Genetic marking and characterization of Tac2-expressing neurons in the central and peripheral nervous system.

BACKGROUND: The neurocircuits that process somatic sensory information in the dorsal horn of the spinal cord are still poorly understood, with one reason being the lack of Cre lines for genetically marking or manipulating selective subpopulations of dorsal horn neurons. Here we describe Tac2-Cre mice that were generated to express the Cre recombinase gene from the Tac2 locus. Tachykinin 2 (Tac2) encodes a neurotransmitter, neurokinin B (NKB). RESULTS: By crossing Tac2-Cre mice with ROSA26-tdTomato reporter mice, we directly visualized Tac2 lineage neurons in the dorsal root ganglia, the dorsal horn of the spinal cord, and many parts of the brain including the olfactory bulb, cerebral cortex, amygdala, hippocampus, habenula, hypothalamus, and cerebellum. This Tac2-Cre allele itself was a null allele for the Tac2 gene. Behavioral analyses showed that Tac2 homozygous null mice responded normally to a series of algogenic (pain-inducing) and pruritic (itch-inducing) stimuli. CONCLUSIONS: Tac2-Cre mice are a useful tool to mark specific subsets of neurons in the sensory ganglia, the dorsal spinal cord, and the brain. These mice can also be used for future genetic manipulations to study the functions of Tac2-expressing neurons or the functions of genes expressed in these neurons.

The interval between Ins2 and Ascl2 is dispensable for imprinting centre function in the murine Beckwith-Wiedemann region.

Imprinted genes are commonly clustered in domains across the mammalian genome, suggesting a degree of coregulation via long-range coordination of their monoallelic transcription. The distal end of mouse chromosome 7 (Chr 7) contains two clusters of imprinted genes within a approximately 1 Mb domain. This region is conserved on human 11p15.5 where it is implicated in the Beckwith-Wiedemann syndrome. In both species, imprinted regulation requires two critical cis-acting imprinting centres, carrying different germline epigenetic marks and mediating imprinted expression in the proximal and distal sub-domains. The clusters are separated by a region containing the gene for tyrosine hydroxylase (Th) as well as a high density of short repeats and retrotransposons in the mouse. We have used the Cre-loxP recombination system in vivo to engineer an interstitial deletion of this approximately 280-kb intervening region previously proposed to participate in the imprinting mechanism or to act as a boundary between the two sub-domains. The deletion allele, Del(7AI), is silent with respect to epigenetic marking at the two flanking imprinting centres. Reciprocal inheritance of Del(7AI) demonstrates that the deleted region, which represents more than a quarter of the previously defined imprinted domain, is associated with intrauterine growth restriction in maternal heterozygotes. In homozygotes, the deficiency behaves as a Th null allele and can be rescued pharmacologically by bypassing the metabolic requirement for TH in utero. Our results show that the deleted interval is not required for normal imprinting on distal Chr 7 and uncover a new imprinted growth phenotype.

Creation and use of a cre recombinase transgenic database.

In many cases, a gene "knockout" results in early embryonic lethality, which obscures the study of potential later functions. In other cases, the "knockout" does not show any phenotype due to the compensation of the gene deficiency by other family members. These limitations have called for further development of the powerful gene-targeting technology. One of the critical tools now being efficiently combined with gene-targeting is site-specific recombination. As the site-specific recombinase technology developed further in the mouse system, it became evident that this tool was going to have a significant impact on the power of mammalian genetics. The number of transgenic mouse lines expressing Cre recombinase with different specificities has steadily increased in the past 15 years and has now surpassed 500. Efficient utilization of this community-generated resource calls for a user-friendly database with all necessary information available about the properties of the Cre transgenic lines. The "CreXmice" database was created to meet these needs and has evolved over the past 4 years from flat file listings of transgenic lines into its current form, a professionally hosted SQL-driven web application. With hundreds of transgenic mouse lines, CreXmice is enriched by its presence on the World Wide Web allowing visitors the opportunity to search or contribute to the global effort by submitting the specific lines being developed by their laboratories.

Epigenetic and phenotypic consequences of a truncation disrupting the imprinted domain on distal mouse chromosome 7.

The distal end of mouse chromosome 7 (Chr 7) contains a large cluster of imprinted genes. In this region two cis-acting imprinting centers, IC1 (H19 DMR) and IC2 (KvDMR1), define proximal and distal subdomains, respectively. To assess the functional independence of IC1 in the context of Chr 7, we developed a recombinase-mediated chromosome truncation strategy in embryonic stem cells and generated a terminal deletion allele, DelTel7, with a breakpoint in between the two subdomains. We obtained germ line transmission of the truncated Chr 7 and viable paternal heterozygotes, confirming the absence of developmentally required paternally expressed genes distal of Ins2. Conversely, maternal transmission of DelTel7 causes a midgestational lethality, consistent with loss of maternally expressed genes in the IC2 subdomain. Expression and DNA methylation analyses on DelTel7 heterozygotes demonstrate the independent imprinting of IC1 in absence of the entire IC2 subdomain. The evolutionarily conserved linkage between the subdomains is therefore not required for IC1 imprinting on Chr 7. Importantly, the developmental phenotype of maternal heterozygotes is rescued fully by a paternally inherited deletion of IC2. Thus, all the imprinted genes located in the region and required for normal development are silenced by an IC2-dependent mechanism on the paternal allele.

Embryonic fibroblasts from mice lacking Tgif were defective in cell cycling.

Holoprosencephaly (HPE) is the most common structural anomaly of the human brain, resulting from incomplete cleavage of the developing forebrain during embryogenesis. Haploinsufficient mutations in the TG-interacting factor (TGIF) gene were previously identified in a subset of HPE families and sporadic patients, and this gene is located within a region of chromosome 18 that is associated with nonrandom chromosomal aberrations in HPE patients. TGIF is a three-amino-acid loop extension (TALE) homeodomain-containing transcription factor that functions both as a corepressor of the transforming growth factor beta (TGF-beta) pathway and as a competitor of the retinoic acid pathway. Here we describe mice deficient in Tgif that exhibited laterality defects and growth retardation and developed kinked tails. Cellular analysis of mutant mouse embryonic fibroblasts (MEFs) demonstrated for the first time that Tgif regulates proliferation and progression through the G1 cell cycle phase. Additionally, wild-type human TGIF was able to rescue this proliferative defect in MEFs. In contrast, a subset of human Tgif mutations detected in HPE patients was unable to rescue the proliferative defect. However, an absence of Tgif did not alter the normal inhibition of proliferation caused by treatment with TGF-beta or retinoic acid. Developmental control of proliferation by Tgif may play a role in the pathogenesis of HPE.

A genetic screen for mutations that affect cranial nerve development in the mouse.

Cranial motor and sensory nerves arise stereotypically in the embryonic hindbrain, act as sensitive indicators of general and region-specific neuronal development, and are directly or indirectly affected in many human disorders, particularly craniofacial syndromes. The molecular genetic hierarchies that regulate cranial nerve development are mostly unknown. Here, we describe the first mouse genetic screen that has used direct immunohistochemical visualization methods to systematically identify genetic loci required for cranial nerve development. After screening 40 pedigrees, we recovered seven new neurodevelopmental mutations. Two mutations model human genetic syndromes. Mutation 7-1 causes facial nerve anomalies and a reduced lower jaw, and is located in a region of conserved synteny with an interval associated with the micrognathia and mental retardation of human cri-du-chat syndrome. Mutation 22-1 is in the Pax3 gene and, thus, models human Waardenburg syndrome. Three mutations cause global axon guidance deficits: one interferes with initial motor axon extension from the neural tube, another causes overall axon defasciculation, and the third affects general choice point selection. Another two mutations affect the oculomotor nerve specifically. Oculomotor nerve development, which is disrupted by six mutations, appears particularly sensitive to genetic perturbations. Phenotypic comparisons of these mutants identifies a "transition zone" that oculomotor axons enter after initial outgrowth and in which new factors govern additional progress. The number of interesting neurodevelopmental mutants revealed by this small-scale screen underscores the promise of similar focused genetic screens to contribute significantly to our understanding of cranial nerve development and human craniofacial syndromes.

Analysis of hindbrain patterning defects caused by the kreisler(enu) mutation reveals multiple roles of Kreisler in hindbrain segmentation.

The embryonic hindbrain is subdivided into eight subunits, termed rhombomeres (r1-r8). The Kreisler (Krml1/MafB/val) transcription factor is expressed in and essential for patterning rhombomeres 5 and 6. Here, we have shown that in the chemically induced kreisler(enu) (kr(enu)) allele, a point mutation in the DNA binding domain abolishes or severely reduces Kreisler-dependent transcription. Comparison of kr(enu)/kr(enu) embryos with those homozygous for the classic kreisler (kr) mutation has reconciled past discrepancies and revealed multiple roles of Kreisler in hindbrain segmentation. These analyses demonstrate that Kreisler is required for maintenance and expansion but not initiation of the Krox20 expressing r5 domain. The differences in the "r5-like" phenotype of kr(enu)/kr(enu) and kr/kr mouse embryos, and zebrafish carrying mutations in the Kreisler orthologue valentino (val) suggest that Kreisler performs many of its r5-specific functions by associating with other proteins. By contrast, kr/kr and kr(enu)/kr(enu) mouse and val-/- zebrafish embryos all exhibit indistinguishable defects in r6 specification. Thus, transcriptionally active Kreisler is required for r6 specification. Unlike mouse kr(enu)/kr(enu) and zebrafish val-/- embryos, kr/kr embryos exhibited anterior defects. We determined that the kr chromosomal inversion caused ectopic Kreisler expression in r3 of kr/kr and kr/+ embryos. Hence, Kreisler regulates maintenance and expansion of r5 and specification of r6 but is not required for r3 development.