Switching on lineage tracers using site-specific recombination.
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Animals↗
Biomedical subjects
S M Dymecki
Publications and source records attributed to S M Dymecki.
Origin of the precerebellar system.
The precerebellar system provides the principal input to the cerebellum and is essential for coordinated motor activity. Using a FLP recombinase-based fate mapping approach, we provide direct evidence in the mouse that this ventral brainstem system derives from dorsally located rhombic neuroepithelium. Moreover, by fate mapping at the resolution of a gene expression pattern, we have uncovered an unexpected subdivision within the precerebellar primordium: embryonic expression of Wnt1 appears to identify the class of precerebellar progenitors that will later project mossy fibers from the brainstem to the cerebellum, as opposed to the class of precerebellar neurons that project climbing fibers. Differential gene expression therefore appears to demarcate two populations within the precerebellar primordium, grouping progenitors by their future type of axonal projection and synaptic partner rather than by final topographical position.
Animals↗
High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP.
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Amino Acid Substitution↗
Combinatorial signaling through BMP receptor IB and GDF5: shaping of the distal mouse limb and the genetics of distal limb diversity.
In this study, we use a mouse insertional mutant to delineate gene activities that shape the distal limb skeleton. A recessive mutation that results in brachydactyly was found in a lineage of transgenic mice. Sequences flanking the transgene insertion site were cloned, mapped to chromosome 3, and used to identify the brachydactyly gene as the type IB bone morphogenetic protein receptor, BmprIB (ALK6). Expression analyses in wild-type mice revealed two major classes of BmprIB transcripts. Rather than representing unique coding RNAs generated by alternative splicing of a single pro-mRNA transcribed from one promoter, the distinct isoforms reflect evolution of two BmprIB promoters: one located distally, driving expression in the developing limb skeleton, and one situated proximally, initiating transcription in neural epithelium. The distal promoter is deleted in the insertional mutant, resulting in a regulatory allele (BmprIB(Tg)) lacking cis-sequences necessary for limb BmprIB expression. Mutants fail to generate digit cartilage, indicating that BMPRIB is the physiologic transducer for the formation of digit cartilage from the skeletal blastema. Expansion of BmprIB expression into the limb through acquisition of these distal cis-regulatory sequences appears, therefore, to be an important genetic component driving morphological diversity in distal extremities. GDF5 is a BMP-related signal, which is also required for proper digit formation. Analyses incorporating both Gdf5 and BmprIB(Tg) alleles revealed that BMPRIB regulates chondrogenesis and segmentation through both GDF5-dependent and -independent processes, and that, reciprocally, GDF5 acts through both IB and other type I receptors. Together, these findings provide in vivo support for the concept of combinatorial BMP signaling, in which distinct outcomes result both from a single receptor being triggered by different ligands and from a single ligand binding to different receptors.
Animals↗
Using Flp-recombinase to characterize expansion of Wnt1-expressing neural progenitors in the mouse.
Here we demonstrate how a Flp recombinase-based tagging system can be used to link temporally distinct developmental events in the mouse. By directly following Flp-mediated DNA rearrangements we have analyzed the adult expansion of embryonic neural progenitors which transiently express the signaling factor Wnt1. We report Wnt1 promoter activity in embryonic cells that give rise to aspects of the adult midbrain, cerebellum, spinal cord, and dorsal root ganglia. These findings show that cells transiently expressing Wnt1 play more than an inductive role during early brain regionalization, giving rise to distinct adult brain regions as well as neural crest derivatives. Moreover, these results reveal two new features of the Flp-FRT system: First, Flp(F70L) can effectively recombine target sites (FRTs) placed in an endogenous locus in a variety of tissues in vivo, despite previous in vitro evidence of thermolability; and second, Flp(F70L) action can be predictably and tightly regulated in the mouse embryo, making it suitable for fate mapping applications. A further advantage of the Flp-FRT system is that marked lineages can ultimately be combined with germline mutations and deficiencies currently being generated using the Cre-loxP recombination system-in this way it should be possible to analyze mutant gene activities directly for their effect on cell fate.
Animals↗
Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice.
Site-specific recombinases are being developed as tools for "in vivo" genetic engineering because they can catalyze precise excisions, integrations, inversions, or translocations of DNA between their distinct recognition target sites. Here it is demonstrated that Flp recombinase can effectively mediate site-specific excisional recombination in mouse embryonic stem cells, in differentiating embryonal carcinoma cells, and in transgenic mice. Broad Flp expression is compatible with normal development, suggesting that Flp can be used to catalyze recombination in most cell types. These properties indicate that Flp can be exploited to make prescribed alterations in the mouse genome.
Animals↗
A modular set of Flp, FRT and lacZ fusion vectors for manipulating genes by site-specific recombination.
Site-specific recombinases can serve as powerful tools to target genetic manipulations to specific cell populations in culture and in the organism. A series of vectors for engineering gene activation, deletion and integration in mammalian cells using Flp recombinase is described here. The vectors are modular in design so that specific cassettes can be linked depending on the application. Using these vectors, efficient Flp-mediated lacZ activation and beta-galactosidase (beta Gal) detection has been demonstrated in mammalian cell culture. These vectors should facilitate using Flp to mark cell populations, as well as to activate, remove or mutate genes in culture and in the mouse.
Amino Acid Sequence↗
Molecular cloning and analysis of cDNA encoding the murine c-yes tyrosine protein kinase.
The cellular yes (c-yes) gene is a member of the class of proto-oncogenes that encode non-receptor tyrosine protein kinases. In this report we describe the isolation of cDNAs that encode the murine c-yes gene product and analysis of the nucleotide sequence of the murine c-yes cDNA clones. The reading frame encodes a protein of 541 amino acids with a calculated molecular mass of 60.63 kDa that is reactive with anti-Yes antisera and possesses protein kinase activity.
Amino Acid Sequence↗
Structure and developmental regulation of the B-lymphoid tyrosine kinase gene blk.
The murine blk gene, which encodes a B-lymphoid-specific tyrosine kinase of the Src family (p55blk), contains 13 exons that span more than 30 kilobases of DNA on chromosome 14. In the first three exons, which encode the 5'-untranslated region and N-terminal amino acid sequence unique to p55blk, the blk gene differs from other members of the src family; in the last 10 exons, the organization of the blk gene is similar to that of other src genes. By primer extension and S1 nuclease protection analyses, we show that blk transcripts initiate from four major sites at the 5'-flank of blk; two sites predominate. The resulting transcripts differ only in the lengths of their 5'-untranslated sequences and encode identical proteins. None of the transcriptional start sites are preceded by consensus TATA elements, AT-rich elements, or extensive GC-rich regions. Expression of blk is regulated during B-cell development: blk RNA is expressed in all pro-B-, pre-B-, and mature B-cell lines examined, but is absent from plasma cell lines. Immunolocalization of p55blk in normal mouse spleen supports these observations: staining is restricted to lymphocytes and is concentrated in regions rich in B-cells; plasma cells and stromal cells are not stained with anti-Blk antibodies. Assays for RNA synthesis in isolated nuclei indicate that the lineage and developmental stage specificities of blk expression are regulated at least in part by changes in its rate of transcription.
Amino Acid Sequence↗
Genetic mapping of the gene for a novel tyrosine kinase, Blk, to mouse chromosome 14.
The gene Blk, which encodes a novel tyrosine kinase expressed preferentially in B-lymphoid cells, was mapped by Southern blot analysis of DNA from the progeny of an intersubspecific backcross. Blk maps to the proximal region of chromosome 14 with the gene order centromere--(Np-1,Tcra)-Blk-sys-Es-10.
Alleles↗
Repression of immunoglobulin enhancers by the helix-loop-helix protein Id: implications for B-lymphoid-cell development.
It has been proposed that the helix-loop-helix (HLH) protein Id serves as a general antagonist of cell differentiation by inhibiting bHLH (HLH with an adjacent stretch of basic amino acids) proteins specifically required for developmental programs (such as MyoD). We show here that ectopic expression of Id represses in vivo activity of the bHLH protein E2-5 (encoded by the E2A gene) and of both the immunoglobulin heavy-chain (IgH) and kappa-light-chain gene enhancers to which E2-5 binds. Id does not affect the activity of the bHLH-zip protein, TFE3, which also binds these enhancers. We examined a large panel of B-cell lines that represent different stages of lymphoid development and found only two that express Id mRNA. The cell lines Ba/F3 and LyD9 have been categorized previously as early B-lymphoid-cell progenitors. Unlike their more mature B-lymphoid-cell counterparts, Ba/F3 and LyD9 cells do not express I mu sterile transcripts, which are indicative of IgH enhancer activity. Moreover, Ba/F3-derived nuclear extracts lack E2-box-binding activity, indicating the absence of free bHLH proteins, and transfected Ba/F3 cells fail to support the activity of the IgH enhancer. Hence, expression of Id correlates inversely with bHLH protein activity and enhancer function in vivo. These results suggest that Id may play a role early in B-lymphoid-cell development to regulate transcription of the IgH locus.
3T3 Cells↗
Specific expression of a tyrosine kinase gene, blk, in B lymphoid cells.
Several pathways of transmembrane signaling in lymphocytes involve protein-tyrosine phosphorylation. With the exception of p56lck, a tyrosine kinase specific to T lymphoid cells that associates with the T cell transmembrane proteins CD4 and CD8, the kinases that function in these pathways are unknown. A murine lymphocyte complementary DNA that represents a new member of the src family has now been isolated and characterized. This complementary DNA, termed blk (for B lymphoid kinase), specifies a polypeptide of 55 kilodaltons that is related to, but distinct from, previously identified retroviral or cellular tyrosine kinases. The protein encoded by blk exhibits tyrosine kinase activity when expressed in bacterial cells. In the mouse and among cell lines, blk is specifically expressed in the B cell lineage. The tyrosine kinase encoded by blk may function in a signal transduction pathway that is restricted to B lymphoid cells.
Amino Acid Sequence↗
Medullary osteogenesis with platinum cathodes.
Studies of electrical stimulation of osteogenesis with stainless steel electrodes have previously established a dose-response relationship between current and bone growth. Examination of the effect of differing geometric current densities resulted in the conclusion that very little electrode surface area was involved in stimulation and led to the design of a multiport "distributive" cathode. A series of experiments were performed to extend these results to wire and multiport platinum electrodes. As before, a current-bone growth dose-response relationship was found. Peak bone growth was greater than for stainless steel. However, peak bone growth occurred at 2.0 microA (versus 20 microA for stainless steel). Correlation studies suggest that small changes in cathodic potential affect bone growth more than similar size changes in current. Finally, the generally benign local host response to platinum suggests that platinum may be a suitable material for chronic indwelling anodes for stimulation of osteogenesis.
Animals↗
Widespread recombinase expression using FLPeR (flipper) mice.
As conditional genetic strategies advance, the need for multiple site-specific recombinase systems has emerged. To meet this need in part, we have targeted the constitutive ROSA26 locus to create a mouse strain with generalized expression of the enhanced version of the site-specific recombinase FLP (FLPe). This strain is designated FLPeR ("flipper"). Using this strain, extensive target gene recombination can be achieved in most tissue types, including cells of the developing germ line. FLPeR mice therefore serve two important functions: as a source of many different FLPe-expressing primary cell lines and as a deleter strain. Moreover, because the FLPeR mouse is a 129-derived strain, a 129 genetic background can be preserved when crossed to most ES cell-derived mice. This enables conditional genetic alterations to be maintained on a standard background, a feature important for obtaining reproducible results and genetically defined controls.
Animals↗