Cascade Of Gene Expression Research Articles

Body plan of turtles: an anatomical, developmental and evolutionary perspective

The evolution of the turtle shell has long been one of the central debates in comparative anatomy. The turtle shell consists of dorsal and ventral parts: the carapace and plastron, respectively. The basic structure of the carapace comprises vertebrae and ribs. The pectoral girdle of turtles sits inside the carapace or the rib cage, in striking contrast to the body plan of other tetrapods. Due to this topological change in the arrangement of skeletal elements, the carapace has been regarded as an example of evolutionary novelty that violates the ancestral body plan of tetrapods. Comparing the spatial relationships of anatomical structures in the embryos of turtles and other amniotes, we have shown that the topology of the musculoskeletal system is largely conserved even in turtles. The positional changes seen in the ribs and pectoral girdle can be ascribed to turtle-specific folding of the lateral body wall in the late developmental stages. Whereas the ribs of other amniotes grow from the axial domain to the lateral body wall, turtle ribs remain arrested axially. Marginal growth of the axial domain in turtle embryos brings the morphologically short ribs in to cover the scapula dorsocaudally. This concentric growth appears to be induced by the margin of the carapace, which involves an ancestral gene expression cascade in a new location. These comparative developmental data allow us to hypothesize the gradual evolution of turtles, which is consistent with the recent finding of a transitional fossil animal, Odontochelys, which did not have the carapace but already possessed the plastron.

The Sound of Silence: Mouse Models for Hearing Loss

Sensorineural hearing loss is one of the most common disabilities in humans. It is estimated that about 278 million people worldwide have slight to extreme hearing loss in both ears, which results in an economic loss for the country and personal loss for the individual. It is thus critical to have a deeper understanding of the causes for hearing loss to better manage and treat the affected individuals. The mouse serves as an excellent model to study and recapitulate some of these phenotypes, identify new genes which cause deafness, and to study their roles in vivo and in detail. Mutant mice have been instrumental in elucidating the function and mechanisms of the inner ear. The development and morphogenesis of the inner ear from an ectodermal layer into distinct auditory and vestibular components depends on well-coordinated gene expression and well-orchestrated signaling cascades within the otic vesicle and interactions with surrounding layers of tissues. Any disruption in these pathways can lead to hearing impairment. This review takes a look at some of the genes and their corresponding mice mutants that have shed light on the mechanism governing hearing impairment (HI) in humans.

Abstract PR5: Human Merkel cell polyomavirus causes Merkel cell carcinoma: Implication of viral etiology in human cancers

Abstract Over 20% of human cancers worldwide are associated with infection. We developed digital transcriptome subtraction (DTS) to identify a previously unregcognized virus, Merkel cell polyomavirus (MCV), in 80% of Merkel cell carcinomas (MCC). MCV is a double-strand DNA virus belonging to the Polyomaviridae family, having conserved early, late and regulatory regions. The polyomavirus early viral tumor (T) antigens play key roles in viral genome replication as well as tumorigenesis. For most polyomaviruses, Large T (LT) stimulate progressions of the cell cycle by binding to a number of cellular control proteins, such as tumor suppressor proteins p53 and the retinoblastoma (pRB). The small T (sT) antigen protein also targets the protein phosphatase 2A (PP2A) family of serine-threonine phosphatases pathway, which may contribute to viral neoplasia. The late region encodes viral capsid proteins (VP1 and VP2) are responsible for the viral particle composition. MCV was found as clonally-integrated virus into MCC and metastasis genomes. We sequenced full-length MCV genomes from 5 of the 8 virus-positive tumors as well as from 1 MCV positive cell line. All tumor-derived sequences have a T antigen truncation mutation that prematurely truncate the MCV LT helicase, rendering the virus nonpermissive. In contrast, full-length MCV genomes isolated from a peripheral blood sample can activate unlicensed replication from an integrated origin—suggesting that secondary mutations to T antigen are required for the integrated virus to survive. We reconstructed a consensus MCV clone, MCV-HF, from 7 MCV genomes. Three days after MCV-HF transfection into 293 cells, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical −40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which LT and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. MCV replication and encapsidation are increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. As a new human tumor virus, MCV can be expected to induce cell transformation in unexpected ways. Knockdown of all T antigen isoforms leads to cell cycle arrest and cell death of MCV-positive MCC cells, showing that the virus is absolutely required for these tumor cells. In transformation studies, overexpression of MCV sT, but not wild-type or tumor-derived MCV LT, causes rodent fibroblast transformation and human fibroblast serum-independent growth. In distinct contrast to SV40, MCV sT-induced cell transformation is independent of its ability to bind and interact with PP2A and heat-shock protein. Instead, MCV sT acts downstream in the mammalian target of rapamycin (mTOR) signaling pathway to target the eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1) and maintain its hyperphosphorylation. Our studies suggest that sT is an new viral oncoprotein that targets dysregulation of cap-dependent translation. In three years since its discovery using human genome project data, new diagnostics based on the Merkel cell polyomavirus have been developed for this enigmatic and severe cancer, and a basic understanding of how MCV promotes MCC has been uncovered. These findings hold promise for development of rational chemotherapeutic targeting of this enigmatic and severe cancer in the near future. This abstract is also presented as Poster B31. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR5.

Crx broadly modulates the pineal transcriptome

Cone-rod homeobox (Crx) encodes Crx, a transcription factor expressed selectively in retinal photoreceptors and pinealocytes, the major cell type of the pineal gland. In this study, the influence of Crx on the mammalian pineal gland was studied by light and electron microscopy and by use of microarray and qRTPCR technology, thereby extending previous studies on selected genes (Furukawa et al. 1999). Deletion of Crx was not found to alter pineal morphology, but was found to broadly modulate the mouse pineal transcriptome, characterized by a>2-fold down-regulation of 543 genes and a>2-fold up-regulation of 745 genes (p<0.05). Of these, one of the most highly up-regulated (18-fold) was Hoxc4, a member of the Hox gene family, members of which are known to control gene expression cascades. During a 24-h period, a set of 51 genes exhibited differential day/night expression in pineal glands of wild-type animals; only eight of these were also day/night expressed in the Crx⁻/⁻ pineal gland. However, in the Crx⁻/⁻ pineal gland 41 genes exhibited differential night/day expression that was not seen in wild-type animals. These findings indicate that Crx broadly modulates the pineal transcriptome and also influences differential night/day gene expression in this tissue. Some effects of Crx deletion on the pineal transcriptome might be mediated by Hoxc4 up-regulation.

Expression profiling reveals differential gene induction underlying specific and non-specific memory for pheromones in mice

Memory for the mating male’s pheromones in female mice is thought to require synaptic changes in the accessory olfactory bulb (AOB). Induction of this memory depends on release of glutamate in response to pheromonal exposure coincident with release of norepinephrine (NE) in the AOB following mating. A similar memory for pheromones can also be induced artificially by local infusion of the GABA A receptor antagonist bicuculline into the AOB. The natural memory formed by exposure to pheromones during mating is specific to the pheromones sensed by the female during mating. In contrast, the artificial memory induced by bicuculline is non-specific and results in the female mice recognizing all pheromones as if they were from the mating male. Although protein synthesis has been shown to be essential for development of pheromone memory, the gene expression cascades critical for memory formation are not known. We investigated changes in gene expression in the AOB using oligonucleotide microarrays during mating-induced pheromone memory (MIPM) as well as bicuculline-induced pheromone memory (BIPM). We found the set of genes induced during MIPM and BIPM are largely non-overlapping and Ingenuity Pathway Analysis revealed that the signaling pathways in MIPM and BIPM also differ. The products of genes induced during MIPM are associated with synaptic function, indicating the possibility of modification at specific synapses, while those induced during BIPM appear to possess neuron-wide functions, which would be consistent with global cellular changes. Thus, these results begin to provide a mechanistic explanation for specific and non-specific memories induced by pheromones and bicuculline infusion respectively.

Myocyte Enhancer Factor 2C, an Osteoblast Transcription Factor Identified by Dimethyl Sulfoxide (DMSO)-enhanced Mineralization

Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem cell-mediated therapies for fracture and other orthopedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of stimulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase activity and extracellular matrix mineralization. Furthermore, similar DMSO-mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1, we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among the numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical, and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased alkaline phosphatase activity, and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. A flow on knockdown of bone-specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c, suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts.

PDX1- and NGN3-mediated in vitro reprogramming of human bone marrow-derived mesenchymal stromal cells into pancreatic endocrine lineages

Reprogramming of multipotent adult bone marrow (BM)-derived mesenchymal stromal/stem cells (MSC) (BM-MSC) represents one of several strategies for cell-based therapy of diabetes. However, reprogramming primary BM-MSC into pancreatic endocrine lineages has not yet been consistently demonstrated. To unravel the role and interaction of key factors governing this process, we used well-characterized telomerase-immortalized human MSC (hMSC-TERT). Pancreatic endocrine differentiation in hMSC-TERT was induced by two major in vitro strategies: (i) endocrine-promoting culture conditions and (ii) ectopic expression of two master regulatory genes of the endocrine lineage, human neurogenin 3 (NGN3) and human pancreatic duodenal homeobox 1 (PDX1). Both approaches triggered pancreatic endocrine gene expression, notably insulin, glucose-transporter 2 and somatostatin. Transgenic overexpression of NGN3 and/or PDX1 proteins not only induced direct target genes, such as NEUROD1 and insulin, and but also triggered parts of the gene expression cascade that is involved in pancreatic endocrine differentiation. Notably, ectopic NGN3 alone was sufficient to initiate the expression of specific beta-cell lineage-associated genes, most importantly PDX1 and insulin. This was demonstrated both transcriptionally by mRNA expression and reporter gene analyzes and at a protein level by Western blotting. Such reprogramming of hMSC-TERT cells induced glucose-insensitive insulin biosynthesis and secretion. Our results indicate that establishment of glucose-dependent insulin secretion in partially reprogrammed human MSC may depend on additional maturation factors. Moreover, hMSC-TERT provides a suitable cell model for investigating further the molecular mechanisms of reprogramming and maturation of adult MSC towards pancreatic endocrine lineages.

Cellular and viral factors regulating Merkel cell polyomavirus replication.

Merkel cell polyomavirus (MCV), a previously unrecognized component of the human viral skin flora, was discovered as a mutated and clonally-integrated virus inserted into Merkel cell carcinoma (MCC) genomes. We reconstructed a replicating MCV clone (MCV-HF), and then mutated viral sites required for replication or interaction with cellular proteins to examine replication efficiency and viral gene expression. Three days after MCV-HF transfection into 293 cells, although replication is not robust, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical ∼40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which large T (LT) and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. VP1 and sT proteins are not detected, and spliced 57kT is markedly diminished, in the replication-defective virus suggesting that early gene splicing and late gene transcription may be dependent on viral DNA replication. MCV replication and encapsidation is increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. Mutation of the MCV LT vacuolar sorting protein hVam6p (Vps39) binding site also enhances MCV replication while exogenous hVam6p overexpression reduces MCV virion production by >90%. Although MCV-HF generates encapsidated wild-type MCV virions, we did not find conditions for persistent transmission to recipient cell lines suggesting that MCV has a highly restricted tropism. These studies identify and highlight the role of polyomavirus DNA replication in viral gene expression and show that viral sT and cellular hVam6p are important factors regulating MCV replication. MCV-HF is a molecular clone that can be readily manipulated to investigate factors affecting MCV replication.

M94 Is Essential for the Secondary Envelopment of Murine Cytomegalovirus

The gene M94 of murine cytomegalovirus (MCMV) as well as its homologues UL16 in alphaherpesviruses is involved in viral morphogenesis. For a better understanding of its role in the viral life cycle, a library of random M94 mutants was generated by modified transposon-based linker scanning mutagenesis. A comprehensive set of M94 mutants was reinserted into the MCMV genome and tested for their capacity to complement the M94 null mutant. Thereby, 34 loss-of-function mutants of M94 were identified, which were tested in a second screen for their capacity to inhibit virus replication. This analysis identified two N-terminal insertion mutants of M94 with a dominant negative effect. We compared phenotypes induced by the conditional expression of these dominant negative M94 alleles with the null phenotype of the M94 deletion. The viral gene expression cascade and the nuclear morphogenesis steps were not affected in either setting. In both cases, however, secondary envelopment did not proceed in the absence of functional M94, and capsids subsequently accumulated in the center of the cytoplasmic assembly complex. In addition, deletion of M94 resulted in a block of cell-to-cell spread. Moreover, the dominant negative mutant of M94 demonstrated a defect in interacting with M99, the UL11 homologue of MCMV.

Quantitative Time-course Profiling of Parasite and Host Cell Proteins in the Human Malaria Parasite Plasmodium falciparum

Studies of the Plasmodium falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intra-erythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade in which most expressed genes exhibit a single transcriptional peak. Because the biochemical and cellular functions of most genes are mediated by the encoded proteins, understanding the relationship between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. Although studies on other organisms show that <50% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on oligonucleotide microarrays and two-dimensional differential gel electrophoresis protein gels. We find that most proteins are represented by more than one isoform, presumably because of post-translational modifications. Like transcripts, most proteins exhibit cyclic abundance profiles with one peak during the IDC, whereas the presence of functionally related proteins is highly correlated. In contrast, the abundance of most parasite proteins peaks significantly later (median 11 h) than the corresponding transcripts and often decreases slowly in the second half of the IDC. Computational modeling indicates that the considerable and varied incongruence between transcript and protein abundance may largely be caused by the dynamics of translation and protein degradation. Furthermore, we present cyclic abundance profiles also for parasite-associated human proteins and confirm the presence of five human proteins with a potential role in antioxidant defense within the parasites. Together, our data provide fundamental insights into transcript-protein relationships in P. falciparum that are important for the correct interpretation of transcriptional data and that may facilitate the improvement and development of malaria diagnostics and drug therapy.

Pulmonary Endpoints (Lung Carcinomas and Asbestosis) Following Inhalation Exposure to Asbestos

Lung carcinomas and pulmonary fibrosis (asbestosis) occur in asbestos workers. Understanding the pathogenesis of these diseases is complicated because of potential confounding factors, such as smoking, which is not a risk factor in mesothelioma. The modes of action (MOA) of various types of asbestos in the development of lung cancers, asbestosis, and mesotheliomas appear to be different. Moreover, asbestos fibers may act differentially at various stages of these diseases, and have different potencies as compared to other naturally occurring and synthetic fibers. This literature review describes patterns of deposition and retention of various types of asbestos and other fibers after inhalation, methods of translocation within the lung, and dissolution of various fiber types in lung compartments and cells in vitro. Comprehensive dose-response studies at fiber concentrations inhaled by humans as well as bivariate size distributions (lengths and widths), types, and sources of fibers are rarely defined in published studies and are needed. Species-specific responses may occur. Mechanistic studies have some of these limitations, but have suggested that changes in gene expression (either fiber-catalyzed directly or by cell elaboration of oxidants), epigenetic changes, and receptor-mediated or other intracellular signaling cascades may play roles in various stages of the development of lung cancers or asbestosis.

Regulation of neurogenesis by extracellular matrix and integrins.

Deciphering the factors that regulate human neural stem cells will greatly aid in their use as models of development and as therapeutic agents. The complex interactions of cells with extracellular matrix (ECM) proteins probably contribute to proper central nervous system development mediating processes which regulate proliferation and differentiation of neural stem/rogenitor cells. Many of these interactions involve transmembrane integrin receptors. Integrins cluster in specific cell-matrix adhesions to provide dynamic links between extracellular and intracellular environments by activation of numerous signal transduction pathways which may influence cell behaviour profoundly by influence on both gene expression and post-transcriptional signalling cascade. In this review we introduced and discussed a number of extracellular and intracellular factors engaged in the transduction of signals induced by cell adhesion to its environment, including matrix components, extracellular proteolytic enzymes, integrins and non-receptor tyrosine kinases.

Unwrapping HDAC1 and HDAC2 functions in Schwann cell myelination

Two studies now show that epigenetic regulation of gene transcription by histone deacetylases (HDACs) 1 and 2 regulates Schwann cell myelination through activation of a cascade of myelin gene expression. Deletion of HDAC1 and HDAC2 in Schwann cells disrupts interactions with axons, blocks myelination and leads to Schwann cell death.

Analysis of the Molecular Mechanisms Governing the Stage-Specific Expression of a Prototypical Housekeeping Gene during Intraerythrocytic Development of P. falciparum

Gene expression during the intraerythrocytic development cycle of the human malarial parasite Plasmodium falciparum is subject to tight temporal control, resulting in a cascade of gene expression to meet the physiological demands of growth, replication, and reinvasion. The roles of the different molecular mechanisms that drive this temporal program of gene expression are poorly understood. Here we report the use of the bxb1 integrase system to reconstitute all aspects of the absolute and temporal control of the prototypical housekeeping gene encoding the proliferating cell nuclear antigen (Pfpcna) around an integrated luciferase reporter cassette. A quantitative analysis of the effect of the serial deletion of 5′ and 3′ genetic elements and sublethal doses of histone deacetylase inhibitors demonstrates that while the absolute control of gene expression could be perturbed, no effect on the temporal control of gene expression was observed. These data provide support for a novel model for the temporal control of potentially hundreds of genes during the intraerythrocytic development of this important human pathogen.

Skeletal development in human

Our understanding of the genetic and molecular control of development in vertebrates has dramatically increased during the last 10 years through the discovery that molecular processes that control development in invertebrates have been conserved during evolution and are also found in vertebrates. Important developmental genes were identified that are not only similar in sequence but also in their molecular function in widely diverged organisms such as C.elegans, Drosophila, zebrafish, mice and man. From Drosophila studies it is now clear that epigenetic development is regulated by cascades of gene expression. Early acting regulatory genes initiate the developmental process and induce the expression of other downstream genes.

A Chinese Herbal Decoction, Danggui Buxue Tang, Stimulates Proliferation, Differentiation and Gene Expression of Cultured Osteosarcoma Cells: Genomic Approach to Reveal Specific Gene Activation

Danggui Buxue Tang (DBT), a Chinese herbal decoction used to treat ailments in women, contains Radix Astragali (Huangqi; RA) and Radix Angelicae Sinensis (Danggui; RAS). When DBT was applied onto cultured MG-63 cells, an increase of cell proliferation and differentiation of MG-63 cell were revealed: both of these effects were significantly higher in DBT than RA or RAS extract. To search for the biological markers that are specifically regulated by DBT, DNA microarray was used to reveal the gene expression profiling of DBT in MG-63 cells as compared to that of RA- or RAS-treated cells. Amongst 883 DBT-regulated genes, 403 of them are specifically regulated by DBT treatment, including CCL-2, CCL-7, CCL-8, and galectin-9. The signaling cascade of this DBT-regulated gene expression was also elucidated in cultured MG-63 cells. The current results reveal the potential usage of this herbal decoction in treating osteoporosis and suggest the uniqueness of Chinese herbal decoction that requires a well-defined formulation. The DBT-regulated genes in the culture could serve as biological responsive markers for quality assurance of the herbal preparation.

Homo sapiens, an unlikely but effective model in Evo-Devo-a review of Quirks of Human Anatomy: an Evo-Devo Look at the Human Body, by Lewis I. Held, Jr.

What can we expect from an Evo-Devo look at the human body? The subject speciesFman rather than the worm or the fruitflyFis one toward which a lot of potential readers will be attracted. But is an Evo-Devo approach to our species really promising? How good canHomo sapiens be as a model species for Evo-Devo? The simple fact of restricting focus to only one species is likely to reduce to a very minimum any hope for a compelling evolutionary argument. In addition, the previous publication record of the author deals solely with pattern formation in Drosophila melanogaster: a model species quite distant from humans. A potential reader might envision a possibly popular but eventually less than academic exercise. All these quibbles, however, are destined to rapidly fade as soon as we start reading from this fascinating book. Held’s prose is brilliant, very often informal but always accurate, and the message it conveys is always clear and well documented, sometimes incredibly so. This is not the first time this author provides a list of references filling roughly one half of the whole book (cf. Held 1992, 2002). In Quirks of Human Anatomy, 152 pages of text and illustrations are followed by 86 pages of references, with 2924 entries, followed in turn by a 22-pages index. Moving into new territory, Held has obviously taken care to gather a very broad and sound background of published information. What, then, of the suitability of Homo sapiens as model species in Evo-Devo? If one is effective, as Held is, in mining the literature for potentially relevant data, there is indeed a lot of evidence to be found there, particularly from the perspective of comparative developmental genetics. To be sure, this comparative evidence differs qualitatively from the body of knowledge we are gathering with flies or worms, that is animals amenable to experimental manipulation. In the case of humans, the comparative dimension is obtained by studying intraspecific variation already present in human populations, in all kinds of pathological (if not monstrous) deviation from the ‘‘normal’’ phenotype. Held’s Evo-Devo approach to the anatomy of the human body, and its many oddities, is nonetheless rooted in a search for genes and gene expression cascades such as those involved, for example, in establishing the characteristic asymmetry of our viscera, the odd crossing of the body midline by optic nerves, or the more or less obvious morphological differences between males and females. A search for genes, of course, because body parts can diverge developmentallyFthat is, ‘‘dissociate’’Fto any great extent only if they have acquired distinct genetic identities. Held admirably also maintains an open eye toward those features, like our fingerprints, which are beyond the control of our genome. This adventurous path amidst the oddities of the human body includes a couple of pages on the presence of nipples in men, or men’s privilege, so to speak, to have a chance of getting bald. Held’s welljustified interest in monsters, for example humans affected by cyclopia, or with completely furry skin, inclusive of hands and feet, opens the door to a related, very intriguing question. How do we determine whether a given kind of monstrosity never observed in humans, but which is nonetheless imaginable, can ever evolve in our species? More precisely, why are some morphologies forbidden in humans, whereas EVOLUTION & DEVELOPMENT 12:6, 647–648 (2010)

P71. Stem cells and gene expression during hemichordate head regeneration

Hemichordate acorn worms grow a new head on the anterior wound surface when the body is transected. In Ptychodera flava, a blastema is evident by 3 days and a new full-sized, functional head differentiates on the cut stump by 12 days. The hemichordate nervous system provides the most ancestral extant example of the dorsal hollow brain, formed by neurulation, characteristic of chordates and humans. Only in hemichordates does such a nervous system regenerate significantly. Animals throughout the phylogenetic scale share similar developmental gene regulatory networks, and our work, along with that of others, supports the conclusion that the same gene regulatory networks occur in hemichordate and chordate neural differentiation. Examination of the regeneration process by whole mount in situ hybridization (WISH) of sequences from Yamanaka pluripotency factors or “stem cell” genes, as defined by genes inducing pluripotency in mammalian somatic cells, and developmental organizer genes reveals a cascade of gene expression associated with the steps of regeneration. A subset of the stem cell genes produce the first WISH signals before a blastema is formed. The second set of stem cell genes begins to generate signal as the blastema is established and this signal persists in the blastema as organizer and early development gene signals appear as differentiation proceeds. These results suggest that activation of cells with regenerative capability involves the early expression of stem cell genes in the cells that will form the blastema. Signal from some stem cell genes continues as the expression of typical embryonic gene regulatory networks are activated for the differentiation of the tissues. In planarians, regeneration is based on mobilization of stem cells residing in the tissue of the animal to form a regeneration blastema. During regeneration of an amphibian limb, a blastema is also formed, apparently by dedifferentiation of tissue cells in the cut limb to stem cells which accumulate to establish the blastema. The nature of the cells and the tissue origin of the cells that participate in hemichordate blastema formation and head regeneration are being investigated. Supported by the PBRC Biomedical Fund UHF#12-209-04.

Pancreatic Stellate Cell Models for Transcriptional Studies of Desmoplasia-Associated Genes

Background: Pancreatic stellate cells are emerging as key players in pathophysiopathological processes underlying the development of pancreatic disease, including pancreatitis and cancer. The cells are scarce in the pancreas making their isolation time and resource use consuming. Methods: Therefore, with the ultimate goal of facilitating mechanistic studies, here we report the isolation, characterization, and immortalization of stellate cell lines from rat and mouse origin. Results: These cell lines display morphological and molecular markers as well as non-tumorigenic characteristics similar to the frequently used hepatic counterparts. In addition, we have tested their robustness as a model for transcriptional regulatory studies. We find that these cells respond well to TGFβ signaling by triggering a distinct cascade of gene expression, some genes overlap with the TGFβ response of LX2 cells. These cells express several key chromatin proteins and epigenetic regulators involved in the regulation of gene expression, including co-repressors such as Sin3A (short-term repression), HP1 (long-term repression), as well as CBP/p300 (activation). Furthermore, these cells are well suited for Gal4-based transcriptional activation and repression assays. Conclusions: The cell model reported here may therefore help fuel investigations in the field of signaling, transcription, and perhaps other studies on similarly exciting cellular processes.

Atypical molecular profile for joint development in the avian costal joint

Development of synovial joints involves generation of cartilaginous anlagen, formation of interzones between cartilage anlagen, and cavitation of interzones to produce fluid filled cavities. Interzone development is not fully understood, but interzones are thought to develop from skeletogenic cells that are inhibited from further chondrogenic development by a cascade of gene expression including Wnt and Bmp family members. We examined the development of the rarely studied avian costal joint to better understand mechanisms of joint development. The costal joint is found within ribs, is morphologically similar to the metatarsophalangeal joint, and undergoes cavitation in a similar manner. In contrast to other interzones, Wnt14/9a, Gdf5, Chordin, Barx1, and Bapx1 are absent from the costal joint interzone, consistent with the absence of active β-catenin and phosphorylated Smad 1/5/8. However Autotaxin and Noggin are expressed. The molecular profile of the costal joint suggests there are alternative mechanisms of interzone development.