Genetic reporter system for oncogenic Igh-Myc translocations in mice.

The Myc-deregulating chromosomal T(12;15)(Igh-Myc) translocation, the hallmark mutation of inflammation- and interleukin 6-dependent mouse plasmacytoma (PCT), is the premier model of cancer-associated chromosomal translocations because it is the only translocation in mice that occurs spontaneously (B lymphocyte lineage) and with predictably high incidence (approximately 85% of PCT), and has a direct counterpart in humans: Burkitt lymphoma t(8;14)(q24;q32) translocation. Here, we report on the development of a genetic system for the detection of T(12;15)(Igh-Myc) translocations in plasma cells of a mouse strain in which an enhanced green fluorescent protein (GFP)-encoding reporter gene has been targeted to Myc. Four of the PCTs that developed in the newly generated translocation reporter mice, designated iGFP(5'Myc), expressed GFP consequent to naturally occurring T(12;15) translocation. GFP expression did not interfere with tumor development or the deregulation of Myc on derivative 12 of translocation, der (12), because the reporter gene was allocated to the reciprocal product of translocation, der (15). Although the described reporter gene approach requires refinement before T(12;15) translocations can be quantitatively detected in vivo, including in B lymphocyte lineage cells that have not yet completed malignant transformation, our findings provide proof of principle that reporter gene tagging of oncogenes in gene-targeted mice can be used to elucidate unresolved questions on the occurrence, distribution and trafficking of cells that have acquired cancer-causing chromosomal translocations of great relevance for humans.

Mouse embryonic stem cells that express a NUP98-HOXD13 fusion protein are impaired in their ability to differentiate and can be complemented by BCR-ABL.

NUP98-HOXD13 (NHD13) fusions have been identified in patients with myelodysplastic syndrome, acute myelogenous leukemia and chronic myeloid leukemia blast crisis. We generated 'knock-in' mouse embryonic stem (ES) cells that express a NHD13 fusion gene from the endogenous murine NUP98 promoter, and used an in vitro differentiation system to differentiate the ES cells to hematopoietic colonies. Replating assays demonstrated that the partially differentiated NHD13 ES cells were immortal, and two of these cultures were transferred to liquid culture. These cell lines are partially differentiated immature hematopoietic cells, as determined by morphology, immunophenotype and gene expression profile. Despite these characteristics, they were unable to differentiate when exposed to high concentrations of erythropoietin (Epo), granulocyte colony-stimulating factor or macrophage colony-stimulating factor. The cell lines are incompletely transformed, as evidenced by their dependence on interleukin 3 (IL-3), and their failure to initiate tumors when injected into immunodeficient mice. We attempted genetic complementation of the NHD13 gene using IL-3 independence and tumorigenicity in immunodeficient mice as markers of transformation, and found that BCR-ABL successfully transformed the cell lines. These findings support the hypothesis that expression of a NHD13 fusion gene impairs hematopoietic differentiation, and that these cell lines present a model system to study the nature of this impaired differentiation.

Pacinian corpuscle development involves multiple Trk signaling pathways.

The development of crural Pacinian corpuscles was explored in neonatal mutant mice lacking nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) or neurotrophin-4 (NT4), or their cognate Trk receptors. Deficits of the corpuscles and their afferents were greatest in NT3, less in BDNF, and least in NT4 null mice. Deletion of NGF or p75(NTR) genes had little or no impact. No Pacinian corpuscles were present in NT3;BDNF and NT3;NT4 double or NT3;BDNF;NT4 triple null mice. Deficits were larger in NT3 than TrkC mutants and were comparable to deficits observed in TrkB or TrkA mutants. Afferents of all corpuscles coexpressed TrkA and TrkB receptors, and some afferents coexpressed all three Trk receptors. Our results suggest that multiple neurotrophins, in particular NT3, regulate the density of crural Pacinian corpuscles, most likely by regulating the survival of sensory neurons. In addition, NT3/TrkB and/or NT3/TrkA signaling plays a greater role than NT3/TrkC signaling in afferents to developing Pacinian corpuscles.

Neurochemical and behavioral deficits consequent to expression of a dominant negative EphA5 receptor.

The Eph family tyrosine kinase receptors and their ligands have been linked to axon guidance and topographic mapping of the developing central nervous system. More specifically, the EphA5 receptor has been shown to play a role in development of hippocamposeptal, retinotectal and thalamocortical projections. Recently, a line of transgenic mice was developed which expresses a truncated EphA5 receptor lacking a functional tyrosine kinase domain. In a previous study, axonal tracing revealed that medial hippocampal axons in this strain projected laterally and ventrally away from their normal target area. In the current study, both transgenic and wild-type controls were evaluated in unconditioned (rotorod and locomotor activity) and conditioned (water maze and active avoidance) behavior tasks which tested hippocampal and striatal functioning. Compared to controls, the transgenic strain did not show differences in rotorod motor activity but did show a transient deficit in spatial navigation ability and a consistent impairment in active avoidance. The dominant-negative mutant receptor also resulted in a decrease in striatal dopamine and serotonin concentrations with no change in hippocampal monoamines. Collectively, these data suggest that animals expressing a truncated EphA5 receptor show deficits related to striatal functioning.

Neural crest stem cell and cardiac endothelium defects in the TrkC null mouse.

TrkC null mice have multiple cardiac malformations. Since neural crest cells participate in cardiac outflow tract septation, the aim of this study was to determine at the cellular level the putative neural crest defect. We have identified three types of progenitor cells: stem cells that undergo self-renewal and can generate many cell types, cells that are restricted in their developmental potentials, and cells that are committed to the smooth muscle cell lineage. In TrkC null mice, there is a greater than 50% decrease in stem cell numbers and an equivalent increase in fate-restricted cells. The outflow tract wall is thickened and the endothelial tube is disorganized. We conclude that deletion of the TrkC gene causes precocious fate restrictions of the neural crest stem cell and a defect of the outflow tract endothelium, both of which may contribute to the outflow tract malformations that occur in TrkC null mice.

Dissection of NT3 functions in vivo by gene replacement strategy.

The development of the peripheral nervous system is governed in part by a family of neurotrophic factors that signal through Trk tyrosine kinase receptors. Neurotrophin 3 (NT3) ablation in mice causes a more severe neuronal phenotype than deletion of its receptor TrkC, suggesting that NT3 acts also through other non-preferred Trk receptors. To study the role of low-affinity ligand receptor interactions in vivo, we have replaced the Nt3 gene with the gene for brain-derived neurotrophic factor (BDNF), a TrkB ligand. As in NT3 and TrkC null mice, the proprioception system of these mutants failed to assemble. However, sensory fiber projections in the embryonic spinal cord suggest chemotropic effects of BDNF in vivo. In the dorsal root ganglia, the developmental dynamic of neuron numbers demonstrates that NT3 is required for activation of TrkB during neurogenesis and that TrkA is required during target tissue innervation. In the inner ear, the ectopic BDNF rescued the severe neuronal deficits caused by NT3 absence, indicating that TrkB and TrkC activate equivalent pathways to promote survival of cochlear neurons. However, specific increased innervation densities suggest unique functions for BDNF and NT3 beyond promoting neuronal survival. This mouse model has allowed the dissection of specific spatiotemporal Trk receptor activation by NT3. Our analysis provides examples of how development can be orchestrated by complex high- and low-affinity interactions between ligand and receptor families.

Cse1l is essential for early embryonic growth and development.

The CSE1L gene, the human homologue of the yeast chromosome segregation gene CSE1, is a nuclear transport factor that plays a role in proliferation as well as in apoptosis. CSE1 and CSE1L are essential genes in Saccharomyces cerevisiae and mammalian cells, as shown by conditional yeast mutants and mammalian cell culture experiments with antisense-mediated depletion of CSE1L. To analyze whether CSE1L is also essential in vivo and whether its absence can be compensated for by other genes or mechanisms, we have cloned the murine CSE1L gene (Cse1l) and analyzed its tissue- and development-specific expression: Cse1l was detected at embryonic day 7.0 (E7.0), E11.0, E15.0, and E17.0, and in adults, high expression was observed in proliferating tissues. Subsequently, we inactivated the Cse1l gene in embryonic stem cells to generate heterozygous and homozygous knockout mice. Mice heterozygous for Cse1l appear normal and are fertile. However, no homozygous pups were born after interbreeding of heterozygous mice. In 30 heterozygote interbreeding experiments, 50 Cse1l wild-type mice and 100 heterozygotes were born but no animal with both Cse1l alleles deleted was born. Embryo analyses showed that homozygous mutant embryos were already disorganized and degenerated by E5.5. This implicates with high significance (P < 0.0001, Pearson chi-square test) an embryonically lethal phenotype of homozygous murine CSE1 deficiency and suggests that Cse1l plays a critical role in early embryonic development.

Neurotrophin-3 modulates noradrenergic neuron function and opiate withdrawal.

Somatic symptoms and aversion of opiate withdrawal, regulated by noradrenergic signaling, were attenuated in mice with a CNS-wide conditional ablation of neurotrophin-3. This occurred in conjunction with altered cAMP-mediated excitation and reduced upregulation of tyrosine hydroxylase in A6 (locus coeruleus) without loss of neurons. Transgene-derived NT-3 expressed by noradrenergic neurons of conditional mutants restored opiate withdrawal symptoms. Endogenous NT-3 expression, strikingly absent in noradrenergic neurons of postnatal and adult brain, is present in afferent sources of the dorsal medulla and is upregulated after chronic morphine exposure in noradrenergic projection areas of the ventral forebrain. NT-3 expressed by non-catecholaminergic neurons may modulate opiate withdrawal and noradrenergic signalling.

Spatial shaping of cochlear innervation by temporally regulated neurotrophin expression.

Previous work suggested qualitatively different effects of neurotrophin 3 (NT-3) in cochlear innervation patterning in different null mutants. We now show that all NT-3 null mutants have a similar phenotype and lose all neurons in the basal turn of the cochlea. To understand these longitudinal deficits in neurotrophin mutants, we have compared the development of the deficit in the NT-3 mutant to the spatial-temporal expression patterns of brain-derived neurotrophic factor (BDNF) and NT-3, using lacZ reporters in each gene and with expression of the specific neurotrophin receptors, trkB and trkC. In the NT-3 mutant, almost normal numbers of spiral ganglion neurons form, but fiber outgrowth to the basal turn is eliminated by embryonic day (E) 13.5. Most neurons are lost between E13.5 and E15.5. During the period preceding apoptosis, NT-3 is expressed in supporting cells, whereas BDNF is expressed mainly in hair cells, which become postmitotic in an apical to basal temporal gradient. During the period of neuronal loss, BDNF is absent from the basal cochlea, accounting for the complete loss of basal turn neurons in the NT-3 mutant. The spatial gradients of neuronal loss in these two mutants appear attributable to spatial-temporal gradients of neurotrophin expression. Our immunocytochemical data show equal expression of their receptors, TrkB and TrkC, in spiral sensory neurons and thus do not relate to the basal turn loss. Mice in which NT-3 was replaced by BDNF show a qualitative normal pattern of innervation at E13.5. This suggests that the pattern of expression of neurotrophins rather than their receptors is essential for the spatial loss of spiral sensory neurons in NT-3 null mutants.

Inactivation of LRG-47 and IRG-47 reveals a family of interferon gamma-inducible genes with essential, pathogen-specific roles in resistance to infection.

The cytokine interferon (IFN)-gamma regulates immune clearance of parasitic, bacterial, and viral infections; however, the underlying mechanisms are poorly understood. Recently, a family of IFN-gamma-induced genes has been identified that encode 48-kD GTP-binding proteins that localize to the endoplasmic reticulum of cells. The prototype of this family, IGTP, has been shown to be required for host defense against acute infections with the protozoan parasite Toxoplasma gondii, but not for normal clearance of the bacterium Listeria monocytogenes and murine cytomegalovirus (MCMV). To determine whether other members of the gene family also play important roles in immune defense, we generated mice that lacked expression of the genes LRG-47 and IRG-47, and examined their responses to representative pathogens. After infection with T. gondii, LRG-47-deficient mice succumbed uniformly and rapidly during the acute phase of the infection; in contrast, IRG-47-deficient mice displayed only partially decreased resistance that was not manifested until the chronic phase. After infection with L. monocytogenes, LRG-47-deficient mice exhibited a profound loss of resistance, whereas IRG-47-deficient mice exhibited completely normal resistance. In addition, both strains displayed normal clearance of MCMV. Thus, LRG-47 and IRG-47 have vital, but distinct roles in immune defense against protozoan and bacterial infections.

Neurotrophin-3 is required for the survival-differentiation of subsets of developing enteric neurons.

Neurotrophin-3 (NT-3) promotes enteric neuronal development in vitro; nevertheless, an enteric nervous system (ENS) is present in mice lacking NT-3 or TrkC. We thus analyzed the physiological significance of NT-3 in ENS development. Subsets of neurons developing in vitro in response to NT-3 became NT-3 dependent; NT-3 withdrawal led to apoptosis, selectively in TrkC-expressing neurons. Antibodies to NT-3, which blocked the developmental response of enteric crest-derived cells to exogenous NT-3, did not inhibit neuronal development in cultures of isolated crest-derived cells but did so in mixed cultures of crest- and non-neural crest-derived cells; therefore, the endogenous NT-3 that supports enteric neuronal development is probably obtained from noncrest-derived mesenchymal cells. In mature animals, retrograde transport of (125)I-NT-3, injected into the mucosa, labeled neurons in ganglia of the submucosal but not myenteric plexus; injections of (125)I-NT-3 into myenteric ganglia, the tertiary plexus, and muscle, labeled neurons in underlying submucosal and distant myenteric ganglia. The labeling pattern suggests that NT-3-dependent submucosal neurons may be intrinsic primary afferent and/or secretomotor, whereas NT-3-dependent myenteric neurons innervate other myenteric ganglia and/or the longitudinal muscle. Myenteric neurons were increased in number and size in transgenic mice that overexpress NT-3 directed to myenteric ganglia by the promoter for dopamine beta-hydroxylase. The numbers of neurons were regionally reduced in both plexuses in mice lacking NT-3 or TrkC. A neuropoietic cytokine (CNTF) interacted with NT-3 in vitro, and if applied sequentially, compensated for NT-3 withdrawal. These observations indicate that NT-3 is required for the normal development of the ENS.

Mutations in Cdh23 cause nonsyndromic hearing loss in waltzer mice.

Mutations at the waltzer (v) locus result in deafness and vestibular dysfunction due to degeneration of the neuroepithelium within the inner ear. Here, we use a positional cloning approach to show that waltzer encodes a novel cadherin (Cdh23), which is most closely related to the Drosophila Fat protein. A single nucleotide deletion in the v(J) allele and a single nucleotide insertion in the v allele are predicted to truncate each protein near the N-terminus and produce a functional null allele. In situ hybridization analysis showed that Cdh23 is expressed in the sensory hair cells of the inner ear, where it has been suggested to be a molecule critical for crosslinking of the stereocilia. In addition, Cdh23 is expressed in the urticulo-saccular foramen,the ductus reuniens, and Reissner's membrane, suggesting that Cdh23 may also be involved in maintaining the ionic composition of the endolymph. Finally, mutations in human CDH23 have recently been described for two loci, DFNB12 and USH1D, which cause nonsyndromic deafness, identifying waltzer as a mouse model for human hearing loss.

A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.

Recently, a highly efficient recombination system for chromosome engineering in Escherichia coli was described that uses a defective lambda prophage to supply functions that protect and recombine a linear DNA targeting cassette with its substrate sequence (Yu et al., 2000, Proc. Natl. Acad. Sci. USA 97, 5978-5983). Importantly, the recombination is proficient with DNA homologies as short as 30-50 bp, making it possible to use PCR-amplified fragments as the targeting cassette. Here, we adapt this prophage system for use in bacterial artificial chromosome (BAC) engineering by transferring it to DH10B cells, a BAC host strain. In addition, arabinose inducible cre and flpe genes are introduced into these cells to facilitate BAC modification using loxP and FRT sites. Next, we demonstrate the utility of this recombination system by using it to target cre to the 3' end of the mouse neuron-specific enolase (Eno2) gene carried on a 250-kb BAC, which made it possible to generate BAC transgenic mice that specifically express Cre in all mature neurons. In addition, we show that fragments as large as 80 kb can be subcloned from BACs by gap repair using this recombination system, obviating the need for restriction enzymes or DNA ligases. Finally, we show that BACs can be modified with this recombination system in the absence of drug selection. The ability to modify or subclone large fragments of genomic DNA with precision should facilitate many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function in the postgenomic era.

Lsh, a SNF2 family member, is required for normal murine development.

Lsh is a member of the SNF2 family of chromatin remodelers, that regulate diverse biological processes such as replication, repair and transcription. Although expression of Lsh is highly tissue specific in adult animals, Lsh mRNA is detectable in multiple tissues during embryogenesis. In order to determine the physiologic role of Lsh during murine development and to assess its unique function in adult mice, we performed targeted deletion of the Lsh gene using homologous recombination in murine embryonic stem cells. Lsh-/- embryos occurred with the expected Mendelian frequency after implantation and during embryogenesis. However, Lsh-/- mice died within a few hours after birth. Furthermore, newborn mice were 22% lower in weight in comparison with their littermates and showed renal lesions. Thus Lsh is a non-redundant member of the SNF2 family and is essential for normal murine development and survival.

Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice.

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive human disease whose clinical features include growth retardation, immunodeficiency, and increased susceptibility to lymphoid malignancies. Cells from NBS patients exhibit gamma-irradiation sensitivity, S-phase checkpoint defects, and genomic instability. Recently, it was demonstrated that this chromosomal breakage syndrome is caused by mutations in the NBS1 gene that result in a total loss of full-length NBS1 expression. Here we report that in contrast to the viability of NBS patients, targeted inactivation of NBS1 in mice leads to early embryonic lethality in utero and is associated with poorly developed embryonic and extraembryonic tissues. Mutant blastocysts showed greatly diminished expansion of the inner cell mass in culture, and this finding suggests that NBS1 mediates essential functions during proliferation in the absence of externally induced damage. Together, our results indicate that the complex phenotypes observed in NBS patients and cell lines may not result from a complete inactivation of NBS1 but may instead result from hypomorphic truncation mutations compatible with cell viability.

Human BRCA1 gene rescues the embryonic lethality of Brca1 mutant mice.

Half of all familial breast cancers are due to mutation in the BRCA1 gene. However, despite its importance, attempts to model BRCA1-induced disease in the mouse have been disappointing. Heterozygous Brca1 knockout mice do not develop mammary tumors and homozygous knockout mice die during embryogenesis from ill-defined causes. Sequence analysis has shown that the coding region, genomic organization, and regulatory sequences of the human and mouse genes are not well conserved. This has raised the question of whether the mouse can serve as an effective model for functional analysis of the human BRCA1 gene. To address this question we have introduced a bacterial artificial chromosome containing the human BRCA1 gene into the germline of Brca1 knockout mice. Surprisingly, we have found that the embryonic lethality of Brca1 knockout mice is rescued by the human transgene. We also show that expression of human BRCA1 transgene mirrors the endogenous murine gene. Our "humanized" transgenic mice can serve as a model system for functional analyses of the human BRCA1 gene. Published 2001 Wiley-Liss, Inc.

Ras-guanine nucleotide exchange factor sos2 is dispensable for mouse growth and development.

The mammalian sos1 and sos2 genes encode highly homologous members of the Son-of-sevenless family of guanine nucleotide exchange factors. They are ubiquitously expressed and play key roles in transmission of signals initiated by surface protein tyrosine kinases that are transduced into the cell through the action of membrane-associated Ras proteins. Recent reports showed that targeted disruption of the sos1 locus results in embryonic lethality. To gain insight into the in vivo function of sos2, we disrupted its catalytic CDC25-H domain by means of gene targeting techniques. Mating among heterozygous sos2(+/-) mice produced viable sos2(-/-) offspring with a normal Mendelian pattern of inheritance, indicating that the loss of sos2 does not interfere with embryo viability in the uterus. Adult homozygous mutant sos2(-/-) mice reached sexual maturity at the same age as their wild-type littermates, and both male and female null mutants were fertile. Histopathological analysis showed no observable differences between mutant and wild-type mice. Our results show that unlike the case for sos1, sos2 gene function is dispensable for normal mouse development, growth, and fertility.