The International journal of developmental biology, 2020
Differential specification of dorsal flight appendages, wing and haltere, in Drosophila provides ... more Differential specification of dorsal flight appendages, wing and haltere, in Drosophila provides an excellent model system to address a number of important questions in developmental biology at the levels of molecules, pathways, tissues, organs, organisms and evolution. Here we discuss the mechanism by which the Hox protein Ubx recognizes and regulates its downstream targets, implications of the same in growth control at cellular and organ level and finally the evolution of haltere from ancestral hindwings in other holometabolous insects.
PTPN11/SHP2, a non-receptor protein tyrosine phosphatase is a prominent target of the receptor ty... more PTPN11/SHP2, a non-receptor protein tyrosine phosphatase is a prominent target of the receptor tyrosine kinase that participates in positive feedback signalling of the human epidermal growth factor receptors and helps in growth and migration. PTPN11/SHP2 is widely believed to be an oncoprotein, although it’s possible tumor-suppressor role is also reported. Our analysis of breast cancer metadata shows, PTPN11/SHP2 copy number loss in luminal A subtype is correlated to poor disease-specific survival and late-stage cancer at diagnosis. Analysis of the level 4 Reverse Phase Protein Array (RPPA) data available on the TCGA database resulted in positive correlations between the lower expression levels of constitutively active variant, the phospho-SHP2-Y542, of PTPN11/SHP2 and larger tumor size and lymph node positivity. We experimentally examined possible negative regulation of growth by PTPN11/SHP2 using MCF10A, a normal breast epithelial cell line. Knock-down of PTPN11/SHP2 resulted in i...
Promoter proximal pausing (PPP) of RNA polymerase II has emerged as a crucial rate-limiting step ... more Promoter proximal pausing (PPP) of RNA polymerase II has emerged as a crucial rate-limiting step in the regulation of gene expression. Regulation of PPP is brought about by complexes 7SK snRNP, P-TEFb (Cdk9/cycT), and the negative elongation factor (NELF), which are highly conserved from Drosophila to humans. Here, we show that RNAi-mediated depletion of bin3 or Hexim of the 7SK snRNP complex or depletion of individual components of the NELF complex enhances Yki-driven growth, leading to neoplastic transformation of Drosophila wing imaginal discs. We also show that increased CDK9 expression cooperates with Yki in driving neoplastic growth. Interestingly, overexpression of CDK9 on its own or in the background of depletion of one of the components of 7SK snRNP or the NELF complex necessarily, and specifically, needed Yki overexpression to cause tumorous growth. Genome-wide gene expression analyses suggested that deregulation of protein homeostasis is associated with tumorous growth of...
We have shown that the expression of the 412 retrotransposon provides a useful early marker for t... more We have shown that the expression of the 412 retrotransposon provides a useful early marker for the development of the gonadal mesoderm in Drosophila embryos. 412 is initially expressed in a set of parasegmentally repeated stripes from parasegments (PS) 2–14 in the mesoderm at the extended germ band stage. During germ band retraction the bulk of 412 expression declines except in dorsolateral clusters of cells in PS10, 11 and 12, where high levels of 412 expression remain. These mesodermal cell clusters are associated with germ cells and subsequently they coalesce, rounding up to form the gonads. The gonadal mesoderm thus appears to originate specifically from three abdominal parasegments, PS10, 11 and 12. We show that the maintenance of high levels of 412 expression in gonadal mesoderm is not induced by contact with germ cells, but rather depends on genetic control by the homeotic genes abdominal-A and Abdominal-B.
Genetic approaches in Drosophila have successfully identified many genes involved in regulation o... more Genetic approaches in Drosophila have successfully identified many genes involved in regulation of growth control as well as genetic interactions relevant to the initiation and progression of cancer in vivo. Here, we report on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: the Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo. We also report on an independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified here are known to be functional in growth control pathways. We also find a number of novel connections to Yki and EGFR driven tissue growth, mostly unique to one of the two. Thus, resourc...
Genetic approaches in Drosophila have successfully identified many genes involved in regulation o... more Genetic approaches in Drosophila have successfully identified many genes involved in regulation of growth control as well as genetic interactions relevant to the initiation and progression of cancer in vivo. Here, we report on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: The Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo. We also report on an independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified here are known to be functional in growth control pathways. We also find a number of novel connections to Yki and EGFR driven tissue growth, mostly unique to one of the two. Thus, resourc...
Scotin is a pro-apoptotic mammalian gene, which is induced upon DNA damage or cellular stress in ... more Scotin is a pro-apoptotic mammalian gene, which is induced upon DNA damage or cellular stress in a p53-dependent manner. In this report, we have used Drosophila as a model system to obtain a preliminary insight into the molecular mechanism of Scotin function, which was further validated using the mammalian system. Targeted expression of Scotin in developing Drosophila induced apoptosis and developmental defects in wings and eyes. Co-expression of Scotin with the anti-apoptotic protein p35, while inhibited the apoptosis in both dividing and non-dividing cells, rescued adult wing or eye phenotypes only when Scotin was expressed in nondividing cells. This suggests that mechanisms of Scotin-induced apoptosis in dividing and non-dividing cells may vary. Suppressorenhancer screen using cell cycle regulators suggested that Scotin may mediate cell cycle arrest at both G 1 /S and G 2 /M phases. Overexpression of Scotin in mammalian cells resulted in mitotic arrest and subsequently apoptosis. Furthermore, a larger proportion of cells overexpressing Scotin showed sequestration of Cyclin B1 in the cytoplasm. These results suggest that one of the ways by which Scotin induces apoptosis is by causing cell cycle arrest.
The International Journal of Developmental Biology, 2011
Thorax closure in Drosophila is a process during adult morphogenesis in which the anterior ends o... more Thorax closure in Drosophila is a process during adult morphogenesis in which the anterior ends of the presumptive notum of the two wing imaginal discs fuse to make a seamless thorax. Similar to dorsal closure during embryogenesis, this process is regulated by Decapentaplegic and JNK signaling pathways. Despite the fact that Peripodial Membrane (PM) cells do not contribute to the formation of any adult structure, they are known to facilitate the process of thorax closure. Here we show that JNK signaling is activated only in a subset of PM cells, known as medial edge cells. While the mechanism that activates JNK signaling specifically in the medial edge cells of the PM is still not understood, the results presented here show that the pair rule gene odd skipped is required to ensure that JNK signaling is not activated anywhere else in the wing disc. Medial edge cells of the PM are elongated in shape, while the remaining PM cells are hexagonal. Down regulation of JNK signaling in the medial edge cells results in defective thorax closure in adult flies. It also causes the transformation of the morphology of medial edge cells into hexagonal shape. Conversely, activation of JNK signaling in hexagonal cells of the PM causes transformation of their morphology to elongated shape. Thus, similar to dorsal closure during embryogenesis, JNKmediated elongation of medial edge cells is functionally correlated to the process of thorax closure.
Proceedings of the National Academy of Sciences, 1991
The expression and subcellular location of porphobilinogen deaminase (PBGD, also known as hydroxy... more The expression and subcellular location of porphobilinogen deaminase (PBGD, also known as hydroxymethylbilane synthase; EC 4.3.1.8), one of the early enzymes of porphyrin synthesis, was investigated in light-grown Euglena and in three cell lines that do not contain chlorophyll: dark-grown Euglena, a streptomycin-bleached mutant, and Astasia longa. In wild-type Euglena, immunogold electron microscopy demonstrated that all the immunodetectable enzyme protein was in the chloroplast. PBGD was shown to be photoregulated, and like many other nuclear-encoded proteins in Euglena, the regulation was at the posttranscriptional level. In the three nonchlorophyllous cell lines, as in light-grown Euglena, a single protein of 40 kDa was detected with antiserum to PBGD. This same antiserum immunoprecipitated a larger precursor protein from the total translation products of poly(A)+ RNA, and a single transcript, which was large enough to encode the precursor, was detected on Northern blots of all f...
Mutations in human Adenomatous Polyposis Coli (APC) gene are associated with both familial and sp... more Mutations in human Adenomatous Polyposis Coli (APC) gene are associated with both familial and sporadic colorectal tumors. APC is known to down regulate bcatenin levels, a transducer of Wnt signaling. The aim of this study is to provide transgenic Drosophila expressing either full-length or truncated forms of human APC (hAPC) protein and methods for using them in functional genomics and drug screening. Consistent with its biochemical properties, targeted expression of either full-length hAPC or its b-catenin binding domain alone negatively regulated the function of the b-catenin homologue, Armadillo (Arm) and thereby, inhibited Wnt/Wg signaling during¯y development. hAPC inhibited Arm function even in the absence of GSK-3b activity, although the latter was required to mediate the degradation of Arm. Consistent with this, hAPC suppressed the phenotypes induced by the over-expression of degradation-resistant forms of Arm. Subsequently, using hAPC-induced eye phenotypes as the assay in a suppressor-enhancer screen, we have identi®ed two new loci in Drosophila, which modulate Wnt/Wg signaling. In addition, an anti-colon cancer drug, indomethacin, speci®cally enhanced hAPC-induced phenotypes.
In both vertebrates and invertebrates, homeotic selector genes confer morphological differences a... more In both vertebrates and invertebrates, homeotic selector genes confer morphological differences along the antero-posterior axis. However, insect wing development is independent of all homeotic gene functions, reflecting the ground plan of an ancestral pterygote, which bore wings on all segments. Dipteran insects such as Drosophila are characterized by a pair of wings in the mesothoracic segment. In all other segments, wing development is essentially repressed by different homeotic genes, although in the metathorax they are modi®ed into a pair of halteres. This necessitates that during development all homeotic genes are to be maintained in a repressed state in wing imaginal discs. In this report we show that (i) the function of the segment polarity gene engrailed (en) is critical to keep the homeotic selector gene Ultrabithorax (Ubx) repressed in wing imaginal discs, (ii) normal levels of En in the posterior compartment of haltere discs, however, are not enough to completely repress Ubx, and (iii) the repression of Ubx by en is independent of Hedgehog signalling through which the longrange signalling of en is mediated during wing development. Finally we provide evidence for a possible mechanism by which en represses Ubx. On the basis of these results we propose that en has acquired two independent functions during the evolution of dorsal appendages. In addition to its well-known function of conferring posterior fate and inducing long-range signalling to pattern the developing appendages, it maintains wing fate by keeping Ubx repressed. [Emerald B. S. and Shashidhara L. S. 2000 Negative regulation of Ultrabithorax expression by engrailed is required for the proper speci®cation of wing development in Drosophila melanogaster. J. Genet. 79, 61±70]
We have used a chromatin ¡mmunopurification approach to identify target genes regulated by the ho... more We have used a chromatin ¡mmunopurification approach to identify target genes regulated by the homeotic gene JJltrabithorax. A monoclonal antibody against the Ultrabithorax gene product is used to immunopurify in vivo Ultrabithorax protein binding sites in embryonic chromatin. The procedure gives an enrichment of sequences with matches to a consensus homeodomain binding site. In one case we have shown that an immunopurified sequence lies within a 4 kb frag
Neurovascular integration during embryonic development is essential for adult physiology. In this... more Neurovascular integration during embryonic development is essential for adult physiology. In this issue of Developmental Cell, Gutnick et al. (2011) report that hypothalamic neurons secrete oxytocin as a guidance cue for endothelial cells to establish their vascular supply-a prerequisite for neuroendocrine secretion from the neurohyophysis in adult life.
Animal growth and development is dependent on reiterative use of key signaling pathways such as H... more Animal growth and development is dependent on reiterative use of key signaling pathways such as Hedgehog (Hh) pathway. It is widely believed that Cubitus-interruptus (Ci) mediates all functions of Hh pathway. Here we report that CG32062, the Drosophila homologue of Ataxin-2 Binding Protein 1 (dA2BP1), functions as a cofactor of Ci to specify intervein region between L3 and L4 veins of the adult wing. Specifically, Ci-mediated transactivation of knot/collier (kn) in this region of the developing wing imaginal disc is dependent on dA2BP1 function. Protein interaction studies and chromatin-immunoprecipiation experiments suggest that Ci helps dA2BP1 to bind kn promoter, which in turn may help Ci to activate kn expression. These results suggest a mechanism by which Ci may activate targets such as kn, which do not have classical Ci/Gli-binding sites.
Background: In Drosophila, segment-specific muscle pattern is thought to be determined by the aut... more Background: In Drosophila, segment-specific muscle pattern is thought to be determined by the autonomous function of homeotic selector genes in the mesoderm in combination with inductive cues from the developing epidermis and nervous system. Here, we have examined the roles of homeotic genes in the patterning of the somatic muscles of the thoracic segments of Drosophila. Results: We determined the expression patterns of homeoproteins in the mesoderm of the thoracic segments during embryonic and adult development. We found that, unlike the mesoderm of the first and third thoracic segments which express Sex combs reduced and Antennapedia (Antp), respectively, the mesoderm of the second thoracic segment does not express any known homeotic selector gene of the Antp or bithorax complex. In animals homozygous for Antp null mutations, the muscles of the second thoracic segment were affected in the embryo, probably as an indirect consequence of its requirement in the ectoderm. Animals that specifically lacked Antp function in the mesoderm, but expressed the gene in the epidermis, developed with a normal muscle pattern in the second thoracic segment. Furthermore, specific ectopic expression of Antp and other homeotic selector genes in the mesoderm of the second thoracic segment respecified its muscle pattern, indicating that these genes are not required autonomously during muscle development in this segment. Finally, we showed that Antp continues to be expressed in the mesoderm of the homeotically transformed third thoracic segment in the 'fourwinged fly', and suggest that this is a likely reason for the failure of flight muscle development in the transformed segment. Conclusions: We present a model for muscle development in the second thoracic segment whereby mesodermal properties are specified entirely by induction, in contrast to muscle development in other segments, where autonomous function for homeotic selector genes is also required.
Although the specific form of an organ is frequently important for its function, the mechanisms u... more Although the specific form of an organ is frequently important for its function, the mechanisms underlying organ shape are largely unknown. In Drosophila, the wings and halteres, homologous appendages of the second and third thoracic segments, respectively, bear different forms: wings are flat whereas halteres are globular and yet both characteristic shapes are essential for a normal flight. The Hox gene Ultrabithorax governs the difference between wing and haltere development, but how Ultrabithorax function in the appendages prevents or allows flat or globular shapes is unknown. Here we show that Ultrabithorax down-regulates Matrix metalloproteinase1 expression in the haltere pouch at early pupal stage, which in turn prevents the rapid clearance of Collagen IV compared to the wing disc. This difference is instrumental in determining cell shape changes, expansion of the disc and apposition of dorsal and ventral layers, all of these phenotypic traits being characteristic of wing pouc...
In the fruitfly Drosophila melanogaster, the differential development of wing and haltere is depe... more In the fruitfly Drosophila melanogaster, the differential development of wing and haltere is dependent on the function of the Hox protein Ultrabithorax (Ubx). Here we compare Ubx-mediated regulation of wing patterning genes between the honeybee, Apis mellifera, the silkmoth, Bombyx mori and Drosophila. Orthologues of Ubx are expressed in the third thoracic segment of Apis and Bombyx, although they make functional hindwings. When over-expressed in transgenic Drosophila, Ubx derived from Apis or Bombyx could suppress wing development, suggesting evolutionary changes at the level of co-factors and/or targets of Ubx. To gain further insights into such events, we identified direct targets of Ubx from Apis and Bombyx by ChIP-seq and compared them with those of Drosophila. While majority of the putative targets of Ubx are species-specific, a considerable number of wing-patterning genes are retained, over the past 300 millions years, as targets in all the three species. Interestingly, many of these are differentially expressed only between wing and haltere in Drosophila but not between forewing and hindwing in Apis or Bombyx. Detailed bioinformatics and experimental validation of enhancer sequences suggest that, perhaps along with other factors, changes in the cis-regulatory sequences of earlier targets contribute to diversity in Ubx function.
In Drosophila, differential development of wing and haltere, which differ in cell size, number an... more In Drosophila, differential development of wing and haltere, which differ in cell size, number and morphology, is dependent on the function of Hox gene Ultrabithorax (Ubx). Here we report our studies on Ubx-mediated regulation of the Fat/Hippo and IIS/dAkt pathways, which control cell number and cell size during development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded, caused considerable increase in haltere size, mainly due to increase in cell number. These phenotypes were also associated with the activation of Akt pathways in developing haltere. Although activation of Akt alone did not affect the cell size or the organ size, we observed dramatic increase in haltere size when Akt was activated in the background where expanded is down regulated. This was associated with the increase in both cell size and cell number. The organ appeared flatter than wildtype haltere and the trichome morphology and spacing resembled that ...
There have been conflicting reports on the requirement of GSK-3b-mediated phosphorylation of the ... more There have been conflicting reports on the requirement of GSK-3b-mediated phosphorylation of the tumor suppressor adenomatous polyposis coli (APC) vis-a`-vis its ability to bind and degrade b-catenin. Using a unique combination of loss of function for Shaggy/GSK-3b and a gain of function for human APC in Drosophila, we show that misexpressed human APC (hAPC) can still sequester Armadillo/b-catenin. In addition, human APC could suppress gain of Wnt/Wingless phenotypes associated with loss of Shaggy/GSK-3b activity, suggesting that sequestered Armadillo/b-catenin is non-functional. Based on these studies, we propose that binding per se of b-catenin by APC does not require phosphorylation by GSK-3b.
The International journal of developmental biology, 2020
Differential specification of dorsal flight appendages, wing and haltere, in Drosophila provides ... more Differential specification of dorsal flight appendages, wing and haltere, in Drosophila provides an excellent model system to address a number of important questions in developmental biology at the levels of molecules, pathways, tissues, organs, organisms and evolution. Here we discuss the mechanism by which the Hox protein Ubx recognizes and regulates its downstream targets, implications of the same in growth control at cellular and organ level and finally the evolution of haltere from ancestral hindwings in other holometabolous insects.
PTPN11/SHP2, a non-receptor protein tyrosine phosphatase is a prominent target of the receptor ty... more PTPN11/SHP2, a non-receptor protein tyrosine phosphatase is a prominent target of the receptor tyrosine kinase that participates in positive feedback signalling of the human epidermal growth factor receptors and helps in growth and migration. PTPN11/SHP2 is widely believed to be an oncoprotein, although it’s possible tumor-suppressor role is also reported. Our analysis of breast cancer metadata shows, PTPN11/SHP2 copy number loss in luminal A subtype is correlated to poor disease-specific survival and late-stage cancer at diagnosis. Analysis of the level 4 Reverse Phase Protein Array (RPPA) data available on the TCGA database resulted in positive correlations between the lower expression levels of constitutively active variant, the phospho-SHP2-Y542, of PTPN11/SHP2 and larger tumor size and lymph node positivity. We experimentally examined possible negative regulation of growth by PTPN11/SHP2 using MCF10A, a normal breast epithelial cell line. Knock-down of PTPN11/SHP2 resulted in i...
Promoter proximal pausing (PPP) of RNA polymerase II has emerged as a crucial rate-limiting step ... more Promoter proximal pausing (PPP) of RNA polymerase II has emerged as a crucial rate-limiting step in the regulation of gene expression. Regulation of PPP is brought about by complexes 7SK snRNP, P-TEFb (Cdk9/cycT), and the negative elongation factor (NELF), which are highly conserved from Drosophila to humans. Here, we show that RNAi-mediated depletion of bin3 or Hexim of the 7SK snRNP complex or depletion of individual components of the NELF complex enhances Yki-driven growth, leading to neoplastic transformation of Drosophila wing imaginal discs. We also show that increased CDK9 expression cooperates with Yki in driving neoplastic growth. Interestingly, overexpression of CDK9 on its own or in the background of depletion of one of the components of 7SK snRNP or the NELF complex necessarily, and specifically, needed Yki overexpression to cause tumorous growth. Genome-wide gene expression analyses suggested that deregulation of protein homeostasis is associated with tumorous growth of...
We have shown that the expression of the 412 retrotransposon provides a useful early marker for t... more We have shown that the expression of the 412 retrotransposon provides a useful early marker for the development of the gonadal mesoderm in Drosophila embryos. 412 is initially expressed in a set of parasegmentally repeated stripes from parasegments (PS) 2–14 in the mesoderm at the extended germ band stage. During germ band retraction the bulk of 412 expression declines except in dorsolateral clusters of cells in PS10, 11 and 12, where high levels of 412 expression remain. These mesodermal cell clusters are associated with germ cells and subsequently they coalesce, rounding up to form the gonads. The gonadal mesoderm thus appears to originate specifically from three abdominal parasegments, PS10, 11 and 12. We show that the maintenance of high levels of 412 expression in gonadal mesoderm is not induced by contact with germ cells, but rather depends on genetic control by the homeotic genes abdominal-A and Abdominal-B.
Genetic approaches in Drosophila have successfully identified many genes involved in regulation o... more Genetic approaches in Drosophila have successfully identified many genes involved in regulation of growth control as well as genetic interactions relevant to the initiation and progression of cancer in vivo. Here, we report on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: the Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo. We also report on an independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified here are known to be functional in growth control pathways. We also find a number of novel connections to Yki and EGFR driven tissue growth, mostly unique to one of the two. Thus, resourc...
Genetic approaches in Drosophila have successfully identified many genes involved in regulation o... more Genetic approaches in Drosophila have successfully identified many genes involved in regulation of growth control as well as genetic interactions relevant to the initiation and progression of cancer in vivo. Here, we report on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: The Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo. We also report on an independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified here are known to be functional in growth control pathways. We also find a number of novel connections to Yki and EGFR driven tissue growth, mostly unique to one of the two. Thus, resourc...
Scotin is a pro-apoptotic mammalian gene, which is induced upon DNA damage or cellular stress in ... more Scotin is a pro-apoptotic mammalian gene, which is induced upon DNA damage or cellular stress in a p53-dependent manner. In this report, we have used Drosophila as a model system to obtain a preliminary insight into the molecular mechanism of Scotin function, which was further validated using the mammalian system. Targeted expression of Scotin in developing Drosophila induced apoptosis and developmental defects in wings and eyes. Co-expression of Scotin with the anti-apoptotic protein p35, while inhibited the apoptosis in both dividing and non-dividing cells, rescued adult wing or eye phenotypes only when Scotin was expressed in nondividing cells. This suggests that mechanisms of Scotin-induced apoptosis in dividing and non-dividing cells may vary. Suppressorenhancer screen using cell cycle regulators suggested that Scotin may mediate cell cycle arrest at both G 1 /S and G 2 /M phases. Overexpression of Scotin in mammalian cells resulted in mitotic arrest and subsequently apoptosis. Furthermore, a larger proportion of cells overexpressing Scotin showed sequestration of Cyclin B1 in the cytoplasm. These results suggest that one of the ways by which Scotin induces apoptosis is by causing cell cycle arrest.
The International Journal of Developmental Biology, 2011
Thorax closure in Drosophila is a process during adult morphogenesis in which the anterior ends o... more Thorax closure in Drosophila is a process during adult morphogenesis in which the anterior ends of the presumptive notum of the two wing imaginal discs fuse to make a seamless thorax. Similar to dorsal closure during embryogenesis, this process is regulated by Decapentaplegic and JNK signaling pathways. Despite the fact that Peripodial Membrane (PM) cells do not contribute to the formation of any adult structure, they are known to facilitate the process of thorax closure. Here we show that JNK signaling is activated only in a subset of PM cells, known as medial edge cells. While the mechanism that activates JNK signaling specifically in the medial edge cells of the PM is still not understood, the results presented here show that the pair rule gene odd skipped is required to ensure that JNK signaling is not activated anywhere else in the wing disc. Medial edge cells of the PM are elongated in shape, while the remaining PM cells are hexagonal. Down regulation of JNK signaling in the medial edge cells results in defective thorax closure in adult flies. It also causes the transformation of the morphology of medial edge cells into hexagonal shape. Conversely, activation of JNK signaling in hexagonal cells of the PM causes transformation of their morphology to elongated shape. Thus, similar to dorsal closure during embryogenesis, JNKmediated elongation of medial edge cells is functionally correlated to the process of thorax closure.
Proceedings of the National Academy of Sciences, 1991
The expression and subcellular location of porphobilinogen deaminase (PBGD, also known as hydroxy... more The expression and subcellular location of porphobilinogen deaminase (PBGD, also known as hydroxymethylbilane synthase; EC 4.3.1.8), one of the early enzymes of porphyrin synthesis, was investigated in light-grown Euglena and in three cell lines that do not contain chlorophyll: dark-grown Euglena, a streptomycin-bleached mutant, and Astasia longa. In wild-type Euglena, immunogold electron microscopy demonstrated that all the immunodetectable enzyme protein was in the chloroplast. PBGD was shown to be photoregulated, and like many other nuclear-encoded proteins in Euglena, the regulation was at the posttranscriptional level. In the three nonchlorophyllous cell lines, as in light-grown Euglena, a single protein of 40 kDa was detected with antiserum to PBGD. This same antiserum immunoprecipitated a larger precursor protein from the total translation products of poly(A)+ RNA, and a single transcript, which was large enough to encode the precursor, was detected on Northern blots of all f...
Mutations in human Adenomatous Polyposis Coli (APC) gene are associated with both familial and sp... more Mutations in human Adenomatous Polyposis Coli (APC) gene are associated with both familial and sporadic colorectal tumors. APC is known to down regulate bcatenin levels, a transducer of Wnt signaling. The aim of this study is to provide transgenic Drosophila expressing either full-length or truncated forms of human APC (hAPC) protein and methods for using them in functional genomics and drug screening. Consistent with its biochemical properties, targeted expression of either full-length hAPC or its b-catenin binding domain alone negatively regulated the function of the b-catenin homologue, Armadillo (Arm) and thereby, inhibited Wnt/Wg signaling during¯y development. hAPC inhibited Arm function even in the absence of GSK-3b activity, although the latter was required to mediate the degradation of Arm. Consistent with this, hAPC suppressed the phenotypes induced by the over-expression of degradation-resistant forms of Arm. Subsequently, using hAPC-induced eye phenotypes as the assay in a suppressor-enhancer screen, we have identi®ed two new loci in Drosophila, which modulate Wnt/Wg signaling. In addition, an anti-colon cancer drug, indomethacin, speci®cally enhanced hAPC-induced phenotypes.
In both vertebrates and invertebrates, homeotic selector genes confer morphological differences a... more In both vertebrates and invertebrates, homeotic selector genes confer morphological differences along the antero-posterior axis. However, insect wing development is independent of all homeotic gene functions, reflecting the ground plan of an ancestral pterygote, which bore wings on all segments. Dipteran insects such as Drosophila are characterized by a pair of wings in the mesothoracic segment. In all other segments, wing development is essentially repressed by different homeotic genes, although in the metathorax they are modi®ed into a pair of halteres. This necessitates that during development all homeotic genes are to be maintained in a repressed state in wing imaginal discs. In this report we show that (i) the function of the segment polarity gene engrailed (en) is critical to keep the homeotic selector gene Ultrabithorax (Ubx) repressed in wing imaginal discs, (ii) normal levels of En in the posterior compartment of haltere discs, however, are not enough to completely repress Ubx, and (iii) the repression of Ubx by en is independent of Hedgehog signalling through which the longrange signalling of en is mediated during wing development. Finally we provide evidence for a possible mechanism by which en represses Ubx. On the basis of these results we propose that en has acquired two independent functions during the evolution of dorsal appendages. In addition to its well-known function of conferring posterior fate and inducing long-range signalling to pattern the developing appendages, it maintains wing fate by keeping Ubx repressed. [Emerald B. S. and Shashidhara L. S. 2000 Negative regulation of Ultrabithorax expression by engrailed is required for the proper speci®cation of wing development in Drosophila melanogaster. J. Genet. 79, 61±70]
We have used a chromatin ¡mmunopurification approach to identify target genes regulated by the ho... more We have used a chromatin ¡mmunopurification approach to identify target genes regulated by the homeotic gene JJltrabithorax. A monoclonal antibody against the Ultrabithorax gene product is used to immunopurify in vivo Ultrabithorax protein binding sites in embryonic chromatin. The procedure gives an enrichment of sequences with matches to a consensus homeodomain binding site. In one case we have shown that an immunopurified sequence lies within a 4 kb frag
Neurovascular integration during embryonic development is essential for adult physiology. In this... more Neurovascular integration during embryonic development is essential for adult physiology. In this issue of Developmental Cell, Gutnick et al. (2011) report that hypothalamic neurons secrete oxytocin as a guidance cue for endothelial cells to establish their vascular supply-a prerequisite for neuroendocrine secretion from the neurohyophysis in adult life.
Animal growth and development is dependent on reiterative use of key signaling pathways such as H... more Animal growth and development is dependent on reiterative use of key signaling pathways such as Hedgehog (Hh) pathway. It is widely believed that Cubitus-interruptus (Ci) mediates all functions of Hh pathway. Here we report that CG32062, the Drosophila homologue of Ataxin-2 Binding Protein 1 (dA2BP1), functions as a cofactor of Ci to specify intervein region between L3 and L4 veins of the adult wing. Specifically, Ci-mediated transactivation of knot/collier (kn) in this region of the developing wing imaginal disc is dependent on dA2BP1 function. Protein interaction studies and chromatin-immunoprecipiation experiments suggest that Ci helps dA2BP1 to bind kn promoter, which in turn may help Ci to activate kn expression. These results suggest a mechanism by which Ci may activate targets such as kn, which do not have classical Ci/Gli-binding sites.
Background: In Drosophila, segment-specific muscle pattern is thought to be determined by the aut... more Background: In Drosophila, segment-specific muscle pattern is thought to be determined by the autonomous function of homeotic selector genes in the mesoderm in combination with inductive cues from the developing epidermis and nervous system. Here, we have examined the roles of homeotic genes in the patterning of the somatic muscles of the thoracic segments of Drosophila. Results: We determined the expression patterns of homeoproteins in the mesoderm of the thoracic segments during embryonic and adult development. We found that, unlike the mesoderm of the first and third thoracic segments which express Sex combs reduced and Antennapedia (Antp), respectively, the mesoderm of the second thoracic segment does not express any known homeotic selector gene of the Antp or bithorax complex. In animals homozygous for Antp null mutations, the muscles of the second thoracic segment were affected in the embryo, probably as an indirect consequence of its requirement in the ectoderm. Animals that specifically lacked Antp function in the mesoderm, but expressed the gene in the epidermis, developed with a normal muscle pattern in the second thoracic segment. Furthermore, specific ectopic expression of Antp and other homeotic selector genes in the mesoderm of the second thoracic segment respecified its muscle pattern, indicating that these genes are not required autonomously during muscle development in this segment. Finally, we showed that Antp continues to be expressed in the mesoderm of the homeotically transformed third thoracic segment in the 'fourwinged fly', and suggest that this is a likely reason for the failure of flight muscle development in the transformed segment. Conclusions: We present a model for muscle development in the second thoracic segment whereby mesodermal properties are specified entirely by induction, in contrast to muscle development in other segments, where autonomous function for homeotic selector genes is also required.
Although the specific form of an organ is frequently important for its function, the mechanisms u... more Although the specific form of an organ is frequently important for its function, the mechanisms underlying organ shape are largely unknown. In Drosophila, the wings and halteres, homologous appendages of the second and third thoracic segments, respectively, bear different forms: wings are flat whereas halteres are globular and yet both characteristic shapes are essential for a normal flight. The Hox gene Ultrabithorax governs the difference between wing and haltere development, but how Ultrabithorax function in the appendages prevents or allows flat or globular shapes is unknown. Here we show that Ultrabithorax down-regulates Matrix metalloproteinase1 expression in the haltere pouch at early pupal stage, which in turn prevents the rapid clearance of Collagen IV compared to the wing disc. This difference is instrumental in determining cell shape changes, expansion of the disc and apposition of dorsal and ventral layers, all of these phenotypic traits being characteristic of wing pouc...
In the fruitfly Drosophila melanogaster, the differential development of wing and haltere is depe... more In the fruitfly Drosophila melanogaster, the differential development of wing and haltere is dependent on the function of the Hox protein Ultrabithorax (Ubx). Here we compare Ubx-mediated regulation of wing patterning genes between the honeybee, Apis mellifera, the silkmoth, Bombyx mori and Drosophila. Orthologues of Ubx are expressed in the third thoracic segment of Apis and Bombyx, although they make functional hindwings. When over-expressed in transgenic Drosophila, Ubx derived from Apis or Bombyx could suppress wing development, suggesting evolutionary changes at the level of co-factors and/or targets of Ubx. To gain further insights into such events, we identified direct targets of Ubx from Apis and Bombyx by ChIP-seq and compared them with those of Drosophila. While majority of the putative targets of Ubx are species-specific, a considerable number of wing-patterning genes are retained, over the past 300 millions years, as targets in all the three species. Interestingly, many of these are differentially expressed only between wing and haltere in Drosophila but not between forewing and hindwing in Apis or Bombyx. Detailed bioinformatics and experimental validation of enhancer sequences suggest that, perhaps along with other factors, changes in the cis-regulatory sequences of earlier targets contribute to diversity in Ubx function.
In Drosophila, differential development of wing and haltere, which differ in cell size, number an... more In Drosophila, differential development of wing and haltere, which differ in cell size, number and morphology, is dependent on the function of Hox gene Ultrabithorax (Ubx). Here we report our studies on Ubx-mediated regulation of the Fat/Hippo and IIS/dAkt pathways, which control cell number and cell size during development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded, caused considerable increase in haltere size, mainly due to increase in cell number. These phenotypes were also associated with the activation of Akt pathways in developing haltere. Although activation of Akt alone did not affect the cell size or the organ size, we observed dramatic increase in haltere size when Akt was activated in the background where expanded is down regulated. This was associated with the increase in both cell size and cell number. The organ appeared flatter than wildtype haltere and the trichome morphology and spacing resembled that ...
There have been conflicting reports on the requirement of GSK-3b-mediated phosphorylation of the ... more There have been conflicting reports on the requirement of GSK-3b-mediated phosphorylation of the tumor suppressor adenomatous polyposis coli (APC) vis-a`-vis its ability to bind and degrade b-catenin. Using a unique combination of loss of function for Shaggy/GSK-3b and a gain of function for human APC in Drosophila, we show that misexpressed human APC (hAPC) can still sequester Armadillo/b-catenin. In addition, human APC could suppress gain of Wnt/Wingless phenotypes associated with loss of Shaggy/GSK-3b activity, suggesting that sequestered Armadillo/b-catenin is non-functional. Based on these studies, we propose that binding per se of b-catenin by APC does not require phosphorylation by GSK-3b.
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Papers by LS SHASHIDHARA