Midline axon guidance and human genetic disorders.

In bilaterally symmetric animals, many axons cross the midline to interconnect the left and right sides of the central nervous system (CNS). This process is critical for the establishment of neural circuits that control the proper integration of information perceived by the organism and the resulting response. While neurons at different levels of the CNS project axons across the midline, the molecules that regulate this process are common to many if not all midline-crossing regions. This article reviews the molecules that function as guidance cues at the midline in the developing vertebrate spinal cord, cortico-spinal tract and corpus callosum. As well, we describe the mutations that have been identified in humans that are linked to axon guidance and midline-crossing defects.

Transcription factor SCL is required for c-kit expression and c-Kit function in hemopoietic cells.

In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the expression of the basic helix-loop-helix transcription factor SCL/Tal1 correlates with that of c-Kit, the receptor for Steel factor (SF) or stem cell factor. To address the possibility that SCL may function upstream of c-kit, we sought to modulate endogenous SCL function in the CD34(+) hemopoietic cell line TF-1, which requires SF, granulocyte/macrophage colony-stimulating factor, or interleukin 3 for survival. Ectopic expression of an antisense SCL cDNA (as-SCL) or a dominant negative SCL (dn-SCL) in these cells impaired SCL DNA binding activity, and prevented the suppression of apoptosis by SF only, indicating that SCL is required for c-Kit-dependent cell survival. Consistent with the lack of response to SF, the level of c-kit mRNA and c-Kit protein was significantly and specifically reduced in as-SCL- or dn-SCL- expressing cells. c-kit mRNA, c-kit promoter activity, and the response to SF were rescued by SCL overexpression in the antisense or dn-SCL transfectants. Furthermore, ectopic c-kit expression in as-SCL transfectants is sufficient to restore cell survival in response to SF. Finally, enforced SCL in the pro-B cell line Ba/F3, which is both SCL and c-kit negative is sufficient to induce c-Kit and SF responsiveness. Together, these results indicate that c-kit, a gene that is essential for the survival of primitive hemopoietic cells, is a downstream target of the transcription factor SCL.

Cooperative interaction between GATA-4 and GATA-6 regulates myocardial gene expression.

Two members of the GATA family of transcription factors, GATA-4 and GATA-6, are expressed in the developing and postnatal myocardium and are equally potent transactivators of several cardiac promoters. However, several in vitro and in vivo lines of evidence suggest distinct roles for the two factors in the heart. Since identification of the endogenous downstream targets of GATA factors would greatly help to elucidate their exact functions, we have developed an adenovirus-mediated antisense strategy to specifically inhibit GATA-4 and GATA-6 protein production in postnatal cardiomyocytes. Expression of several endogenous cardiac genes was significantly down-regulated in cells lacking GATA-4 or GATA-6, indicating that these factors are required for the maintenance of the cardiac genetic program. Interestingly, transcription of some genes like the alpha- and beta-myosin heavy-chain (alpha- and beta-MHC) genes was preferentially regulated by GATA-4 due, in part, to higher affinity of GATA-4 for their promoter GATA element. However, transcription of several other genes, including the atrial natriuretic factor and B-type natriuretic peptide (ANF and BNP) genes, was similarly down-regulated in cardiomyocytes lacking one or both GATA factors, suggesting that GATA-4 and GATA-6 could act through the same transcriptional pathway. Consistent with this, GATA-4 and GATA-6 were found to colocalize in postnatal cardiomyocytes and to interact functionally and physically to provide cooperative activation of the ANF and BNP promoters. The results identify for the first time bona fide in vivo targets for GATA-4 and GATA-6 in the myocardium. The data also show that GATA factors act in concert to regulate distinct subsets of genes, suggesting that combinatorial interactions among GATA factors may differentially control various cellular processes.

Cardiac tissue enriched factors serum response factor and GATA-4 are mutual coregulators.

Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal alpha-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.

The developmentally regulated avian protein IFAPa-400 is transitin.

Transitin and IFAPa-400 are developmentally regulated high M(r) proteins expressed transiently in early chick embryogenesis. Both are associated with radially oriented fibers in the developing CNS and with various neural and myogenic tissues before their down-regulation at later stages. Previous studies have shown that IFAPa-400 colocalized and copurified with intermediate filament proteins and recent molecular cloning has indicated that transitin is a member of this family of cytoskeletal proteins. Here, we provide evidence that IFAPa-400 and transitin are the same protein. The sequence of a composite cDNA corresponding to more than 700 amino acids of IFAPa-400 carboxy-terminal extremity is identical to that of transitin. Both proteins exhibit identical apparent M(r) and isoelectric point. Immunopurified IFAPa-400 reacts with different antibodies to transitin and vice-versa. The patterns of expression of both proteins show a perfect coincidence at the tissue level. At the subcellular level, most antibodies to IFAPa-400/transitin decorate a typical intermediate filament network. However, monoclonal antibody A2B11, at the origin of transitin identification, exhibits a staining more typical of a cortical component, suggesting that different populations of transitin exist within the cell.

Postoperative radiotherapy for chondrosarcoma of the L1 vertebral body: a case report.

A 51-year-old man presented with a low-grade chondrosarcoma of the L1 vertebral body, which had been completely resected. Radiotherapy was indicated; however, the close proximity of critical structures such as the spinal cord and the kidneys complicated the delivery of the high radiation dose required. In this paper we present the clinical and technical aspects of the radiotherapy technique used to treat this patient; we also describe the patient immobilization and radiation dose verification techniques used. The patient was treated with 18 MV photons using multiple field arc rotations with fields asymmetric with respect to the rotational axis of the collimator head. The spinal cord was aligned along the horizontal axis of the linear accelerator isocenter, and the patient was immobilized using a 10 degrees styrofoam wedge combined with an Alpha Cradle mold. In this manner, the patient was stabilized in a comfortable position, which facilitated the daily check of the isocenter position within the patient. Radiation dose verification was carried out with films in an anthropomorphic phantom and with an ionization chamber in a water phantom. These measurements confirmed the accuracy of the isodose distributions calculated for the treatment with asymmetric fields in the arc therapy mode. The use of this technique in conjunction with the positioning device permitted the delivery of 64 Gy to the L1 vertebral body with relatively low doses to the critical structures, amounting to 32 Gy at the surface of the spinal cord and less than 16 Gy to half of both kidneys.

[Imaging of a foreign body in the submandibular space]. / Imagerie d'un corps étranger de la loge sous-maxillaire.

Foreign bodies in the oral cavity are infrequent in children. In spite of a negative clinical exploration, US and CT showed the suction cup of a rubber-tipped dart in the left submandibular space of a 3 year-old child. Surgery could be guided by these investigations.

Distal truncation of KCC3 in non-French Canadian HMSN/ACC families.

BACKGROUND: Hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC) is a severe and progressive autosomal recessive polyneuropathy. Mutations in the potassium-chloride cotransporter 3 gene (KCC3) were identified as responsible for HMSN/ACC in the French Canadian (FC) population. In the present study, the authors were interested in finding new mutations in non-FC populations, assessing the activity of mutant proteins and refining genotype-phenotype correlations. METHODS: The authors screened KCC3 for mutations using direct sequencing in six non-FC HMSN/ACC families. They then assessed the functionality of the most common mutant protein using a flux assay in Xenopus laevis oocytes. RESULTS: The authors identified mutations in exon 22 of KCC3: a novel mutation (del + 2994-3003; E1015X) in one family, as well as a known mutation (3031C-->T; R1011X) found in five unrelated families and associated with two different haplotypes. The function of the cotransporter was abolished, although a limited amount of mutant proteins were correctly localized at the membrane. CONCLUSIONS: KCC3 mutations in exon 22 constitute a recurrent mutation site for hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), regardless of ethnic origin, and are the most common cause of HMSN/ACC in the non-French Canadian (FC) families analyzed so far. Therefore, for genetic analysis, exon 22 screening should be prioritized in non-FC populations. Finally, the R1011X mutation leads to the abrogation of KCC3's function in Xenopus laevis oocytes, likely due to impaired transit of the cotransporter.

GATA transcription factors and cardiac development.

Three members of the GATA family of transcription factors, GATA-4, -5, and -6, are expressed in the developing heart. One family member, GATA-5, is restricted to the endocardium while the other two, GATA-4 and -6, are present in the myocardium where they apparently fulfil distinct functions. The mechanisms underlying GATA factor specificity are not fully understood but may involve interaction with other tissue-restricted or ubiquitous co-factors. Thus, combinatorial interaction among GATA factors or between GATA factors and other co-factors may differentially control various stages of cardiogenesis.

GATA-dependent recruitment of MEF2 proteins to target promoters.

The myocyte enhancer factor-2 (MEF2) proteins are MADS-box transcription factors that are essential for differentiation of all muscle lineages but their mechanisms of action remain largely undefined. In mammals, the earliest site of MEF2 expression is the heart where the MEF2C isoform is detectable as early as embryonic day 7.5. Inactivation of the MEF2C gene causes cardiac developmental arrest and severe downregulation of a number of cardiac markers including atrial natriuretic factor (ANF). However, most of these promoters contain no or low affinity MEF2 binding sites and they are not significantly activated by any MEF2 proteins in heterologous cells suggesting a dependence on a cardiac-enriched cofactor for MEF2 action. We provide evidence that MEF2 proteins are recruited to target promoters by the cell-specific GATA transcription factors, and that MEF2 potentiates the transcriptional activity of this family of tissue-restricted zinc finger proteins. Functional MEF2/GATA-4 synergy involves physical interaction between the MEF2 DNA-binding domain and the carboxy zinc finger of GATA-4 and requires the activation domains of both proteins. However, neither MEF2 binding sites nor MEF2 DNA binding capacity are required for transcriptional synergy. The results unravel a novel pathway for transcriptional regulation by MEF2 and provide a molecular paradigm for elucidating the mechanisms of action of MEF2 in muscle and non-muscle cells.

[Irrigation-lavage of the peritoneum. Use of a new type of drain]. / Irrigation-lavage du péritoine. utilisation d'un nouvaeu drain évacauteur.

A newly conceived voiding through drain is presented. It seems efficient in maintaining permeability of the drainage tube, without exclusion and thus allows for long standing irrigation and washout of the peritoneal cavity.

The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors.

The tissue-restricted GATA-4 transcription factor and Nkx2-5 homeodomain protein are two early markers of precardiac cells. Both are essential for heart formation, but neither can initiate cardiogenesis. Overexpression of GATA-4 or Nkx2-5 enhances cardiac development in committed precursors, suggesting each interacts with a cardiac cofactor. We tested whether GATA-4 and Nkx2-5 are cofactors for each other by using transcription and binding assays with the cardiac atrial natriuretic factor (ANF) promoter_the only known target for Nkx2-5. Co-expression of GATA-4 and Nkx2-5 resulted in synergistic activation of the ANF promoter in heterologous cells. The synergy involves physical Nkx2-5-GATA-4 interaction, seen in vitro and in vivo, which maps to the C-terminal zinc finger of GATA-4 and a C-terminus extension; similarly, a C-terminally extended homeodomain of Nkx2-5 is required for GATA-4 binding. Structure/function studies suggest that binding of GATA-4 to the C-terminus autorepressive domain of Nkx2-5 may induce a conformational change that unmasks Nkx2-5 activation domains. GATA-6 cannot substitute for GATA-4 for interaction with Nkx2-5. This interaction may impart functional specificity to GATA factors and provide cooperative crosstalk between two pathways critical for early cardiogenesis. Given the co-expression of GATA proteins and NK2 class members in other tissues, the GATA/Nkx partnership may represent a paradigm for transcription factor interaction during organogenesis.

Gonococcal growth factors in tissue cultures.

Growth of Neisseria gonorrhoeae in the presence of Rhesus monkey kidney (RE2) cells containing Eagle's minimal essential medium (MEM) 'conditioned' by 18-20 h of contact with RE2 cells began 3-4 h after inoculation and by 16-20 h had reached a maximum. When 'conditioned' medium (CM) was removed from the tissue cells and replaced with fresh medium a 5-6 h lag period occurred before multiplication of gonococci began and by 16 h growth was also near a maximum. CM removed from tissue cells supported the growth of six strains of N. gonorrhoeae. Three strains failed to grow in CM but multiplied when tissue cells were present. None of the strains grew in the presence of tissue cells killed by heating or in tissue-culture medium alone. Of the six strains of N. gonorrhoeae which grew in CM, three kept as their major (90%) population T1 colonies, while the remaining strains produced T1 organisms and T1A, T3, and T4 colonial variants. These results indicate that tissue cells secrete growth factors which support the growth and maintenance of T1 colonial forms of certain strains of N. gonnorhoeae.

Tissue-specific GATA factors are transcriptional effectors of the small GTPase RhoA.

Rho-like GTPases play a pivotal role in the orchestration of changes in the actin cytoskeleton in response to receptor stimulation, and have been implicated in transcriptional activation, cell growth regulation, and oncogenic transformation. Recently, a role for RhoA in the regulation of cardiac contractility and hypertrophic cardiomyocyte growth has been suggested but the mechanisms underlying RhoA function in the heart remain undefined. We now report that transcription factor GATA-4, a key regulator of cardiac genes, is a nuclear mediator of RhoA signaling and is involved in the control of sarcomere assembly in cardiomyocytes. Both RhoA and GATA-4 are essential for sarcomeric reorganization in response to hypertrophic growth stimuli and overexpression of either protein is sufficient to induce sarcomeric reorganization. Consistent with convergence of RhoA and GATA signaling, RhoA potentiates the transcriptional activity of GATA-4 via a p38 MAPK-dependent pathway that phosphorylates GATA-4 activation domains and GATA binding sites mediate RhoA activation of target cardiac promoters. Moreover, a dominant-negative GATA-4 protein abolishes RhoA-induced sarcomere reorganization. The identification of transcription factor GATA-4 as a RhoA mediator in sarcomere reorganization and cardiac gene regulation provides a link between RhoA effects on transcription and cell remodeling.

A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease.

Heterozygous Tbx5(del/+) mice were generated to study the mechanisms by which TBX5 haploinsufficiency causes cardiac and forelimb abnormalities seen in Holt-Oram syndrome. Tbx5 deficiency in homozygous mice (Tbx5(del/del)) decreased expression of multiple genes and caused severe hypoplasia of posterior domains in the developing heart. Surprisingly, Tbx5 haploinsufficiency also markedly decreased atrial natriuretic factor (ANF) and connexin 40 (cx40) transcription, implicating these as Tbx5 target genes and providing a mechanism by which 50% reduction of T-box transcription factors cause disease. Direct and cooperative transactivation of the ANF and cx40 promoters by Tbx5 and the homeodomain transcription factor Nkx2-5 was also demonstrated. These studies provide one potential explanation for Holt-Oram syndrome conduction system defects, suggest mechanisms for intrafamilial phenotypic variability, and account for related cardiac malformations caused by other transcription factor mutations.