- Email: [email protected]
Regulatory Differences in Natal Down Development between Altricial and Precocial Birds
Abstracts
the evolution of higher organisms, giving them de novo protein coding sequence and/or transcriptional regulatory elements such as enhancer. Here, we report that LTRs of MaLR, mouse endogenous retrovirus (ERV), carry the binding sites of Tbx6, an evolutionary conserved family of T-box transcription factors. Whole-genome sequence analysis identified over 3,000 clusters of multiple Tbx6 binding sites. Twenty seven percent of these potential Tbx6 binding sites were located within MaLR LTRs (ORR1A, ORR1B, ORR1C and ORR1D). Gel-shift assay showed Tbx6 bound strongly to these LTRs in vitro. RT-PCR revealed that 4 genes (Twist2, Pitx2, Oscp1 and Nfxl1), laying adjacent to LTRs bearing clusters of Tbx6 binding sites, were down-regulated in Tbx6-/- embryos at 8 days postcoitum. These data indicate that at least some of Tbx6 binding sites locate within LTRs are biologically active, regulating the expression of nearby genes. LTRs bearing Tbx6 binding cluster near the 4 genes above were conserved in mouse and rat genome but not in other vertebrates, implying they participate rodent-specific fine-tuning of gene expression in embryogenesis. doi:10.1016/j.mod.2017.04.300
PS3.95 Higher Expression of Key Developmental Genes in Drosophila melanogaster with Accelerated Development Namita Chauhan, Namita Agrawal, Mallikarjun Shakarad University of Delhi, India The ability of biological systems to maintain stability of the phenotype despite genetic, environmental or physiological perturbations is ‘developmental robustness’. The final phenotype is an outcome of regulated coordination between various developmental and molecular mechanisms. In Drosophila melanogaster the correct patterning and development is guaranteed by alignment of gene expression patterns in tissues at specific events such as molting and pupariation either by modulating the development rates or the release of hormones in response to environmental and physiological variations. Thus, plasticity in the rate of pattern progression as well as the level at which the genes are expressed might be an important contributing factor in regulating the robustness of development. To gain a better insight, we studied the developmental robustness in D. melanogaster populations selected for faster pre-adult development. These populations show rapid development compared to their ancestors with a ~22% reduction in egg to eclosion development time with significant reductions in larval durations. Despite a significant reduction in development duration, the flies from the selected populations exhibit stability of phenotype, albeit significantly small in size. We compared the onset of various transition events, their duration and the expression patterns of genes in both the populations. Our results suggest that the reduced development time in selected flies is accompanied by a higher expression of developmental genes to ensure correct pattern formation at similar development stage that is being attained early in these populations compared to their ancestral controls. doi:10.1016/j.mod.2017.04.301
PS3.96 A conserved regulatory program controls lateral plate mesoderm emergence Christopher Hess, Karin D. Prummel, Susan Nieuwenhuize, Elena Chiavacci, Sibylle Burger, Alexa Burger, Anastasia Felker, Christian Mosimann Institute of Molecular Life Sciences, University of Zürich, Switzerland
S113
Cardiovascular progenitors arise along with kidney, smooth muscle, and limb lineages from lateral plate mesoderm (LPM). How LPM is specified within the mesoderm and what molecular features connect its distinct cell fates remain vaguely defined, in part due to the LPM’s dynamic architecture. By combining live imaging, cisregulatory element analysis, and cross-species reporter assays, we charted the emergence of the LPM and trace its control back to an evolutionarily conserved regulatory program. From live imaging and genetic lineage tracing, we found a pan-LPM enhancer in the zebrafish draculin (drl) gene that is selectively active in all presumptive LPM progenitors from mid-gastrulation to early somitogenesis. The drl pan-LPM enhancer is necessary and sufficient for driving LPM-specific expression, suggesting a common upstream activity in all LPM progenitors. Panoramic SPIM of drl reporters captured the dynamic LPM formation from a restricted mesendoderm territory during gastrulation to its subsequent bilateral condensation. In cross-species assays, we uncovered LPM-specific activity of the zebrafish drl pan-LPM enhancer in early lamprey and chicken embryos. We further corroborated LPM-specific cross-species activity of additional pan-LPM enhancers isolated from additional gene loci. These results suggest that an ancient, evolutionarily conserved program controls LPM emergence. To define this program, we used Cas9 RNP-based crispant analysis, reporter assays, and ChIP to identify a transcription factor combination centered on the mesendoderm regulators Eomes/FoxH1 that is sufficient to drive pan-LPM enhancer activity. Distinct from other mesoderm lineages, these factors demarcate a dedicated mesendoderm progenitor field that restricts the emergence of LPM progenitors to their future lateral position. Altogether, our work reveals that the complete LPM segregates from other mesendoderm already early on, possibly representing an ancient cell fate domain within the primordial vertebrate embryo. Our data provide a framework for charting the earliest emergence and the evolutionary basis of the diverse LPM fates. doi:10.1016/j.mod.2017.04.302
PS3.97 Regulatory Differences in Natal Down Development between Altricial and Precocial Birds Chih-Kuan Chenb, Pei-Chi Sua, Siao-Man Wub, I-Ming Liua, Wen-Hsiung Lib, Chen Siang Nga a
National Tsing Hua University, Hsinchu, Taiwan Biodiversity Research Center, Taipei, Taiwan
b
Birds can be classified as altricial or precocial. The hatchlings of altricial birds are almost naked and relatively immobile, whereas those of precocial birds are covered with natal down and immediately or quickly mobile. The precocial developmental type is considered the ancestral state. Such evolutionary divergence is thought to reflect adaptation to different environments, but the molecular basis of nakedness is unclear. We have used altricial zebra finch and precocial chicken as models for identifying causative elements of natal down differences between altricial and precocial hatchlings and found that the FGF/MAPK signaling pathway is involved in natal down growth suppression and that FGF16 is a candidate upstream signaling suppressor. Natal down suppression in altricial birds could have evolved more than once, giving us an outstanding opportunity to study if convergent phenotypes can be made by similar genes or pathways. We extend the study to investigate the expression patterns of genes involved in early feather development and FGF/MAPK pathways in the embryos of budgerigar, duck, rock pigeon using in situ hybridization and immunohistochemical staining. Our preliminary data suggested that
S114
Abstracts
the suppression of natal down could be due to different molecular and cellular mechanisms in budgerigars whose chicks are also naked.
doi:10.1016/j.mod.2017.04.303
PS3.98 Antagonistic BMP-cWNT signaling in the cnidarian Nematostella vectensis: Implications for the evolution of mesoderm Naveen Wijesenaa, David Simmonsb, Mark Martindaleb a
Rajarata University of Sri Lanka, Mihintale, Sri Lanka Whitney Lab, University of Florida, Saint Augustine, United States
b
Gastrulation was arguably the key evolutionary innovation that enabled metazoan diversification, leading to the formation of distinct germ layers and specialized tissues. Differential gene expression specifying cell fate is governed by the inputs of intracellular and/or extracellular signals. Beta-catenin/Tcf and the TGF-beta Bone Morphogenetic Protein (BMP) provide critical molecular signaling inputs during germ layer specification in bilaterian metazoans but there has beenno direct experimental evidence for a specific role for BMP signaling during endomesoderm specification in the early branching metazoan Nematostella vectensis (ananthozoan cnidarian),. Using forward transcriptomics, we show that Betacatenin/Tcf signaling and BMP2/4 signaling provide differential inputs into the cnidarian endomesodermal gene regulatory network at the onset of gastrulation (24hpf) inNematostella vectensis. Surprisingly, Beta-catenin/Tcf signaling and BMP2/4 signaling regulate a subset of common downstream target genes in the GRN in opposite ways, leading to the spatial and temporal differentiation of fields of cells in the developing embryo. Thus, we show that regulatory interactions between Beta-catenin/Tcf signaling and BMP2/4 signaling are required for the specification and determination of different embryonic regions and the patterning of the oralaboral axis in Nematostella. We also show functionally that the conserved “kernel” of the bilaterian heart mesoderm GRN is operational in Nematostella vectensis, which reinforces the hypothesis that the endoderm and mesoderm in triploblastic bilaterians evolved from the bifunctional endomesoderm (gastrodermis) of a diploblastic ancestor, and that slow rhythmic contractions might have been one of the earliest functions of mesoderm.
doi:10.1016/j.mod.2017.04.304
PS3.99 Cellular and molecular mechanisms regulating limb position in vertebrates Chloe Moreaua,b,c, Nicolas Denansd, Olivier Pourquiée, Jerome Grosa,b a
Institut Pasteur, Paris, France URA2578, CNRS, France c Cellule Pasteur UPMC, Paris, France d Stanford School of Medicine, United States e Harvard Medical School, Boston, United States b
During vertebrate development, limbs emerge from the trunk of the embryo as buds that progressively elongate into fully developed
appendages. Whereas the molecular and genetic interactions underlying limb initiation, outgrowth and patterning have been greatly elucidated, how limbs reproducibly form at specific locations along the body axis still remains unclear. The position of limbs is very consistent within one species but is highly variable among vertebrates. Interestingly, a number of studies have shown that this variation is associated with variations in the expression pattern of Hox genes. Because of their role in patterning embryonic axes, these genes have long remained good candidates in regulating the position of the limbs. Here, we hypothesize that the positional information of limb progenitor cells is acquired during the early steps of gastrulation and we explore the role of Hox genes in this process. Combining classical embryology techniques with molecular and live imaging approaches in the chick embryo, we aim at characterizing the cellular and molecular events underlying the formation of the Lateral Plate Mesoderm (LPM, i.e. the mesodermal compartment giving rise to limbs) and in particular the role of Hox genes in patterning the LPM into limb-forming and non-limb-forming domains, during the gastrulation process.
doi:10.1016/j.mod.2017.04.305
PS3.100 Single-cell RNA-seq unveils divergent modes of chemoresistance in squamous cell carcinoma Ankur Sharmaa, Vibhor Kumara, Elaine Yiqun Caob, Hui Sun Leongb, Muhammad Hakimullaha, Neeraja Ramakrishnana, Xiaoqian Zhanga, Fui Teen Chongb, Shumei Chiaa, Matan Thangavelu Thangavelua, Angeline Mei Lin Wonga, Xue Lin Kwangb, Daniel Shao-Weng Tanb, Giridharan Periyasamya, N. Gopalakrishna Iyerb, Ramanuj DasGuptaa a
Genome Institute of, Singapore National Cancer Centre, Singapore
b
Intra-tumor heterogeneity (ITH) generates distinct cellular populations that contribute to tumor maintenance, progression, and drug-resistance. However, the contribution of individual cells to chemotherapy-resistance remains poorly understood. We generated single-cell mRNA sequencing profile of cisplatin-resistant squamous cell carcinoma (SCC) cells. Here we demonstrate that retention of native cellular-states is the principle mode of drug-resistance, a phenomenon we term ‘cellular inertia’. Interestingly, in comparison to other cancers ‘cellular-plasticity’ manifesting as epithelial-tomesenchymal transition (EMT) was observed to be a minor source of drug-resistance. These observations were also corroborated in cisplatin treated oral SCC patients. Importantly, gene expression signature from cellular plasticity-induced drug-resistant models correlated with poor survival. Synthetic lethal screens revealed a critical function of chromatin remodelers in EMT-associated acquired drug resistance. Specifically, we demonstrate the role of BRD4mediated H3K27 acetylation as an adaptive response to chemotherapy, which could be reversed by JQ1. In summary, our study provides critical insights into the divergent modes of drug resistance, and paves the way for therapeutic modalities involving epigenetic targeting of tumor evolution and ITH.
doi:10.1016/j.mod.2017.04.306
the evolution of higher organisms, giving them de novo protein coding sequence and/or transcriptional regulatory elements such as enhancer. Here, we report that LTRs of MaLR, mouse endogenous retrovirus (ERV), carry the binding sites of Tbx6, an evolutionary conserved family of T-box transcription factors. Whole-genome sequence analysis identified over 3,000 clusters of multiple Tbx6 binding sites. Twenty seven percent of these potential Tbx6 binding sites were located within MaLR LTRs (ORR1A, ORR1B, ORR1C and ORR1D). Gel-shift assay showed Tbx6 bound strongly to these LTRs in vitro. RT-PCR revealed that 4 genes (Twist2, Pitx2, Oscp1 and Nfxl1), laying adjacent to LTRs bearing clusters of Tbx6 binding sites, were down-regulated in Tbx6-/- embryos at 8 days postcoitum. These data indicate that at least some of Tbx6 binding sites locate within LTRs are biologically active, regulating the expression of nearby genes. LTRs bearing Tbx6 binding cluster near the 4 genes above were conserved in mouse and rat genome but not in other vertebrates, implying they participate rodent-specific fine-tuning of gene expression in embryogenesis. doi:10.1016/j.mod.2017.04.300
PS3.95 Higher Expression of Key Developmental Genes in Drosophila melanogaster with Accelerated Development Namita Chauhan, Namita Agrawal, Mallikarjun Shakarad University of Delhi, India The ability of biological systems to maintain stability of the phenotype despite genetic, environmental or physiological perturbations is ‘developmental robustness’. The final phenotype is an outcome of regulated coordination between various developmental and molecular mechanisms. In Drosophila melanogaster the correct patterning and development is guaranteed by alignment of gene expression patterns in tissues at specific events such as molting and pupariation either by modulating the development rates or the release of hormones in response to environmental and physiological variations. Thus, plasticity in the rate of pattern progression as well as the level at which the genes are expressed might be an important contributing factor in regulating the robustness of development. To gain a better insight, we studied the developmental robustness in D. melanogaster populations selected for faster pre-adult development. These populations show rapid development compared to their ancestors with a ~22% reduction in egg to eclosion development time with significant reductions in larval durations. Despite a significant reduction in development duration, the flies from the selected populations exhibit stability of phenotype, albeit significantly small in size. We compared the onset of various transition events, their duration and the expression patterns of genes in both the populations. Our results suggest that the reduced development time in selected flies is accompanied by a higher expression of developmental genes to ensure correct pattern formation at similar development stage that is being attained early in these populations compared to their ancestral controls. doi:10.1016/j.mod.2017.04.301
PS3.96 A conserved regulatory program controls lateral plate mesoderm emergence Christopher Hess, Karin D. Prummel, Susan Nieuwenhuize, Elena Chiavacci, Sibylle Burger, Alexa Burger, Anastasia Felker, Christian Mosimann Institute of Molecular Life Sciences, University of Zürich, Switzerland
S113
Cardiovascular progenitors arise along with kidney, smooth muscle, and limb lineages from lateral plate mesoderm (LPM). How LPM is specified within the mesoderm and what molecular features connect its distinct cell fates remain vaguely defined, in part due to the LPM’s dynamic architecture. By combining live imaging, cisregulatory element analysis, and cross-species reporter assays, we charted the emergence of the LPM and trace its control back to an evolutionarily conserved regulatory program. From live imaging and genetic lineage tracing, we found a pan-LPM enhancer in the zebrafish draculin (drl) gene that is selectively active in all presumptive LPM progenitors from mid-gastrulation to early somitogenesis. The drl pan-LPM enhancer is necessary and sufficient for driving LPM-specific expression, suggesting a common upstream activity in all LPM progenitors. Panoramic SPIM of drl reporters captured the dynamic LPM formation from a restricted mesendoderm territory during gastrulation to its subsequent bilateral condensation. In cross-species assays, we uncovered LPM-specific activity of the zebrafish drl pan-LPM enhancer in early lamprey and chicken embryos. We further corroborated LPM-specific cross-species activity of additional pan-LPM enhancers isolated from additional gene loci. These results suggest that an ancient, evolutionarily conserved program controls LPM emergence. To define this program, we used Cas9 RNP-based crispant analysis, reporter assays, and ChIP to identify a transcription factor combination centered on the mesendoderm regulators Eomes/FoxH1 that is sufficient to drive pan-LPM enhancer activity. Distinct from other mesoderm lineages, these factors demarcate a dedicated mesendoderm progenitor field that restricts the emergence of LPM progenitors to their future lateral position. Altogether, our work reveals that the complete LPM segregates from other mesendoderm already early on, possibly representing an ancient cell fate domain within the primordial vertebrate embryo. Our data provide a framework for charting the earliest emergence and the evolutionary basis of the diverse LPM fates. doi:10.1016/j.mod.2017.04.302
PS3.97 Regulatory Differences in Natal Down Development between Altricial and Precocial Birds Chih-Kuan Chenb, Pei-Chi Sua, Siao-Man Wub, I-Ming Liua, Wen-Hsiung Lib, Chen Siang Nga a
National Tsing Hua University, Hsinchu, Taiwan Biodiversity Research Center, Taipei, Taiwan
b
Birds can be classified as altricial or precocial. The hatchlings of altricial birds are almost naked and relatively immobile, whereas those of precocial birds are covered with natal down and immediately or quickly mobile. The precocial developmental type is considered the ancestral state. Such evolutionary divergence is thought to reflect adaptation to different environments, but the molecular basis of nakedness is unclear. We have used altricial zebra finch and precocial chicken as models for identifying causative elements of natal down differences between altricial and precocial hatchlings and found that the FGF/MAPK signaling pathway is involved in natal down growth suppression and that FGF16 is a candidate upstream signaling suppressor. Natal down suppression in altricial birds could have evolved more than once, giving us an outstanding opportunity to study if convergent phenotypes can be made by similar genes or pathways. We extend the study to investigate the expression patterns of genes involved in early feather development and FGF/MAPK pathways in the embryos of budgerigar, duck, rock pigeon using in situ hybridization and immunohistochemical staining. Our preliminary data suggested that
S114
Abstracts
the suppression of natal down could be due to different molecular and cellular mechanisms in budgerigars whose chicks are also naked.
doi:10.1016/j.mod.2017.04.303
PS3.98 Antagonistic BMP-cWNT signaling in the cnidarian Nematostella vectensis: Implications for the evolution of mesoderm Naveen Wijesenaa, David Simmonsb, Mark Martindaleb a
Rajarata University of Sri Lanka, Mihintale, Sri Lanka Whitney Lab, University of Florida, Saint Augustine, United States
b
Gastrulation was arguably the key evolutionary innovation that enabled metazoan diversification, leading to the formation of distinct germ layers and specialized tissues. Differential gene expression specifying cell fate is governed by the inputs of intracellular and/or extracellular signals. Beta-catenin/Tcf and the TGF-beta Bone Morphogenetic Protein (BMP) provide critical molecular signaling inputs during germ layer specification in bilaterian metazoans but there has beenno direct experimental evidence for a specific role for BMP signaling during endomesoderm specification in the early branching metazoan Nematostella vectensis (ananthozoan cnidarian),. Using forward transcriptomics, we show that Betacatenin/Tcf signaling and BMP2/4 signaling provide differential inputs into the cnidarian endomesodermal gene regulatory network at the onset of gastrulation (24hpf) inNematostella vectensis. Surprisingly, Beta-catenin/Tcf signaling and BMP2/4 signaling regulate a subset of common downstream target genes in the GRN in opposite ways, leading to the spatial and temporal differentiation of fields of cells in the developing embryo. Thus, we show that regulatory interactions between Beta-catenin/Tcf signaling and BMP2/4 signaling are required for the specification and determination of different embryonic regions and the patterning of the oralaboral axis in Nematostella. We also show functionally that the conserved “kernel” of the bilaterian heart mesoderm GRN is operational in Nematostella vectensis, which reinforces the hypothesis that the endoderm and mesoderm in triploblastic bilaterians evolved from the bifunctional endomesoderm (gastrodermis) of a diploblastic ancestor, and that slow rhythmic contractions might have been one of the earliest functions of mesoderm.
doi:10.1016/j.mod.2017.04.304
PS3.99 Cellular and molecular mechanisms regulating limb position in vertebrates Chloe Moreaua,b,c, Nicolas Denansd, Olivier Pourquiée, Jerome Grosa,b a
Institut Pasteur, Paris, France URA2578, CNRS, France c Cellule Pasteur UPMC, Paris, France d Stanford School of Medicine, United States e Harvard Medical School, Boston, United States b
During vertebrate development, limbs emerge from the trunk of the embryo as buds that progressively elongate into fully developed
appendages. Whereas the molecular and genetic interactions underlying limb initiation, outgrowth and patterning have been greatly elucidated, how limbs reproducibly form at specific locations along the body axis still remains unclear. The position of limbs is very consistent within one species but is highly variable among vertebrates. Interestingly, a number of studies have shown that this variation is associated with variations in the expression pattern of Hox genes. Because of their role in patterning embryonic axes, these genes have long remained good candidates in regulating the position of the limbs. Here, we hypothesize that the positional information of limb progenitor cells is acquired during the early steps of gastrulation and we explore the role of Hox genes in this process. Combining classical embryology techniques with molecular and live imaging approaches in the chick embryo, we aim at characterizing the cellular and molecular events underlying the formation of the Lateral Plate Mesoderm (LPM, i.e. the mesodermal compartment giving rise to limbs) and in particular the role of Hox genes in patterning the LPM into limb-forming and non-limb-forming domains, during the gastrulation process.
doi:10.1016/j.mod.2017.04.305
PS3.100 Single-cell RNA-seq unveils divergent modes of chemoresistance in squamous cell carcinoma Ankur Sharmaa, Vibhor Kumara, Elaine Yiqun Caob, Hui Sun Leongb, Muhammad Hakimullaha, Neeraja Ramakrishnana, Xiaoqian Zhanga, Fui Teen Chongb, Shumei Chiaa, Matan Thangavelu Thangavelua, Angeline Mei Lin Wonga, Xue Lin Kwangb, Daniel Shao-Weng Tanb, Giridharan Periyasamya, N. Gopalakrishna Iyerb, Ramanuj DasGuptaa a
Genome Institute of, Singapore National Cancer Centre, Singapore
b
Intra-tumor heterogeneity (ITH) generates distinct cellular populations that contribute to tumor maintenance, progression, and drug-resistance. However, the contribution of individual cells to chemotherapy-resistance remains poorly understood. We generated single-cell mRNA sequencing profile of cisplatin-resistant squamous cell carcinoma (SCC) cells. Here we demonstrate that retention of native cellular-states is the principle mode of drug-resistance, a phenomenon we term ‘cellular inertia’. Interestingly, in comparison to other cancers ‘cellular-plasticity’ manifesting as epithelial-tomesenchymal transition (EMT) was observed to be a minor source of drug-resistance. These observations were also corroborated in cisplatin treated oral SCC patients. Importantly, gene expression signature from cellular plasticity-induced drug-resistant models correlated with poor survival. Synthetic lethal screens revealed a critical function of chromatin remodelers in EMT-associated acquired drug resistance. Specifically, we demonstrate the role of BRD4mediated H3K27 acetylation as an adaptive response to chemotherapy, which could be reversed by JQ1. In summary, our study provides critical insights into the divergent modes of drug resistance, and paves the way for therapeutic modalities involving epigenetic targeting of tumor evolution and ITH.
doi:10.1016/j.mod.2017.04.306