Differential activities of the RET tyrosine kinase receptor isoforms during mammalian embryogenesis.

The RET receptor tyrosine kinase has a critical role in kidney organogenesis and the development of the enteric nervous system. Two major isoforms, RET9 and RET51, differ in the amino acid sequence of the C-terminal tail as a result of alternative splicing. To determine the roles of these isoforms in vivo, we used targeted mutagenesis to generate mice that express either RET9 or RET51. Monoisoformic RET9 mice, which lack RET51, are viable and appear normal. In contrast, monoisoformic RET51 animals, which lack RET9, have kidney hypodysplasia and lack enteric ganglia from the colon. To study the differential activities of the two RET isoforms further, we generated transgenic mice expressing ligand-dependent and constitutively active forms of RET9 or RET51 under the control of the Hoxb7 regulatory sequences. Such RET9 transgenes are capable of rescuing the kidney agenesis in RET-deficient mice or causing kidney hypodysplasia in wild-type animals. In contrast, similar RET51 transgenes fail to rescue the kidney agenesis or cause hypodysplasia. Our findings show that RET9 and RET51 have different signaling properties in vivo and define specific temporal and spatial requirements of c-Ret function during renal development and histogenesis of the enteric nervous system.

Homeodomain proteins Mox1 and Mox2 associate with Pax1 and Pax3 transcription factors.

Mox1 and Mox2 homeobox genes have been shown to be critical in axial skeleton and in limb muscle development respectively. Pax1 and Pax3 gene products are also implicated in these processes. Mox and Pax expression patterns are highly overlapping both spatially and temporally during embryonic development. We show here for the first time that Mox proteins physically interact with Pax1 and Pax3 using the yeast two-hybrid protein interaction assay as well as in vitro biochemical assays. There is a strong preference of Mox1 to associate with Pax1 rather than Pax3 and of Mox2 to associate with Pax3 rather than Pax1. The observed interactions are mediated through the homeodomain of Mox.

Isolation of the avian homologue of the homeobox gene Mox2 and analysis of its expression pattern in developing somites and limbs.

We have isolated the cDNA of avian Mox2 and analyzed its expression pattern during somitogenesis and limb bud formation. Mox2 plays an important role in limb muscle differentiation in the mouse. Mox2 is expressed in the somites of developing chick embryos and in presumptive migrating myoblasts from the dermomyotome to the limb buds. It is also expressed in the ventral and dorsal part of limb buds and is associated with non-proliferating myoblasts. Significant differences were observed in chick and mouse expression patterns, namely in the chick dermomyotome and limb.

Genomewide genetic linkage analysis confirms the presence of susceptibility loci for schizophrenia, on chromosomes 1q32.2, 5q33.2, and 8p21-22 and provides support for linkage to schizophrenia, on chromosomes 11q23.3-24 and 20q12.1-11.23.

We have performed genetic linkage analysis in 13 large multiply affected families, to test the hypothesis that there is extensive heterogeneity of linkage for genetic subtypes of schizophrenia. Our strategy consisted of selecting 13 kindreds containing multiple affected cases in three or more generations, an absence of bipolar affective disorder, and a single progenitor source of schizophrenia with unilineal transmission into the branch of the kindred sampled. DNA samples from these families were genotyped with 365 microsatellite markers spaced at approximately 10-cM intervals across the whole genome. We observed LOD scores >3.0 at five distinct loci, either in the sample as a whole or within single families, strongly suggesting etiological heterogeneity. Heterogeneity LOD scores >3.0 in the sample as a whole were found at 1q33.2 (LOD score 3.2; P=.0003), 5q33.2 (LOD score 3.6; P=.0001), 8p22.1-22 (LOD score 3.6; P=.0001), and 11q21 (LOD score 3.1; P=.0004). LOD scores >3.0 within single pedigrees were found at 4q13-31 (LOD score 3.2; P=.0003) and at 11q23.3-24 (LOD score 3.2; P=.0003). A LOD score of 2.9 was also found at 20q12.1-11.23 within in a single family. The fact that other studies have also detected LOD scores >3.0 at 1q33.2, 5q33.2, 8p21-22 and 11q21 suggests that these regions do indeed harbor schizophrenia-susceptibility loci. We believe that the weight of evidence for linkage to the chromosome 1q22, 5q33.2, and 8p21-22 loci is now sufficient to justify intensive investigation of these regions by methods based on linkage disequilibrium. Such studies will soon allow the identification of mutations having a direct effect on susceptibility to schizophrenia.

Mox2 is a component of the genetic hierarchy controlling limb muscle development.

The skeletal muscles of the limbs develop from myogenic progenitors that originate in the paraxial mesoderm and migrate into the limb-bud mesenchyme. Among the genes known to be important for muscle development in mammalian embryos are those encoding the basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs; MyoD, Myf5, myogenin and MRF4) and Pax3, a paired-type homeobox gene that is critical for the development of limb musculature. Mox1 and Mox2 are closely related homeobox genes that are expressed in overlapping patterns in the paraxial mesoderm and its derivatives. Here we show that mice homozygous for a null mutation of Mox2 have a developmental defect of the limb musculature, characterized by an overall reduction in muscle mass and elimination of specific muscles. Mox2 is not needed for the migration of myogenic precursors into the limb bud, but it is essential for normal appendicular muscle formation and for the normal regulation of myogenic genes, as demonstrated by the downregulation of Pax3 and Myf5 but not MyoD in Mox2-deficient limb buds. Our findings show that the MOX2 homeoprotein is an important regulator of vertebrate limb myogenesis.

New DNA markers with increased informativeness show diminished support for a chromosome 5q11-13 schizophrenia susceptibility locus and exclude linkage in two new cohorts of British and Icelandic families.

Genetic linkage of schizophrenia to markers at 5q11.2-13.3 had been reported previously in five Icelandic and two British families, but attempts at replication in independent samples have been unsuccessful. We report here an update on the diagnoses and results of linkage analyses using newer highly polymorphic microsatellite markers at or near the loci D5S76 and D5S39 in the original sample of pedigrees and in two new family samples from Iceland and from Britain. The new results show a reduction in evidence for linkage in the original sample and evidence against linkage in the two new family samples. Although it is possible that a rare locus is present, perhaps in the region 5p14.1-13.1 rather than 5q11.2-13.3, it appears most likely that the original positive lod scores represent an exaggeration of the 'true' lod scores due to random effects and that the small lod scores we now obtain could have arisen by chance.

Linkage study of the D5 dopamine receptor gene (DRD5) in multiplex Icelandic and English schizophrenia pedigrees.

OBJECTIVE: The authors investigated the possibility that genetic variation or mutation of the dopamine D5 receptor gene might modify susceptibility to schizophrenia. METHOD: Twenty-three Icelandic and English pedigrees containing multiple cases of schizophrenia were genotyped by using a highly informative microsatellite for the D5 dopamine receptor gene DRD5. RESULTS: By means of three different affection models, negative lod scores were obtained under assumptions of autosomal dominant and recessive inheritance. There was no evidence for locus heterogeneity. Nonparametric extended relative pair analysis also produced negative results. CONCLUSIONS: These data indicate that mutations of the D5 dopamine receptor gene are not a major cause of schizophrenia in these pedigrees. Because of the probable existence of locus heterogeneity, the D5 receptor gene may be of etiologic importance in other families with schizophrenia.

Exclusion of linkage of schizophrenia to the gene for the dopamine D2 receptor (DRD2) and chromosome 11q translocation sites.

There have been previous reports of a 1q43;11q21 translocation cosegregating with schizophrenia and a 9p22;11q22.3 translocation cosegregating with manic depression. In addition, the genes for the dopamine D2 receptor and for tyrosinase both map to chromosome 11q. Three 11q DNA markers were used to investigate 23 pedigrees containing multiple cases of schizophrenia. Strongly negative lod scores were obtained, providing evidence against linkage over a 70 cM region which included both translocation sites and both candidate genes.

Isolation of the human MOX2 homeobox gene and localization to chromosome 7p22.1-p21.3.

We have isolated and characterized cDNA clones encoding a novel human homeobox gene, MOX2, the homologue of the murine mox-2 gene. The MOX2 protein contains all of the characteristic features of Mox-2 proteins of other vertebrate species, namely the homeobox, the polyhistidine stretch, and a number of potential serine/threonine phosphorylation sites. The homeodomain of MOX2 protein is identical to all other vertebrate species reported so far (rodents and amphibians). Outside the homeodomain, Mox-2 proteins share a high degree of identity, except for a few amino acid differences encountered between the human and the rodent polypeptides. A polyhistidine stretch of 12 amino acids in the N terminal region of the protein is also conserved among humans, rodents, and (only partly) amphibians. The chromosomal position of MOX2 was assigned to 7p22.1-p21.3.

The Marfan syndrome gene locus as a favoured locus for susceptibility to schizophrenia.

Marfan syndrome (MS) is a rare autosomal dominant disorder of connective tissue with manifestations in the cardiovascular, ocular and skeletal systems. Genetic linkage analysis with random probes has mapped the MS locus to 15q21.1. There have been several reports of Marfan syndrome co-segregating with schizophrenia within families, which suggest that a common genetic factor may be shared between schizophrenia susceptibility and MS. This could be due to a cytogenetic abnormality affecting both genetic loci or due to co-segregation of two disease loci near each other on the same chromosome. We tested this hypothesis by using genetic linkage analysis with multiplex families. Using three genetic markers spanning the MS locus, we were unable to find evidence of linkage with schizophrenia across the Marfan syndrome locus on chromosome 15.

Expression of the c-ret proto-oncogene during mouse embryogenesis.

The c-ret proto-oncogene encodes a receptor tyrosine kinase whose normal function has yet to be determined. To begin to investigate the potential role of this gene in vertebrate development, we have isolated cDNA clones representing the murine c-ret gene, and have analyzed the pattern of expression during mouse embryogenesis, using northern blotting, in situ hybridization to histological sections and whole-mount hybridization histochemistry. c-ret transcripts were detected beginning at day 8.5 of embryogenesis, and were observed in a number of cell lineages in the developing peripheral and central nervous systems, as well as in the excretory system. In the cranial region at day 8.5-9.5, c-ret mRNA was restricted to a population of neural crest cells migrating from rhombomere 4 and forming the anlage of the facioacoustic ganglion, as well as to a closely associated domain of surface ectoderm and pharyngeal endoderm. At later stages (10.5-14.5 days), c-ret mRNA was observed in all cranial ganglia. In the peripheral nervous system of the trunk, c-ret was expressed in the autonomic ganglia and in subsets of cells in the dorsal root ganglia. In the enteric nervous system, c-ret was expressed in the presumptive enteric neuroblasts of the vagal crest (day 9.0-11.5), and in the myenteric ganglia of the gut (day 13.5-14.5). c-ret mRNA was observed in several regions of the central nervous system, including the undifferentiated neuroepithelial cells of the ventral neural tube (8.5 days), the motor neurons in the spinal cord and the hindbrain (10.5-14.5 days), the embryonic neuroretina (day 13.5) and the layers of the postnatal retina containing ganglion, amacrine and horizontal cells. Outside the nervous system, c-ret was expressed in the nephric (Wolffian) duct at day 8.5-10.5, the ureteric bud epithelium (but not the surrounding metanephric mesenchyme) at day 11.0-11.5, and the growing tips of the renal collecting ducts (but not the previously formed, subcortical portions of the collecting ducts, or the mesenchyme-derived renal vesicles) at day 13.5-17.5. Our results suggest that the c-ret gene may encode the receptor for a factor involved in the proliferation, migration, differentiation or survival of a variety of neuronal cell lineages, as well as in inductive interactions during organogenesis of the kidney.

Cloning of the human dopamine D5 receptor gene and identification of a highly polymorphic microsatellite for the DRD5 locus that shows tight linkage to the chromosome 4p reference marker RAF1P1.

We identified a cosmid clone with exact sequence homology to part of the human dopamine D5 receptor gene (DRD5) after screening a cosmid library with the human DRD1 gene. The dopamine D5 receptor was mapped to chromosome 4p15.1-p15.3 by in situ hybridization and using a somatic cell hybrid panel. We report here the further localization of the DRD5 gene following identification of a highly polymorphic dinucleotide repeat sequence in the cosmid clone. The microsatellite (D5(CT/GT/GA)n) had 12 alleles with a polymorphic information content value of 0.77. Linkage analysis in 39 CEPH pedigrees demonstrated tight linkage to the chromosome 4p reference marker RAF1P1 (Zmaxf 20.66 at theta f 0.05 and ZmaxM 16.57 at theta m 0.07).

Microsatellite polymorphisms for chromosome 5 bands q11.2-q13.3.

The genes for spinal muscular atrophy (SMA) and a possible subtype of schizophrenia (SCZD1) have been mapped to chromosome 5q11.2-q13.3. DNA markers have been mapped to 5q11.2-q13.3 using a hybrid cell line deleted for this region [Gilliam et al., Genomics 1989;5:940-944]. Genomic lambda clones for these markers facilitated the identification of highly polymorphic microsatellites. A total of ten microsatellites were identified and sequenced. Of these, seven were found to be polymorphic. Four had polymorphism information content values > 0.7. New polymorphic microsatellites were sequenced for D5S76, D5S125, D5S39, D5S127 and HEX-B. Two-point and multipoint analysis in non-CEPH pedigrees confirmed that the microsatellites were in tight linkage with each other. These new microsatellites will increase the efficiency of linkage analysis for these disorders.

Linkage disequilibrium between two highly polymorphic microsatellites.

The PCR was used to amplify genomic DNA from two microsatellite (dC-dA)n.(dG-dT)n sequences found to be present in the same chromosome 5 genomic clone. Analysis of the haplotype frequencies of these two interspersed repeat sequences in individuals showed strong allelic association or linkage disequilibrium. Six alleles were found for p599 (CA)n with a PIC value of 0.71 and 8 alleles were seen for lambda 599 (CA)n with a PIC value of 0.74. The two microsatellites are separated by approximately 7 kb. Analysis of the length variations for the two microsatellites showed that they were positively correlated, a finding that has no obvious explanation. The strong linkage disequilibrium found demonstrates stability during evolution for these novel markers. Therefore they should be powerful new tools for studying genetic drift and admixture of populations. Furthermore, disequilibrium data from microsatellites can be used in the fine mapping and cloning of disease genes.

A cosmid clone for the 5HT1A receptor (HTR1A) reveals a TaqI RFLP that shows tight linkage to dna loci D5S6, D5S39, and D5S76.

A human neuroreceptor clone (G21), which was isolated by cross-hybridization with the human clone for the beta 2-adrenergic receptor, has recently been shown to encode the gene for the 5HT1A receptor (HTR1A) subtype. In situ hybridization to human metaphase chromosomes mapped the G21 sequence to chromosome 5 at bands 5q11.2-q13. The clone G21 recognizes a SacI RFLP with low heterozygosity (0.13). To increase the informativeness of the HTR1A locus we have isolated two new cosmid clones containing the receptor gene. No polymorphic microsatellites were present in the cosmids. However, one cosmid revealed a new TaqI RFLP that showed tight linkage to new highly polymorphic microsatellites for the loci D5S76, D5S39, and D5S6 in seven British and Icelandic reference pedigrees (maximum LOD of 13.2 with D5S76).

Stereospecific effect of flupenthixol on neuroreceptor gene expression.

An experimental method to test the hypothesis that antipsychotic (neuroleptic) agents influence gene expression in the mouse brain has been developed using the cis and trans stereoisomers of flupenthixol. The cis form of the drug is known to be clinically effective against some of the psychotic symptoms of schizophrenia as opposed to the trans isomer which is relatively inactive. A 2- to 3-fold increase in the abundance of dopamine 2 receptor mRNA was observed in the cis treated mice after a period of ten weeks. No change was observed in the expression of the dopamine D2 receptor gene upon treatment with the trans isomer. No change in the amount of 5-HT1A, 5-HT1C, alpha 1 adrenergic, beta 1 and beta 2 adrenergic neuroreceptor mRNA was found in the mice treated with active drug. The results show a long-term adaptation to D2 antagonism at the level of gene expression which occurs over a similar time scale to that of the clinical response to neuroleptic treatment of schizophrenia.

Recent and future molecular genetic research into schizophrenia.

The difficulties anticipated in the application of molecular genetics to schizophrenia research have not prevented the first successful localization of a susceptibility gene for a subtype of schizophrenia. It is argued that this approach is the most useful of the possible molecular genetic strategies because it leads both to enhanced clinical genetic investigation and to further recombinant DNA research to clone and sequence schizophrenia susceptibility mutations. Future recombinant DNA research can now use long-range mapping and cloning techniques such as the chromosome walking/jumping approach and the strategy of cloning brain-specific cDNAs from brain mRNA. The identification of carriers for high-risk studies and the genetic validation of diagnosis appear to be the most promising clinical developments. Prenatal counseling will only become widely feasible when much more is known about the extent of heterogeneity of linkage in schizophrenia.