Forward genetic screen of mouse reveals dominant missense mutation in the P/Q-type voltage-dependent calcium channel, CACNA1A.

Dominant mutations of the P/Q-type Ca(2+) channel (CACNA1A) underlie several human neurological disorders, including episodic ataxia type 2, familial hemiplegic migraine 1 (FHM1) and spinocerebellar ataxia 6, but have not been found previously in the mouse. Here we report the first dominant ataxic mouse model of Cacna1a mutation. This Wobbly mutant allele of Cacna1a was identified in an ethylnitrosourea (ENU) mutagenesis dominant behavioral screen. Heterozygotes exhibit ataxia from 3 weeks of age and have a normal life span. Homozygotes have a righting reflex defect from postnatal day 8 and later develop severe ataxia and die prematurely. Both heterozygotes and homozygotes exhibit cerebellar atrophy with focal reduction of the molecular layer. No obvious loss of Purkinje cells or decrease in size of the granule cell layer was observed. Real-time polymerase chain reaction revealed altered expression levels of Cacna1g, Calb2 and Th in Wobbly cerebella, but Cacna1a messenger RNA and protein levels were unchanged. Positional cloning revealed that Wobbly mice have a missense mutation leading to an arginine to leucine (R1255L) substitution, resulting in neutralization of a positively charged amino acid in repeat III of voltage sensor segment S4. The dominance of the Wobbly mutation more closely resembles patterns of CACNA1A mutation in humans than previously described mouse recessive mutants (tottering, leaner, rolling Nagoya and rocker). Positive-charge neutralization in S4 has also been shown to underlie several cases of human dominant FHM1 with ataxia. The Wobbly mutant thus highlights the importance of the voltage sensor and provides a starting point to unravel the neuropathological mechanisms of this disease.

Molecular genetics of the early development of hindbrain serotonergic neurons.

The serotonergic (5HT) system plays a key role in modulating behaviors, such as appetite and anxiety and has been implicated in many human disorders of mood and mind. Recent studies have begun to identify the signaling molecules and transcriptional cascades governing 5HT neuron development in the hindbrain. Already at early stages, local differences in requirements of 5HT neuron development have become apparent. These studies point toward cryptic heterogeneity amongst 5HT neurons and suggest that 5HT neuron determination and differentiation may be more flexible and less absolute biologic processes than might have been expected. Ultimately, the intrinsic heterogeneity and environmental sensitivity of 5HT neurons may help explain the variability observed in some human behavioral disorders, such as autism spectrum disorder, and the less predictable behavioral consequences of fetal alcohol syndrome.

Gene-trap mutagenesis: past, present and beyond.

Although at least 35,000 human genes have been sequenced and mapped, adequate expression or functional information is available for only approximately 15% of them. Gene-trap mutagenesis is a technique that randomly generates loss-of-function mutations and reports the expression of many mouse genes. At present, several large-scale, gene-trap screens are being carried out with various new vectors, which aim to generate a public resource of mutagenized embryonic stem (ES) cells. This resource now includes more than 8,000 mutagenized ES-cell lines, which are freely available, making it an appropriate time to evaluate the recent advances in this area of genomic technology and the technical hurdles it has yet to overcome.

Regulation of mouse lens fiber cell development and differentiation by the Maf gene.

Maf is a basic domain/leucine zipper domain protein originally identified as a proto-oncogene whose consensus target site in vitro, the T-MARE, is an extended version of an AP-1 site normally recognized by Fos and Jun. Maf and the closely related family members Neural retina leucine zipper (Nrl), L-Maf, and Krml1/MafB have been implicated in a wide variety of developmental and physiologic roles; however, mutations in vivo have been described only for Krml1/MafB, in which a loss-of-function causes abnormalities in hindbrain development due to failure to activate the Hoxa3 and Hoxb3 genes. We have used gene targeting to replace Maf coding sequences with those of lacZ, and have carried out a comprehensive analysis of embryonic expression and the homozygous mutant phenotype in the eye. Maf is expressed in the lens vesicle after invagination, and becomes highly upregulated in the equatorial zone, the site at which self-renewing anterior epithelial cells withdraw from the cell cycle and terminally differentiate into posterior fiber cells. Posterior lens cells in Maf(lacZ) mutant mice exhibit failure of elongation at embryonic day 11.5, do not express (&agr;)A- and all of the (beta)-crystallin genes, and display inappropriately high levels of DNA synthesis. This phenotype partially overlaps with those reported for gene targeting of Prox1 and Sox1; however, expression of these genes is grossly normal, as is expression of Eya1, Eya2, Pax6, and Sox2. Recombinant Maf protein binds to T-MARE sites in the (alpha)A-, (beta)B2-, and (beta)A4-crystallin promoters but fails to bind to a point mutation in the (alpha)A-crystallin promoter that has been shown previously to be required for promoter function. Our results indicate that Maf directly activates many if not all of the (beta)-crystallin genes, and suggest a model for coordinating cell cycle withdrawal with terminal differentiation.

Human KRML (MAFB): cDNA cloning, genomic structure, and evaluation as a candidate tumor suppressor gene in myeloid leukemias.

Members of the MAF family of basic region/leucine zipper transcription factors can affect transcription in either a positive or a negative fashion, depending on their partner protein(s) and the context of the target promoter. The KRML (MAFB) transcriptional regulator plays a pivotal role in regulating lineage-specific hematopoiesis by repressing ETS1-mediated transcription of erythroid-specific genes in myeloid cells. In previous studies, we mapped the human KRML gene within a genomic contig on human chromosome 20, bands q11.2-q13.1. We have isolated the human cDNA containing the full-length predicted open reading frame (ORF). Multiple KRML transcripts of approximately 1.8 and approximately 3 kb, which differ in the length of the 3' untranslated region, are ubiquitously expressed in hematopoietic tissues and encode a protein with 323 amino acids (MW 35,832). The protein has 84% identity and 92% similarity to the murine protein. The ORF of the human KRML gene contains no introns, and the gene spans approximately 3 kb. KRML maps within the smallest commonly deleted segment in malignant myeloid disorders characterized by a deletion of 20q; however, we detected no mutations of KRML in leukemia cells with loss of 20q. Thus, KRML is unlikely to be involved in the pathogenesis of malignant myeloid disorders characterized by abnormalities of chromosome 20.

Expression of Zkrml2, a homologue of the Krml1/val segmentation gene, during embryonic patterning of the zebrafish (Danio rerio).

We have identified Zkrml2, a novel homologue of the segmentation gene Krml/val in zebrafish (Danio rerio). Zkrml2 shows 72% and 92% identity in its basic leucine zipper domain with mouse Krml1 and zebrafish val, respectively. Zkrml2 is expressed coincident with MyoD throughout the somites starting at the three somite stage, becomes restricted to the dermomyotome, and subsequently disappears. Transient expression is also detected in the reticulospinal and oculomotor neurons. Zkrml2 maps to the Oregon linkage group 11 (Boston Linkage group 14) with no mapped zebrafish mutations nearby.

Equivalence in the genetic control of hindbrain segmentation in fish and mouse.

The vertebrate hindbrain is subdivided into a series of rhombomeres whose segmental organization serves to pattern the architecture and innervation of the developing head. The zebrafish gene valentino is required cell-autonomously in the development of rhombomeres 5 and 6, and valentino mutants lack visible hindbrain segmentation caudal to the r3/4 boundary (Moens, C. B., Yan, Y.-L., Appel, B., Force, A. G., and Kimmel, C. B. (1996) Development 122, 3981-3990). Here we show that valentino is the zebrafish homologue of the mouse segmentation gene kreisler, which encodes a bZip transcription factor. The valentino gene is expressed in a manner consistent with its proposed role in subdividing rhombomeres 5 and 6 from their common precursor 'proto-segment' in the presumptive hindbrain, a process that we also demonstrate is reflected in the normal order of appearance of rhombomere boundaries. As well as having similar phenotypes with respect to visible hindbrain segmentation and patterns of marker gene expression, valentino and kreisler mutants have similar pharyngeal arch and inner ear defects, consistent with a conserved role for this gene in hindbrain segmentation and in patterning of the head periphery.

The mouse segmentation gene kr encodes a novel basic domain-leucine zipper transcription factor.

The mouse kreisler (kr) mutation causes segmentation abnormalities in the caudal hindbrain and defective inner ear development. Based on an inversion discovered in the original kr allele, we selected a candidate cDNA highly expressed in the developing caudal hindbrain. This cDNA encodes a basic domain-leucine zipper (bZIP) transcription factor and was confirmed to represent the kr gene by analysis of a second kr allele, generated by chemical mutagenesis, in which a serine is substituted for an asparagine residue conserved in the DNA-binding domain of all known bZIP family members. The identity, expression, and mutant phenotype of kr indicate an early role in axial patterning and provide insights into the molecular and embryologic mechanisms that govern hindbrain and otic development.

Cloning of the mouse agouti gene predicts a secreted protein ubiquitously expressed in mice carrying the lethal yellow mutation.

The mouse agouti gene controls the deposition of yellow and black pigment in developing hairs. Several dominant alleles, including lethal yellow (Ay), result in the exclusive production of yellow pigment and have pleiotropic effects that include obesity and increased tumor susceptibility. In an interspecific backcross, we established genetic limits for the agouti gene and found that the Ay and the lethal non-agouti (ax) allele were not separated from a previously identified probe at the breakpoint of the Is1GsO chromosomal rearrangement. Using the Is1GsO probe, we isolated the agouti gene, and find that it has the potential to code for a secreted protein expressed in hair follicles and the epidermis, and that the level of expression correlates with the synthesis of yellow pigment. In the Ay mutation, there is a chromosomal rearrangement that results in the production of a chimeric RNA expressed in nearly every tissue of the body. The 5' portion of this chimeric RNA contains highly expressed novel 5' sequences, but the 3' portion retains the protein-coding potential of the nonmutant allele. We speculate that dominant pleiotropic effects of Ay are caused by ectopic activation of a signaling pathway similar to that used during normal hair growth.

Altered rhombomere-specific gene expression and hyoid bone differentiation in the mouse segmentation mutant, kreisler (kr).

Rhombomeres appear transiently in the vertebrate hindbrain shortly after neurulation and are thought to represent embryologic compartments in which the expression of different combinations of genes leads to segment-specific differentiation of the developing hindbrain, the cranial ganglia, and the branchial arches. To determine the extent to which gene expression is related to the formation of visible rhombomere boundaries, we have examined, by in situ hybridization, the expression of five rhombomere-specific genes in mouse embryos homozygous for the kreisler (kr) mutation, in which rhombomeres 4-7 are replaced by a smooth morphologically unsegmented neural tube. Using molecular probes specific for Hoxb-1 (Hox-2.9), Hoxb-3 (Hox-2.7), Hoxb-4 (Hox-2.6), Krox-20, or Fgf-3 (Int-2), we found that the kr mutation affects the expression of all the genes we examined, but, surprisingly, the altered patterns of expression are not restricted to that portion of the mutant hindbrain which is morphologically abnormal. Rostral expression boundaries of Hoxb-3 and Hoxb-4 are displaced from their normal positions at r4/5 and r6/7 to the approximate positions of r3/4 and r4/5, respectively. The expression domains of Krox-20 and Fgf-3 are also displaced in a rostral direction and the intensity of Fgf-3 hybridization is greatly reduced. The expression domain of Hoxb-1 is affected differently from the other genes in kr/kr embryos; its rostral boundary at r3/4 is intact but the caudal boundary is displaced from its normal location at r4/5 to the approximate position of r5/6. Because boundaries of gene expression for Hoxb-1 and Hoxb-4 are found in a region of the kr/kr hindbrain that lacks visible rhombomeres, establishment of regional identity, as reflected by differential gene expression, does not require overt segmentation. To investigate whether the altered patterns of gene expression we observed in the kr/kr embryonic hindbrain are associated with morphologic changes in the adult, we examined neural crest-derived tissues of the second and third branchial arches, which normally arise from rhombomeres 4 and 6, respectively. We found that the hyoid bone in kr/kr animals exhibited an accessory process on the greater horn (a third arch structure) most easily explained by ectopic development of a second arch structure (the hyoid lesser horn) in an area normally derived from the third arch.