minibrain: a new protein kinase family involved in postembryonic neurogenesis in Drosophila.

The development of the adult central nervous system of Drosophila requires a precise and reproducible pattern of neuroblast proliferation during postembryonic neurogenesis. We show here that mutations in the minibrain (mnb) gene cause an abnormal spacing of neuroblasts in the outer proliferation center (opc) of larval brain, with the implication that mnb opc neuroblasts produce less neuronal progeny than do wild type. As a consequence, the adult mnb brain exhibits a specific and marked size reduction of the optic lobes and central brain hemispheres. The insufficient number of distinct neurons in mnb brains is correlated with specific abnormalities in visual and olfactory behavior. The mnb gene encodes a novel, cell type-specific serine-threonine protein kinase family that is expressed and required in distinct neuroblast proliferation centers during postembryonic neurogenesis. The mnb kinases share extensive sequence similarities with kinases involved in the regulation of cell division.

Functional recovery of troponin I in a Drosophila heldup mutant after a second site mutation.

To identify proteins that interact in vivo with muscle components we have used a genetic approach based on the isolation of suppressors of mutant alleles of known muscle components. We have applied this system to the case of troponin I (TnI) in Drosophila and its mutant allele heldup2 (hdp2). This mutation causes an alanine to valine substitution at position 116 after a single nucleotide change in a constitutive exon. Among the isolated suppressors, one of them results from a second site mutation at the TnI gene itself. Muscles endowed with TnI mutated at both sites support nearly normal myofibrillar structure, perform notably well in wing beating and flight tests, and isolated muscle fibers produce active force. We show that the structural and functional recovery in this suppressor does not result from a change in the stoichiometric ratio of TnI isoforms. The second site suppression is due to a leucine to phenylalanine change within a heptameric leucine string motif adjacent to the actin binding domain of TnI. These data evidence a structural and functional role for the heptameric leucine string that is most noticeable, if not specific, in the indirect flight muscle.

A tropomyosin-2 mutation suppresses a troponin I myopathy in Drosophila.

A suppressor mutation, D53, of the held-up(2) allele of the Drosophila melanogaster Troponin I (wupA) gene is described. D53, a missense mutation, S185F, of the tropomyosin-2, Tm2, gene fully suppresses all the phenotypic effects of held-up(2), including the destructive hypercontraction of the indirect flight muscles (IFMs), a lack of jumping, the progressive myopathy of the walking muscles, and reductions in larval crawling and feeding behavior. The suppressor restores normal function of the IFMs, but flight ability decreases with age and correlates with an unusual, progressive structural collapse of the myofibrillar lattice starting at the center. The S185F substitution in Tm2 is close to a troponin T binding site on tropomyosin. Models to explain suppression by D53, derived from current knowledge of the vertebrate troponin-tropomyosin complex structure and functions, are discussed. The effects of S185F are compared with those of two mutations in residues 175 and 180 of human alpha-tropomyosin 1 which cause familial hypertrophic cardiomyopathy (HCM).

The behaving brain of a fly.

The use of Drosophila as a suitable system to answer behavioural questions is usually based on the availability of mutant phenotypes. Indeed, the 'single-gene' approach to behaviour was a very illuminating strategy in practical and conceptual terms, and served to prove that the genetic mechanisms sustaining behaviour could be analysed. However, the essence of neurogenetics goes for beyond the utilitarian use of mutants as tools to dissect behavior. Our main contention is that the study of the genetic basis of behaviour requires the study of genomes, rather than single genes, and their functional organization. Here, we use two aspects of behaviour, olfaction and movement control, as examples to illustrate the intricate, albeit understandable, relationship between the genome and behaviour. At present, these examples offer only a glimpse into this relationship. Further progress might be reached if studies on the regulation of functionally related genes are undertaken. On these grounds, it appears that the answer to many fundamental questions about behaviour that are amendable to experimentation might come from the work on Drosophila, providing that the required multidisciplinary efforts are focused on this organism.

The haplolethal region at the 16F gene cluster of Drosophila melanogaster: structure and function.

Extensive aneuploid analyses had shown the existence of a few haplolethal (HL) regions and one triplolethal region in the genome of Drosophila melanogaster. Since then, only two haplolethals, 22F1-2 and 16F, have been directly linked to identified genes, dpp and wupA, respectively. However, with the possible exception of dpp, the actual bases for this dosage sensitivity remain unknown. We have generated and characterized dominant-lethal mutations and chromosomal rearrangements in 16F and studied them in relation to the genes in the region. This region extends along 100 kb and includes at least 14 genes. The normal HL function depends on the integrity of a critical 4-kb window of mostly noncoding sequences within the wupA transcription unit that encodes the muscle protein troponin I (TNI). All dominant lethals are breakpoints within that window, which prevent the functional expression of TNI and other adjacent genes in the proximal direction. However, independent mutations in these genes result in recessive lethal phenotypes only. We propose that the HL at 16F represents a long-range cis regulatory region that acts upon a number of functionally related genes whose combined haploidy would yield the dominant-lethal effect.

[Aortic coarctation and postcoarctation mycotic aneurysm in a woman 20 weeks pregnant: anesthetic management]. / Coartación aórtica y aneurisma micótico postcoartación en gestante de 20 semanas: manejo anestésico.

A 28-year-old woman, 20 weeks pregnant, was diagnosed with aortic coarctation and postcoarctation mycotic aneurysm. After anesthetic induction, blood pressure was monitored in the radial artery of the right arm and the femoral artery of the right leg for two purposes: to verify hemodynamic stability as required in this type of operation and to determine the pressure gradient between the upper and lower limbs, which was approximately 40 mm Hg. To prevent spinal cord ischemia, an intradural catheter was inserted into the fourth and fifth lumbar space for spinal fluid drainage. A double lumen tube was used for intubation so that the left lung could be blocked, and a centrifugal pump was used instead of extracorporeal circulation. The aneurysm was resected through a left thoracotomy and an aortic prosthesis was placed. Satisfactory outcome was indicated by resolution of the pressure gradient, and fetal viability was verified by ultrasound. The mother suffered no neurological complications and the pregnancy continued to term uneventfully. We review the anesthetic procedure to follow in such cases.

Histological findings of birdshot chorioretinopathy in an eye with ciliochoroidal melanoma.

PURPOSE: To describe the histological findings of birdshot chorioretinopathy. DESIGN/PARTICIPANT: This is a case study of a single patient who has both birdshot chorioretinopathy and ciliochoroidal melanoma. METHODS: A 55-year-old woman who was HLA-A29 positive and had birdshot chorioretinopathy had a large ciliochoroidal melanoma (T4b N0 M0) and underwent enucleation. OUTCOME MEASURES: Using histopathology, we hope to further define the pathological findings in an eye with both birdshot chorioretinopathy and coexistant ciliochoroidal melanomas. RESULTS: The eye showed a ciliochoroidal melanoma. In addition, elsewhere, there were multiple choroidal nodules of lymphocytes that showed the presence of CD3-positive cells, which also stained for CD4 or CD8. There were only a few CD20-positive B cells and rare CD68-positive histiocytes. No granulomas were present. DISCUSSION: To our knowledge, there are only two previous reports describing the histological findings in birdshot chorioretinopathy: one that was HLA-A29 negative showing choroidal granulomas and another that was HLA-A29 positive exhibiting histological findings similar to our case. Incidentally, the latter case had a history of cutaneous melanoma. CONCLUSION: Birdshot chorioretinopathy is a nongranulomatous nodular infiltration of the choroid.

The expression of Ultrabithorax (Ubx) during development of the nervous system of Drosophila.

Using a double-staining technique with the neuron-specific monoclonal antibody 22C10 and the anti-Ubx monoclonal antibody FP 3-38 we describe the development of landmark cells in the nervous system of Drosophila. The staining with MAb 22C10 provides an internal system of reference that allows a precise localization of the most prominent Ubx active cells. The expression of Ubx seems to initiate at the segmental border in the hypoderm and the homologous region in the neuromeres. Also, the extent of Ubx expression follows quantitative and qualitative changes during embryonic development.

The pattern of early neuronal differentiation in Drosophila melanogaster.

Based on the staining of Drosophila embryos with neuron-specific monoclonal antibodies we describe the differentiation of the earliest neurons in the central nervous system. The metameric array undergoes a number of changes during development that distinguish several morphological units: metameres, neuromeres and ganglia. Neural landmarks in the CNS and the periphery are identified on the bases of their developmental history, axonal projection and segment specificity. A time sequence of differentiation starting in the posterior gnathal and anterior thoracic anlage has been found.

Single neuron mosaics of the drosophila gigas mutant project beyond normal targets and modify behavior.

gigas is a lethal mutant that differentiates enlarged cells, including the nucleus. This trait manifests only after the completion of the mitotic program. We have taken advantage of this phenotype to test in vivo the capacity of normal target cells to arrest the growth of mutant sensory axons. Single neuron connectivity changes have been analyzed in mosaics after horseradish peroxidase retrograde tracings. A mutant mechanoreceptor neuron, growing over a genetically normal substrate, contacts its normal target, and in addition projects to novel areas of the CNS. The mutant axon does terminate its growth eventually, and the new additional targets that are reached correspond to mechanoreceptor domains in other ganglia, indicating that this territorial constraint is operational in the mutant. gigas neurons maintain their stereotyped profile and represent an expanded version of the normal branching pattern. The ultrastructure of the invading projections does not reveal gliotic or necrotic reactions from the new cell contacts. The functional consequences of the connectivity changes produced by the mutant mechanoreceptors have been studied in grooming behavior. Mosaic flies carrying a single gigas mechanoreceptor show modified, albeit context-coherent, grooming responses after stimulation of the mutant bristle, whereas the response from neighboring normal sensory neurons remains unchanged. All of these experiments indicate that target recognition and growth arrest are two dissectible processes of neural development, and they highlight the autonomous features of the growth cone during pathfinding.

The presynaptic cell determines the number of synapses in the Drosophila optic ganglia.

The role of the pre- and postsynaptic cells in determining the number of synapses has been investigated in retina mosaics of the gigas (gig) mutant of Drosophila. Mutant photoreceptors are two to three times larger than those of the wild type, while adjacent cells in the mosaic retina and the lamina are normal in size. Serial electron microscope reconstructions of mosaic lamina cartridges show that gig photoreceptors establish more synapses upon lamina neurons than the normal photoreceptors do. By contrast, the number of feedback synapses that lamina neurons make onto gig photoreceptors does not increase. The increment in the number of synapses correlates positively with the increment of presynaptic cell membrane, resulting in constancy of synapse density. The phototactic response of flies bearing a gig eye is abnormal, indicating that the extra synapses are functional.

The level of DLDB/CHIP controls the activity of the LIM homeodomain protein apterous: evidence for a functional tetramer complex in vivo.

The LIM homeodomain (LIM-HD) protein Apterous (Ap) and its cofactor DLDB/CHIP control dorso- ventral (D/V) patterning and growth of Drosophila wing. To investigate the molecular mechanisms of Ap/CHIP function we altered their relative levels of expression and generated mutants in the LIM1, LIM2 and HD domains of Ap, as well as in the LIM-interacting and self-association domains of CHIP. Using in vitro and in vivo assays we found that: (i) the levels of CHIP relative to Ap control D/V patterning; (ii) the LIM1 and LIM2 domains differ in their contributions to Ap function; (iii) Ap HD mutations cause weak dominant negative effects; (iv) overexpression of ChipDeltaSAD mutants mimics Ap lack-of-function, and this dominant negative phenotype is caused by titration of Ap because it can be rescued by adding extra Ap; and (v) overexpression of ChipDeltaLID mutants also causes an Ap lack-of-function phenotype, but it cannot be rescued by extra Ap. These results support the model that the Ap-CHIP active complex in vivo is a tetramer.

The 28-kDa protein whose phosphorylation is induced by protein kinase C activators in MCF-7 cells belongs to the family of low molecular mass heat shock proteins and is the estrogen-regulated 24-kDa protein.

We have previously reported the presence of a 28-kDa protein in human mammary adenocarcinoma MCF-7 cells, whose phosphorylation by phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and permeant diacylglycerol 1,2-dioctanoyl-sn-glycerol was correlated to growth arrest induced by the protein kinase C (PKC) activators. We now investigate the possible identity of this protein with the estrogen-regulated "24-kDa" protein shown as related to the mammalian heat shock protein 27 (Fuqua, S. A. W., Blum-Salingaros, M., and McGuire, W. L. (1989) Cancer Res 49, 4126-4129). 32P-Labeled 28-kDa protein from TPA-treated MCF-7 cells was immunoprecipitated with a 24-kDa-specific monoclonal antibody. Immunoblots from cell extracts fractionated by two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis demonstrated that TPA induced the conversion of a 28-kDa isoform "a" (pI 6.7) to a more acidic isoform "b" (pI 6.2). Two-dimensional gel analysis of [3H]leucine-labeled MCF-7 cell extracts demonstrated that conversely to TPA, which induced only phosphorylation of 28-kDa protein, heat shock induced both synthesis (increase of isoform a) and phosphorylation (conversion of isoforms a to b) of the protein. 32P labeling of MCF-7 cells allowed demonstration of the presence of an extra phosphoisoform "c" (pI 5.9) upon TPA as well as heat shock treatment. When cells were pretreated with the bisindolylmaleimide GF109203X, a selective inhibitor of PKC, the heat shock-induced phosphorylation was unchanged, while the TPA effect was almost abolished, suggesting that the heat shock-activated protein kinase was very likely different from PKC. However, peptide mapping of the 28-kDa phosphoprotein suggested identical sites of phosphorylation upon TPA and heat shock stimulation. Partial amino acid sequencing of the 28-kDa protein revealed identity with both the 24-kDa protein and the mammalian HSP27. The fact that estrogens and PKC, respectively, regulate expression and phosphorylation of this 24/28-kDa protein strongly argues for its key role in MCF-7 cell proliferation and differentiation.

Shaker encodes a family of putative potassium channel proteins in the nervous system of Drosophila.

The Shaker locus of Drosophila contains a very large transcription unit. It is expressed predominantly in the nervous system by multiple, differential as well as alternative, splicing mechanisms into different, but functionally related proteins. The structure of the Shaker transcription unit and the properties of the encoded Shaker protein family provide a molecular basis for A channel diversity in excitable cells.

Troponin I is encoded in the haplolethal region of the Shaker gene complex of Drosophila.

We have analyzed one of the nine complementation groups that constitute the haplolethal (HL) region of the Shaker gene complex (ShC). Five mutations, including a dominant lethal, define this complementation group: HL I. Mutant phenotypes show abnormal embryogenesis with structural defects in the nervous system and aberrant degeneration of specific adult muscles in addition to altered action potentials. HL I encodes a family of proteins with extensive homology to invertebrate troponin I (TnI). Members of this family are brought about by two alternative and two mutually exclusive exons in conjunction with two differential polyadenylation sites. Transcription analysis indicates that some isoforms are adult specific and others are synthesized throughout development, except during early metamorphosis. Certain isoforms of Drosophila TnI are expressed in specific muscles. The specificity of mutant phenotypes suggests a functional role of particular TnI isoforms in the development and the mature activity of muscle and nervous systems.

Conservation of the expression and function of apterous orthologs in Drosophila and mammals.

The Drosophila apterous (ap) gene encodes a protein of the LIM-homeodomain family. Many transcription factors of this class have been conserved during evolution; however, the functional significance of their structural conservation is generally not known. ap is best known for its fundamental role as a dorsal selector gene required for patterning and growth of the wing, but it also has other important functions required for neuronal fasciculation, fertility, and normal viability. We isolated mouse (mLhx2) and human (hLhx2) ap orthologs, and we used transgenic animals and rescue assays to investigate the conservation of the Ap protein during evolution. We found that the human protein LHX2 is able to regulate correctly ap target genes in the fly, causes the same phenotypes as Ap when ectopically produced, and most importantly rescues ap mutant phenotypes as efficiently as the fly protein. In addition, we found striking similarities in the expression patterns of the Drosophila and murine genes. Both mLhx2 and ap are expressed in the respective nerve cords, eyes, olfactory organs, brain, and limbs. These results demonstrate the conservation of Ap protein function across phyla and argue that aspects of its expression pattern have also been conserved from a common ancestor of insects and vertebrates.

Identification of genes that modify ataxin-1-induced neurodegeneration.

A growing number of human neurodegenerative diseases result from the expansion of a glutamine repeat in the protein that causes the disease. Spinocerebellar ataxia type 1 (SCA1) is one such disease-caused by expansion of a polyglutamine tract in the protein ataxin-1. To elucidate the genetic pathways and molecular mechanisms underlying neuronal degeneration in this group of diseases, we have created a model system for SCA1 by expressing the full-length human SCA1 gene in Drosophila. Here we show that high levels of wild-type ataxin-1 can cause degenerative phenotypes similar to those caused by the expanded protein. We conducted genetic screens to identify genes that modify SCA1-induced neurodegeneration. Several modifiers highlight the role of protein folding and protein clearance in the development of SCA1. Furthermore, new mechanisms of polyglutamine pathogenesis were revealed by the discovery of modifiers that are involved in RNA processing, transcriptional regulation and cellular detoxification. These findings may be relevant to the treatment of polyglutamine diseases and, perhaps, to other neurodegenerative diseases, such as Alzheimer's and Parkinson's disease.