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Biomedical subjects

A Chandrasekhar

Publications and source records attributed to A Chandrasekhar.

15 recordsLinked to original sources
PubMed2004

Hedgehog and Fgf signaling pathways regulate the development of tphR-expressing serotonergic raphe neurons in zebrafish embryos.

Serotonin (5HT) plays major roles in the physiological regulation of many behavioral processes, including sleep, feeding, and mood, but the genetic mechanisms by which serotonergic neurons arise during development are poorly understood. In the present study, we have investigated the development of serotonergic neurons in the zebrafish. Neurons exhibiting 5HT-immunoreactivity (5HT-IR) are detected from 45 h postfertilization (hpf) in the ventral hindbrain raphe, the hypothalamus, pineal organ, and pretectal area. Tryptophan hydroxylases encode rate-limiting enzymes that function in the synthesis of 5HT. As part of this study, we cloned and analyzed a novel zebrafish tph gene named tphR. Unlike two other zebrafish tph genes (tphD1 and tphD2), tphR is expressed in serotonergic raphe neurons, similar to tph genes in mammalian species. tphR is also expressed in the pineal organ where it is likely to be involved in the pathway leading to synthesis of melatonin. To better understand the signaling pathways involved in the induction of the serotonergic phenotype, we analyzed tphR expression and 5HT-IR in embryos in which either Hh or Fgf signals are abrogated. Hindbrain 5HT neurons are severely reduced in mutants lacking activity of either Ace/Fgf8 or the transcription factor Noi/Pax2.1, which regulates expression of ace/fgf8, and probably other genes encoding signaling proteins. Similarly, serotonergic raphe neurons are absent in embryos lacking Hh activity confirming a conserved role for Hh signals in the induction of these cells. Conversely, over-activation of the Hh pathway increases the number of serotonergic neurons. As in mammals, our results are consistent with the transcription factors Nk2.2 and Gata3 acting downstream of Hh activity in the development of serotonergic raphe neurons. Our results show that the pathways involved in induction of hindbrain serotonergic neurons are likely to be conserved in all vertebrates and help establish the zebrafish as a model system to study this important neuronal class.

Animals↗
PubMed1999

Molecular cloning, expression, and activity of zebrafish semaphorin Z1a.

Semaphorins/collapsins are a large family of secreted and cell surface molecules that are thought to guide growth cones to their targets. Although some members are clearly repulsive to specific growth cones in vitro, the in vivo role of many of these molecules in vertebrate embryos is still unclear. As a first step towards clarifying the in vivo role of semaphorins/collapsins, we analyzed semaZ1a in the simple and well-characterized zebrafish embryo. SemaZ1a is a secreted molecule that is highly homologous to Sema III/D/collapsin-1, and it can collapse chick dorsal root ganglion growth cones in vitro. It is expressed in highly specific patterns within the developing embryo, which suggests that it influences outgrowth by a variety of growth cones including those of the posterior lateral line ganglion. Consistent with this hypothesis, the peripherally extending growth cones of posterior lateral line neurons retract and partially collapse during normal outgrowth.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and developmental expression of a zebrafish axonal glycoprotein similar to TAG-1.

TAG-1 is a mammalian cell adhesion molecule of the immunoglobulin superfamily that is expressed transiently by a subset of neurons and serves as a fertile substrate for neurite outgrowth in vitro (Furley, A.H., Morton, S.B., Manalo, D., Karagogeos, S., Dodd, H., Jessell, T.M., 1990 The axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth promoting activity. Cell 61, 157-170). In order to examine the in vivo function of this molecule, we have cloned a zebrafish tag1-like cDNA and analyzed its expression patterns. tag1 Is expressed transiently by specific subsets of neurons when they are projecting their axons or when they are migrating. The specific and dynamic pattern of expression of zebrafish tag1 is consistent with its proposed role in axon guidance and cell migration.

Animals↗
PubMed1999

The zebrafish detour gene is essential for cranial but not spinal motor neuron induction.

The zebrafish detour (dtr) mutation generates a novel neuronal phenotype. In dtr mutants, most cranial motor neurons, especially the branchiomotor, are missing. However, spinal motor neurons are generated normally. The loss of cranial motor neurons is not due to aberrant hindbrain patterning, failure of neurogenesis, increased cell death or absence of hh expression. Furthermore, activation of the Hh pathway, which normally induces branchiomotor neurons, fails to induce motor neurons in the dtr hindbrain. Despite this, not all Hh-mediated regulation of hindbrain development is abolished since the regulation of a neural gene by Hh is intact in the dtr hindbrain. Finally, dtr can function cell autonomously to induce branchiomotor neurons. These results suggest that detour encodes a component of the Hh signaling pathway that is essential for the induction of motor neurons in the hindbrain but not in the spinal cord and that dtr function is required for the induction of only a subset of Hh-mediated events in the hindbrain.

Animals↗
PubMed1998

Role of sonic hedgehog in branchiomotor neuron induction in zebrafish.

The role of zebrafish hedgehog genes in branchiomotor neuron development was analyzed by examining mutations that affect the expression of the hedgehog genes and by overexpressing these genes in embryos. In cyclops mutants, reduction in sonic hedgehog (shh) expression, and elimination of tiggy-winkle hedgehog (twhh) expression, correlated with reductions in branchiomotor neuron populations. Furthermore, branchiomotor neurons were restored in cyclops mutants when shh or twhh was overexpressed. These results suggest that Shh and/or Twhh play an important role in the induction of branchiomotor neurons in vivo. In sonic-you (syu) mutants, where Shh activity was reduced or eliminated due to mutations in shh, branchiomotor neurons were reduced in number in a rhombomere-specific fashion, but never eliminated. Similarly, spinal motor neurons were reduced, but not eliminated, in syu mutants. These results demonstrate that Shh is not solely responsible for inducing branchiomotor and spinal motor neurons, and suggest that Shh and Twhh may function as partially redundant signals for motor neuron induction in zebrafish.

Animals↗
PubMed1997

Development of branchiomotor neurons in zebrafish.

The mechanisms underlying neuronal specification and axonogenesis in the vertebrate hindbrain are poorly understood. To address these questions, we have employed anatomical methods and mutational analysis to characterize the branchiomotor neurons in the zebrafish embryo. The zebrafish branchiomotor system is similar to those in the chick and mouse, except for the location of the nVII and nIX branchiomotor neurons. Developmental analyses of genes expressed by branchiomotor neurons suggest that the different location of the nVII neurons in the zebrafish may result from cell migration. To gain insight into the mechanisms underlying the organization and axonogenesis of these neurons, we examined the development of the branchiomotor pathways in neuronal mutants. The valentino b337 mutation blocks the formation of rhombomeres 5 and 6, and severely affects the development of the nVII and nIX motor nuclei. The cyclops b16 mutation deletes ventral midline cells in the neural tube, and leads to a severe disruption of most branchiomotor nuclei and axon pathways. These results demonstrate that rhombomere-specific cues and ventral midline cells play important roles in the development of the branchiomotor pathways.

Animals↗
PubMed1995

A mutation that depresses cGMP phosphodiesterase activity in Dictyostelium affects cell motility through an altered chemotactic signal.

The streamer F (StmF) mutant of Dictyostelium discoideum is defective in cGMP-phosphodiesterase activity. In early aggregation territories, when individual cells chemotax toward aggregation centers prior to streaming, the average periodicity of surging of StmF cells is half that of wild-type cells. In addition, in the period between surges, which has been interpreted to include the peak and back of the wave, StmF cells abnormally remain nonmotile and retain their elongate shape. In contrast, in the period between surges wild-type cells form pseudopods randomly around their cell perimeter and take on an amorphous shape. Using a newly developed protocol for vitally staining and tracking individual mutant cells in unstained wild-type aggregation territories and individual wild-type cells in unstained StmF aggregation territories, we have found that mutant cells behave normally (i.e., like their predominant wild-type neighbors) in the deduced back of waves generated by wild-type cells. Conversely, wild-type cells behave aberrantly (i.e., like their predominant StmF neighbors) in the deduced back of waves generated by mutant cells. These results suggest that the defective behavior of StmF cells in early StmF aggregation territories is not due to a single cell defect in responsiveness, but, rather, is due either to the genesis of an aberrant cAMP wave or to the accumulation of a molecule which interferes with normal behavior in the back of the wave.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

Ponticulin plays a role in the positional stabilization of pseudopods.

Ponticulin is a 17-kD glycoprotein that represents a major high affinity link between the plasma membrane and the cortical actin network of Dictyostelium. To assess the role of ponticulin in pseudopod extension and retraction, the motile behavior of two independently generated mutants lacking ponticulin was analyzed using computer-assisted two- and three-dimensional motion analysis systems. More than half of the lateral pseudopods formed off the substratum by ponticulin-minus cells slipped relative to the substratum during extension and retraction. In contrast, all pseudopods formed off the substratum by wild-type cells were positionally fixed in relation to the substratum. Ponticulin-minus cells also formed a greater proportion of both anterior and lateral pseudopods off the substratum and absorbed a greater proportion of lateral pseudopods into the uropod than wild-type cells. In a spatial gradient of cAMP, ponticulin-minus cells were less efficient in tracking the source of chemoattractant. Since ponticulin-minus cells extend and retract pseudopods with the same time course as wild-type cells, these behavioral defects in ponticulin-minus cells appear to be the consequence of pseudopod slippage. These results demonstrate that pseudopods formed off the substratum by wild-type cells are positionally fixed in relation to the substratum, that ponticulin is required for positional stabilization, and that the loss of ponticulin and the concomitant loss of positional stability of pseudopods correlate with a decrease in the efficiency of chemotaxis.

Actins↗
PubMed1995

Functional analysis of the promoter of the phase-specific WH11 gene of Candida albicans.

Candida albicans WO-1 switches spontaneously, frequently, and reversibly between a hemispherical white and a flat gray (opaque) colony-forming phenotype. This transition affects a number of morphological and physiological parameters and involves the activation and deactivation of phase-specific genes. The WH11 gene is transcribed in the white but not the opaque phase. A chimeric WH11-firefly luciferase gene containing the 5' upstream region of WH11 was demonstrated to be under phase regulation regardless of the site of integration, and a series of promoter deletion constructs was used to delineate two white-phase-specific transcription activation domains. Gel retardation experiments with the individual distal or proximal domain and white-phase or opaque-phase protein extract demonstrated the formation of one distal white-phase-specific complex and two proximal white-phase-specific complexes. Specific subfragments were tested for their ability to compete with the entire domain in the formation of complexes with white-phase protein extract in order to map the proximal domain sequence involved in white-phase-specific complex formation. Our results indicate that white-phase-specific transcription of WH11 is positively regulated by trans-acting factors interacting with two cis-acting activation sequences in the WH11 promoter.

Animals↗
PubMed1992

Biological and molecular correlates between induced dedifferentiation and spore germination in Dictyostelium.

When developing cultures of Dictyostelium discoideum are disaggregated at any time prior to cell wall formation and challenged to reinitiate development, amoebae will progress through the original sequence of morphogenetic stages, but the second time through they will do so in roughly one-tenth the original time, a process known as 'rapid recapitulation'. However, if disaggregated cells are suspended in nutrient medium, they enter a program of dedifferentiation during which they lose the capacity to rapidly recapitulate after an 80 minute lag period in a process known as 'erasure'. Here we show that cells that have completed the morphogenetic program and emerge from spore coats in the process of germination have also erased. In addition, the germination-specific 270 gene family is expressed during induced dedifferentiation in a unique fashion, and a germination-defective mutant exhibits a dramatic delay in erasure without concomitant defects in the program of gene regulation accompanying induced dedifferentiation. These results suggest for the first time that induced dedifferentiation and spore germination share some common processes in converting cells from a developmental to vegetative state.

Animals↗
PubMed1992

Induction chemotherapy in non-metastatic high grade osteo sarcomas--results of pilot study at Cancer Institute (WIA), Madras.

Twelve patients with high grade osteosarcomas of the extremities were treated with two cycles of induction chemotherapy using adriamycin and cis-platinum and sandwich radiation between the two cycles (4000 rads). Ten patients underwent amputation or disarticulation, two patients had wide excision followed by endoprosthesis. The specimen was assessed for grade of necrosis. The Disease Free Survival at a minimum follow-up period of 26 months and median follow-up period of 35.5%. All the five patients who developed distant metastases had shown only a grade I necrosis in the tumour.

Adolescent↗
PubMed1990

Developmental mechanisms regulating the rapid decrease in a cohesion glycoprotein mRNA in Dictyostelium function primarily at the level of mRNA degradation.

During the morphogenetic program in Dictyostelium discoideum, the transcript of the gene encoding the cohesion glycoprotein gp80 accumulates to a maximum level between 4 and 6 hr, (just prior to the onset of aggregation), remains high between 6 and 10 hr (the ripple to loose aggregate stages), and then decreases to less than 10% of the maximum level between 10 and 12 hr (the tight aggregate stage). The level of gp80 transcript also decreases precipitously at the time of the erasure event in the program of dedifferentiation, or when cAMP is added to a dedifferentiating cell population prior to the erasure event. In the dedifferentiation-defective mutant HI4, the cAMP-stimulated system for rapidly reducing the level of gp80 transcript is intact, but the mechanism functioning at the time of the erasure event is defective, demonstrating that the two reduction mechanisms are dissociable. By comparing the levels of gp80 transcript with the levels of in vitro transcription of the gene in isolated nuclei, it is demonstrated that the rapid reduction of gp80 transcript immediately after aggregation and immediately after addition of 10(-4) M cAMP are the result of increased transcript degradation. The rapid reduction of gp80 transcript at the erasure event may also be due to increased transcript degradation, but transcriptional regulation cannot be completely ruled out in this case.

Cell Adhesion Molecules↗
PubMed1989

Dictyostelium erasure mutant HI4 abnormally retains development-specific mRNAs during dedifferentiation.

The Dictyostelium mutant HI4 progresses through morphogenesis normally, but is defective in the reverse program of dedifferentiation. In contrast to dedifferentiating wild-type cells, HI4 cells retain the capacity to rapidly reaggregate well after the "erasure event" employing a nonchemotactic aggregation mechanism involving random collisions and cohesion. They also do not lose contact sites A (gp80) at the prescribed time in the dedifferentiation program. HI4 cells accumulate transcripts of the cysteine protease gene CP2 (formerly referred to as 16G1) and the cohesion glycoprotein gene gp80 at the correct times in the morphogenetic program, but abnormally retain these transcripts at high levels well after the prescribed times at which they are lost in wild-type cells during the reverse program of dedifferentiation. The retention of these mRNAs in HI4 cells after the erasure event is not due to abnormal maintenance of a high level of intracellular cAMP during dedifferentiation. The rapid reduction in the level of gp80 transcript which can be effected by the addition of cAMP prior to the erasure event in wild-type cells is also retained by HI4 cells well after the erasure event. The results suggest that cells possess at least two mechanisms for the reduction of gp80 transcript. One involves the immediate response to cAMP and may function during the forward program of development. The second functions specifically during the reverse program of dedifferentiation. It is this latter, erasure-specific mechanism which is selectively defective in the HI4 variant.

Blotting, Northern↗
PubMedDate not supplied

Connexin expression and cell coupling fail to reverse the v-src transformed growth characteristics of a Cx43-/- cell line.

Gap junctions, composed of connexins, have been shown to suppress transformation in a variety of malignancies and transformed cell types. In addition, transforming factors such as the src oncogene have been shown to directly phosphorylate some connexins (e.g., Cx43) and inhibit coupling. To investigate the role of gap junctions in cell transformsation by v-src, we utilized a clonal cell line derived from Cx43 knockout mice (KoA) that was immortalized, but not transformed. Transfection by v-src induced a marked transformed phenotype characterized by growth in low serum and anchorage-independent conditions. Subsequent transfections by Cx43, Cx32 or vector alone were then tested for their effects on growth. Activity of pp60v-src was confirmed in all transfectants as well as the ability of pp60v-src to phosphorylate Cx43 in several clones. Despite the documented effect of pp60v-src on Cx43 channel closure, modest coupling was still retained in many of the Cx43 and Cx32 transfectants. However, none of the four Cx43 transfected clones showed significant inhibitory effects on proliferation in either anchorage-independent or low serum growth conditions. Of the Cx32 clones, only one in five showed effects on growth in both assays, which was the same ratio observed for the control transfectants. Thus, based on the levels of expression achieved, which were comparable to endogenous levels in established cell lines, neither Cx43 nor Cx32 serve as effective suppressors of the transformed growth phenotype of this v-src expressing cell line.

Animals↗