Fate of midbrain dopaminergic neurons controlled by the engrailed genes.

Deficiencies in neurotransmitter-specific cell groups in the midbrain result in prominent neural disorders, including Parkinson's disease, which is caused by the loss of dopaminergic neurons of the substantia nigra. We have investigated in mice the role of the engrailed homeodomain transcription factors, En-1 and En-2, in controlling the developmental fate of midbrain dopaminergic neurons. En-1 is highly expressed by essentially all dopaminergic neurons in the substantia nigra and ventral tegmentum, whereas En-2 is highly expressed by a subset of them. These neurons are generated and differentiate their dopaminergic phenotype in En-1/En-2 double null mutants, but disappear soon thereafter. Use of an En-1/tau-LacZ knock-in mouse as an autonomous marker for these neurons indicates that they are lost, rather than that they change their neurotransmitter phenotype. A single allele of En-1 on an En-2 null background is sufficient to produce a wild type-like substantia nigra and ventral tegmentum, whereas in contrast a single allele of En-2 on an En-1 null background results in the survival of only a small proportion of these dopaminergic neurons, a finding that relates to the differential expression of En-1 and En-2. Additional findings indicate that En-1 and En-2 regulate expression of alpha-synuclein, a gene that is genetically linked to Parkinson's disease. These findings show that the engrailed genes are expressed by midbrain dopaminergic neurons from their generation to adulthood but are not required for their specification. However, the engrailed genes control the survival of midbrain dopaminergic neurons in a gene dose-dependent manner. Our findings also suggest a link between engrailed and Parkinson's disease.

Evx1 is a postmitotic determinant of v0 interneuron identity in the spinal cord.

Interneurons in the ventral spinal cord are essential for coordinated locomotion in vertebrates. During embryogenesis, the V0 and V1 classes of ventral interneurons are defined by expression of the homeodomain transcription factors Evx1/2 and En1, respectively. In this study, we show that Evx1 V0 interneurons are locally projecting intersegmental commissural neurons. In Evx1 mutant embryos, the majority of V0 interneurons fail to extend commissural axons. Instead, they adopt an En1-like ipsilateral axonal projection and ectopically express En1, indicating that V0 interneurons are transfated to a V1 identity. Conversely, misexpression of Evx1 represses En1, suggesting that Evx1 may suppress the V1 interneuron differentiation program. Our findings demonstrate that Evx1 is a postmitotic determinant of V0 interneuron identity and reveal a critical postmitotic phase for neuronal determination in the developing spinal cord.

Kidney development in cadherin-6 mutants: delayed mesenchyme-to-epithelial conversion and loss of nephrons.

During nephrogenesis, dynamic changes in the expression of cell adhesion molecules are evident as epithelial structures differentiate from the induced mesenchyme. The cadherins are thought to play an important role in the metanephric mesenchyme, when cells aggregate to form the renal vesicle, a polarized epithelial structure which eventually fuses with the ureteric bud to generate a continuous nascent nephron. We have generated and analyzed mice with a targeted mutation in the gene encoding cadherin-6 (Cad-6), a type II cadherin expressed during early stages of nephrogenesis. These mice are viable and fertile, and they complete both early and late aspects of nephrogenesis. However, upon closer examination in vitro and in vivo, a fraction of the induced metanephric mesenchyme in Cad-6 mutant kidneys fails to form a fully polarized epithelium on schedule. Moreover, a significant number of the renal vesicles in Cad-6 mutant kidneys apparently fail to fuse to the ureteric bud. These alterations in epithelialization and fusion apparently lead to a loss of nephrons in the adult. These studies support the idea that cadherins play an essential role in the formation of epithelial structures and underscore the importance of timing in orchestrating the morphogenesis of complex epithelial tissues.

Engrailed-1 and netrin-1 regulate axon pathfinding by association interneurons that project to motor neurons.

During early development, multiple classes of interneurons are generated in the spinal cord including association interneurons that synapse with motor neurons and regulate their activity. Very little is known about the molecular mechanisms that generate these interneuron cell types, nor is it known how axons from association interneurons are guided toward somatic motor neurons. By targeting the axonal reporter gene &tgr;-lacZ to the En1 locus, we show the cell-type-specific transcription factor Engrailed-1 (EN1) defines a population of association neurons that project locally to somatic motor neurons. These EN1 interneurons are born early and their axons pioneer an ipsilateral longitudinal projection in the ventral spinal cord. The EN1 interneurons extend axons in a stereotypic manner, first ventrally, then rostrally for one to two segments where their axons terminate close to motor neurons. We show that the growth of EN1 axons along a ventrolateral pathway toward motor neurons is dependent on netrin-1 signaling. In addition, we demonstrate that En1 regulates pathfinding and fasciculation during the second phase of EN1 axon growth in the ventrolateral funiculus (VLF); however, En1 is not required for the early specification of ventral interneuron cell types in the embryonic spinal cord.

Genetic linkage of HLA-class II locus to mite-specific IgE immune responsiveness.

BACKGROUND: IgE response to common inhalant allergens seems to be the major determinant of the development of atopic rhinitis and asthma but it has been difficult to demonstrate genetic control of the IgE response. OBJECTIVE: To investigate genetic linkage between specific IgE reactions to purified aero-allergens (grass, birch, cat, mite) and the HLA-class II locus. METHODS: DNA-based HLA-class II typing was performed for determination of DRB1, DQB1 and DPB1 alleles. Linkage was studied by the affected sibpair method and the extended transmission disequilibrium test in 100 children from 40 nuclear families selected from a homogeneous population in south-western Germany. RESULTS: Significant linkage of mite-specific IgE response to HLA-DPB (P = 0.00001), HLA-DRB (0.02) and HLA-DQB (P = 0.001) was revealed by sibpair analysis of MHC class II alleles and confirmed by the extended transmission disequilibrium test for HLA-DRB (P = 0.01) and HLA-DPB (P = 0.04). No consistent significant linkage between the HLA-class II locus and IgE response to grass pollen, birch pollen, and cat dander could be demonstrated. CONCLUSION: The findings are consistent with the existence of one or more genes in the HLA-class II region modifying the IgE immune response to common environmental allergens.

PAX2 is expressed in multiple spinal cord interneurons, including a population of EN1+ interneurons that require PAX6 for their development.

Members of the PAX family of transcription factors are candidates for controlling cell identity in the spinal cord. We have morphologically analyzed cells that express one of these transcription factors, PAX2, demonstrating multiple interneuron cell types express PAX2. Two ventral populations of PAX2-expressing interneurons in the spinal cord are marked by coexpression of the transcription factors, EN1 and EVX1. Interestingly, the expression domains of PAX2, EN1 and EVX1 in postmitotic neurons correlate closely with those of Pax6 and Pax7 in the ventricular zone, implicating these patterning genes in the regulation of PAX2, EN1 and EVX1. We show that one of these patterning genes, Pax6, is required for the correct specification of ventral PAX2+ interneurons that coexpress EN1. These results demonstrate that the early activity of patterning genes in the ventricular zone determines interneuron identity in the spinal cord.

Identification of cis-acting elements as DNase I hypersensitive sites in lysozyme gene chromatin.

DNase I hypersensitive sites in chromatin of eukaryotic cells mark the positions of multifactorial cis-acting elements. Mapping DH sites by indirect end labeling is a convenient procedure used for identifying regulatory elements within extensive regions of chromatin and for gaining information about their functional specificity as well as their fine structure.

Dynamic chromatin: the regulatory domain organization of eukaryotic gene loci.

It is hypothesized that nuclear DNA is organized in topologically constrained loop domains defining basic units of higher order chromatin structure. Our studies are performed in order to investigate the functional relevance of this structural subdivision of eukaryotic chromatin for the control of gene expression. We used the chicken lysozyme gene locus as a model to examine the relation between chromatin structure and gene function. Several structural features of the lysozyme locus are known: the extension of the region of general DNAasel sensitivity of the active gene, the location of DNA-sequences with high affinity for the nuclear matrix in vitro, and the position of DNAasel hypersensitive chromatin sites (DHSs). The pattern of DHSs changes depending on the transcriptional status of the gene. Functional studies demonstrated that DHSs mark the position of cis-acting regulatory elements. Additionally, we discovered a novel cis-activity of the border regions of the DNAasel sensitive domain (A-elements). By eliminating the position effect on gene expression usually observed when genes are randomly integrated into the genome after transfection, A-elements possibly serve as punctuation marks for a regulatory chromatin domain. Experiments using transgenic mice confirmed that the complete structurally defined lysozyme gene domain behaves as an independent regulatory unit, expressing the gene in a tissue specific and position independent manner. These expression features were lost in transgenic mice carrying a construct, in which the A-elements as well as an upstream enhancer region were deleted, indicating the lack of a locus activation function on this construct. Experiments are designed in order to uncover possible hierarchical relationships between the different cis-acting regulatory elements for stepwise gene activation during cell differentiation. We are aiming at the definition of the basic structural and functional requirements for position independent and high level gene expression. The result of these experiments will have important consequences for random gene transfer with predictable and reproducible expression of transgenes.