimeric embryos tended to form secondary neural tubes. These secondary neural tubes were entirely fgfr1(delta tmk)/fgfr1(delta tmk) cell derived. The adoption of ectopic neural fate suggests that normal morphogenetic movement through the streak is essential not only for proper mesodermal patterning but also for correct determination of mesodermal/neurectodermal cell fates.

A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo.

Morphogenesis depends on the precise control of basic cellular processes such as cell proliferation and differentiation. Wnt5a may regulate these processes since it is expressed in a gradient at the caudal end of the growing embryo during gastrulation, and later in the distal-most aspect of several structures that extend from the body. A loss-of-function mutation of Wnt5a leads to an inability to extend the A-P axis due to a progressive reduction in the size of caudal structures. In the limbs, truncation of the proximal skeleton and absence of distal digits correlates with reduced proliferation of putative progenitor cells within the progress zone. However, expression of progress zone markers, and several genes implicated in distal outgrowth and patterning including Distalless, Hoxd and Fgf family members was not altered. Taken together with the outgrowth defects observed in the developing face, ears and genitals, our data indicates that Wnt5a regulates a pathway common to many structures whose development requires extension from the primary body axis. The reduced number of proliferating cells in both the progress zone and the primitive streak mesoderm suggests that one function of Wnt5a is to regulate the proliferation of progenitor cells.

T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification.

Wnt3a encodes a signal that is expressed in the primitive streak of the gastrulating mouse embryo and is required for paraxial mesoderm development. In its absence cells adopt ectopic neural fates. Embryos lacking the T-box-containing transcription factors, Brachyury or Tbx6, also lack paraxial mesoderm. Here we show that Brachyury is specifically down-regulated in Wnt3a mutants in cells fated to form paraxial mesoderm. Transgenic analysis of the T promoter identifies T (Brachyury) as a direct transcriptional target of the Wnt signaling pathway. Our results suggest that Wnt3a, signaling via Brachyury, modulates a balance between mesodermal and neural cell fates during gastrulation.

Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice.

The receptor tyrosine kinase Flk-1 (ref. 1) is believed to play a pivotal role in endothelial development. Expression of the Flk-1 receptor is restricted to endothelial cells and their embryonic precursors, and is complementary to that of its ligand, vascular endothelial growth factor (VEGF), which is an endothelial-specific mitogen. Highest levels of flk-1 expression are observed during embryonic vasculogenesis and angiogenesis, and during pathological processes associated with neovascularization, such as tumour angiogenesis. Because flk-1 expression can be detected in presumptive mesodermal yolk-sac blood-island progenitors as early as 7.0 days postcoitum, Flk-1 may mark the putative common embryonic endothelial and haematopoietic precursor, the haemangioblast, and thus may also be involved in early haematopoiesis. Here we report the generation of mice deficient in Flk-1 by disruption of the gene using homologous recombination in embryonic stem (ES) cells. Embryos homozygous for this mutation die in utero between 8.5 and 9.5 days post-coitum, as a result of an early defect in the development of haematopoietic and endothelial cells. Yolk-sac blood islands were absent at 7.5 days, organized blood vessels could not be observed in the embryo or yolk sac at any stage, and haematopoietic progenitors were severely reduced. These results indicate that Flk-1 is essential for yolk-sac blood-island formation and vasculogenesis in the mouse embryo.

A model for the modulation of muscle cell determination and differentiation by growth factors.

In an adult organism three principal types of muscle tissue can be found: skeletal, smooth, and cardiac. While each display subtle differences, for the most part they express a common set of genes that are representative of differentiated muscle. Several in vitro muscle cell lines have provided clues as to how the developmental programs of muscle cell proliferation, determination, and differentiation are controlled. In this paper we will explore recent advances in our understanding of how growth factors, acting through specific signal transduction pathways, control muscle gene expression. The transcription of muscle genes is controlled by specific cis-acting regulatory sequences. We will discuss how growth factors may exert their effects on muscle genes by modulating the expression of nuclear DNA-binding proteins that directly regulate muscle gene expression.