Life Sciences Commons^™

Developing Inside A Layer Of Germs—A Potential Role For Multiciliated Surface Cells In Vertebrate Embryos, Ryan R. Kerney

Biology Faculty Publications

This paper reviews current research on the microbial life that surrounds vertebrate embryos. Several clades are believed to develop inside sterile—or near-sterile—embryonic microhabitats, while others thrive within a veritable zoo of microbial life. The occurrence of embryo-associated microbes in some groups, but not others, is an under-appreciated transition (possibly transitions) in vertebrate evolution. A lack of comparable studies makes it currently impossible to correlate embryo-associated microbiomes with other aspects of vertebrate evolution. However, there are embryonic features that should instruct a more targeted survey. This paper concludes with a hypothesis for the role of multiciliated surface cells in amphibian and …

Larval Anatomy Of Monotypic Painted Ant Nest Frogs Lithodytes Lineatus Reveals Putative Homoplasies With The Leptodactylus Pentadactylus Group (Anura: Leptodactylidae), Filipe A.C Do Nascimento, Rafael O. De Sá, Paulo C. De A. Garcia

Biology Faculty Publications

The morphological diversity of anuran larvae made them an important source of information for evolutionary and systematic studies. For the monotypic frog genus Lithodytes, which has an interesting taxonomic history, including its past synonymizing with Adenomera and its placement as a subgenus of Leptodactylus, the information provided from its larvae can help to understand its systematics interrelationships and also provide insights about its evolutionary trajectories. Herein, we provide a detailed description of the larval morphology of Lithodytes lineatus, including novel data of internal morphology (buccopharyngeal cavity and skeleton), and discuss some morphological and evolutionary aspects in relation …

Connective Tissue Fibroblasts From Highly Regenerative Mammals Are Refractory To Ros-Induced Cellular Senescence, Sandeep Saxena, Hemendra J. Vekaria, Patrick G. Sullivan, Ashley W. Seifert

Biology Faculty Publications

A surveillance system in mammals constantly monitors cell activity to protect against aberrant proliferation in response to damage, injury and oncogenic stress. Here we isolate and culture connective tissue fibroblasts from highly regenerative mammals (Acomys and Oryctolagus) to determine how these cells interpret signals that normally induce cellular senescence in non-regenerating mammals (Mus and Rattus). While H₂O₂ exposure substantially decreases cell proliferation and increases p53, p21, p16, and p19 in cells from mice and rats, cells from spiny mice and rabbits are highly resistant to H₂O₂. Quantifying oxygen consumption …

Fgf-Signaling Is Compartmentalized Within The Mesenchyme And Controls Proliferation During Salamander Limb Development, Sruthi Purushothaman, Ahmed Elewa, Ashley W. Seifert

Biology Faculty Publications

Although decades of studies have produced a generalized model for tetrapod limb development, urodeles deviate from anurans and amniotes in at least two key respects: their limbs exhibit preaxial skeletal differentiation and do not develop an apical ectodermal ridge (AER). Here, we investigated how Sonic hedgehog (Shh) and Fibroblast growth factor (Fgf) signaling regulate limb development in the axolotl. We found that Shh-expressing cells contributed to the most posterior digit, and that inhibiting Shh-signaling inhibited Fgf8 expression, anteroposterior patterning, and distal cell proliferation. In addition to lack of a morphological AER, we found that salamander …

Nell2 Regulates The Contralateral-Versus-Ipsilateral Visual Projection As A Domain-Specific Positional Cue, Chizu Nakamoto, Elaine Durward, Masato Horie, Masaru Nakamoto

Biology Faculty Publications

In mammals with binocular vision, retinal ganglion cell (RGC) axons from each eye project to eye-specific domains in the contralateral and ipsilateral dorsal lateral geniculate nucleus (dLGN), underpinning disparity-based stereopsis. Although domain-specific axon guidance cues that discriminate contralateral and ipsilateral RGC axons have long been postulated as a key mechanism for development of the eye-specific retinogeniculate projection, the molecular nature of such cues has remained elusive. Here, we show that the extracellular glycoprotein Nell2 (neural epidermal growth factor-like-like 2) is expressed in the dorsomedial region of the dLGN, which ipsilateral RGC axons terminate in and contralateral axons avoid. In Nell2 …

The Folic Acid Metabolism Gene Mel-32/Shmt Is Required For Normal Cell Cycle Lengths In Caenorhabditis Elegans, Kalin D. Konrad, Raquel A. Campbell, Vanessa Thiel, Jessica Sullivan-Brown

Biology Faculty Publications

Neural tube defects are common and serious birth defects in which the brain and/or spinal cord are exposed outside the body. Supplementation of foods with folic acid, an essential vitamin, is linked to a lower risk of neural tube defects; however, the mechanisms by which folic acid influence neural tube defect risk are unclear. Our research seeks to identify the basic cellular roles of known folic acid metabolism genes during morphogenesis using the roundworm Caenorhabditis elegans (C. elegans) as a simple model system. Here, we used live imaging to characterize defects in embryonic development when mel-32 is depleted. mel-32 is …

Butyl Benzyl Phthalate (Bbp) Induces Caudal Defects During Embryonic Development, Nicole M. Roy, Ewelina Zambrzycka, Jenna Santangelo

Biology Faculty Publications

Butyl benzyl phthalate (BBP) is commonly added during the manufacturing of plastics to increase flexibility and elasticity. However, BBP leaches off of plastic and environment presence has been detected in soil, groundwater and sediment potentially effecting organisms in the environment. Given the widespread uses of BBP in household, consumer goods and the presence of BBP in the environment, studies on developmental toxicity are needed. Here, we use a zebrafish model to investigate the early developmental toxicity of BBP. We treated gastrula staged embryos with increasing concentrations of BBP and noted concentration-dependent defects in caudal tail development, but the effect was …

Deep Ancestry Of Programmed Genome Rearrangement In Lampreys, Vladimir A. Timoshevskiy, Ralph T. Lampman, Jon E. Hess, Laurie L. Porter, Jeramiah J. Smith

Biology Faculty Publications

In most multicellular organisms, the structure and content of the genome is rigorously maintained over the course of development. However some species have evolved genome biologies that permit, or require, developmentally regulated changes in the physical structure and content of the genome (programmed genome rearrangement: PGR). Relatively few vertebrates are known to undergo PGR, although all agnathans surveyed to date (several hagfish and one lamprey: Petromyzon marinus) show evidence of large scale PGR. To further resolve the ancestry of PGR within vertebrates, we developed probes that allow simultaneous tracking of nearly all sequences eliminated by PGR in P. marinus and …

A Linkage Map For The Newt Notophthalmus Viridescens: Insights In Vertebrate Genome And Chromosome Evolution, Melissa C. Keinath, S. Randal Voss, Panagiotis A. Tsonis, Jeramiah J. Smith

Biology Faculty Publications

Genetic linkage maps are fundamental resources that enable diverse genetic and genomic approaches, including quantitative trait locus (QTL) analyses and comparative studies of genome evolution. It is straightforward to build linkage maps for species that are amenable to laboratory culture and genetic crossing designs, and that have relatively small genomes and few chromosomes. It is more difficult to generate linkage maps for species that do not meet these criteria. Here, we introduce a method to rapidly build linkage maps for salamanders, which are known for their enormous genome sizes. As proof of principle, we developed a linkage map with thousands …

Macrophages Are Necessary For Epimorphic Regeneration In African Spiny Mice, Jennifer Simkin, Thomas R. Gawriluk, John C. Gensel, Ashley W. Seifert

Biology Faculty Publications

How the immune system affects tissue regeneration is not well understood. In this study, we used an emerging mammalian model of epimorphic regeneration, the African spiny mouse, to examine cell-based inflammation and tested the hypothesis that macrophages are necessary for regeneration. By directly comparing inflammatory cell activation in a 4 mm ear injury during regeneration (Acomys cahirinus) and scarring (Mus musculus), we found that both species exhibited an acute inflammatory response, with scarring characterized by stronger myeloperoxidase activity. In contrast, ROS production was stronger and more persistent during regeneration. By depleting macrophages during injury, we demonstrate …

Transciptome Analysis Illuminates The Nature Of The Intracellular Interaction In A Vertebrate-Algal Symbiosis, John A. Burns, Huanjia Zhang, Elizabeth M. Hill, Eunsoo Kim, Ryan R. Kerney

Biology Faculty Publications

During embryonic development, cells of the green alga Oophila amblystomatis enter cells of the salamander Ambystoma maculatum forming an endosymbiosis. Here, using de novo dual-RNA seq, we compared the host salamander cells that harbored intracellular algae to those without algae and the algae inside the animal cells to those in the egg capsule. This two-by-two-way analysis revealed that intracellular algae exhibit hallmarks of cellular stress and undergo a striking metabolic shift from oxidative metabolism to fermentation. Culturing experiments with the alga showed that host glutamine may be utilized by the algal endosymbiont as a primary nitrogen source. Transcriptional changes in …

Mating Patterns And Post-Mating Isolation In Three Cryptic Species Of The Engystomops Petersi Species Complex, Paula A. Trillo, Andrea E. Narvaez, Santiago R. Ron, Kim L. Hoke

Biology Faculty Publications

Determining the extent of reproductive isolation in cryptic species with dynamic geographic ranges can yield important insights into the processes that generate and maintain genetic divergence in the absence of severe geographic barriers. We studied mating patterns, propensity to hybridize in nature and subsequent fertilization rates, as well as survival and development of hybrid F₁ offspring for three nominal species of the Engystomops petersi species complex in Yasuní National Park, Ecuador. We found at least two species in four out of six locations sampled, and 14.3% of the wild pairs genotyped were mixed-species (heterospecific) crosses. We also found reduced …

Transposon-Mediated Stable Suppression Of Gene Expression In The Developing Chick Retina, Masaru Nakamoto, Chizu Nakamoto

Biology Faculty Publications

The embryonic chick has long been a favorite model system for in vivo studies of vertebrate development. However, a major technical limitation of the chick embryo has been the lack of efficient loss-of-function approaches for analyses of gene functions. Here, we describe a methodology in which a transgene encoding artificial microRNA sequences is introduced into embryonic chick retinal cells by in ovo electroporation and integrated into the genome using the Tol2 transposon system. We show that this methodology can induce potent and stable suppression of gene expression. This technique therefore provides a rapid and robust loss-of-function approach for studies of …

Evolution Of The Vertebrate Claudin Gene Family: Insights From A Basal Vertebrate, The Sea Lamprey, Christian Mukendi, Nicholas Dean, Rushil Lala, Jeramiah J. Smith, Marianne E Bronner, Natalya V. Nikitina

Biology Faculty Publications

Claudins are major constituents of tight junctions, contributing both to their intercellular sealing and selective permeability properties. While claudins and claudin-like molecules are present in some invertebrates, the association of claudins with tight junctions has been conclusively documented only in vertebrates. Here we report the sequencing, phylogenetic analysis and comprehensive spatiotemporal expression analysis of the entire claudin gene family in the basal extant vertebrate, the sea lamprey. Our results demonstrate that clear orthologues to about half of all mammalian claudins are present in the lamprey, suggesting that at least one round of whole genome duplication contributed to the diversification of …

Nel Positively Regulates The Genesis Of Retinal Ganglion Cells By Promoting Their Differentiation And Survival During Development, Chizu Nakamoto, Soh-Leh Kuan, Amy Findlay, Elaine Durward, Zhufeng Ouyang, Masaru Nakamoto, Ewa Zakrewska, Takuma Endo

Biology Faculty Publications

For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1–like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine …

Germline Transgenic Methods For Tracking Cells And Testing Gene Function During Regeneration In The Axolotl, Shahryar Khattak, Maritta Schuez, Tobias Richter, Dunja Knapp, Saori L. Haigo, Tatiana Sandoval-Guzmán, Kristyna Hradlikova, Annett Duemmler, Ryan R. Kerney, Elly M. Tanaka

Biology Faculty Publications

The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum(axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly …

Unexpected Phylogenetic Positions Of The Genera Rupirana And Crossodactylodes Reveal Insights Into The Biogeography And Reproductive Evolution Of Leptodactylid Frogs, Antoine Foquet, Boris Leonardo Blotto, Maximiliano Manuel Maronna, Vanessa Kruth Verdade, Flora Acuña Juncá, Rafael O. De Sá, Miguel Trefaut Rodrigues

Biology Faculty Publications

Despite major progress in deciphering the amphibian tree of life by molecular phylogenetics, we identified two questions remaining to be answered regarding relationships within Hyloidea, the clade of South American origin that comprises most extant anuran diversity. A few genera like Rupirana and Crossodactylodes have enigmatic phylogenetic positions, and relationships among major lineages within some families like Leptodactylidae remain ambiguous. To resolve these specific questions we used two approaches (1) a complete matrix approach representing >6.6 kb, including most major Hyloidea lineages (61 terminals) combining different methods of phylogenetic reconstruction and measures of node support; and (2) a supermatrix approach …

Cartilage On The Move: Cartilage Lineage Tracing During Tadpole Metamorphosis, Ryan R. Kerney, Alison L. Brittain, Brian K. Hall, Daniel R. Buchholz

Biology Faculty Publications

The reorganization of cranial cartilages during tadpole metamorphosis is a set of complex processes. The fates of larval cartilage-forming cells (chondrocytes) and sources of adult chondrocytes are largely unknown. Individual larval cranial cartilages may either degenerate or remodel, while many adult cartilages appear to form de novo during metamorphosis. Determining the extent to which adult chondrocytes/cartilages are derived from larval chondrocytes during metamorphosis requires new techniques in chondrocyte lineage tracing. We have developed two transgenic systems to label cartilage cells throughout the body with fluorescent proteins. One system strongly labels early tadpole cartilages only. The other system inducibly labels forming …

Rapid Range Expansion In The Great Plains Narrow-Mouthed Toad (Gastrophryne Olivacea) And A Revised Taxonomy For North American Microhylids, Jeffrey W. Streicher, Christian L. Cox, Jonathan A. Campbell, Eric N. Smith, Rafael O. De Sá

Biology Faculty Publications

We investigated genetic variation within the Great Plains narrow-mouthed toad, Gastrophryne olivacea, across its geographic range in the United States and Mexico. An analysis of mitochondrial DNA (mtDNA) from 105 frogs revealed remarkably low levels of genetic diversity in individuals inhabiting the central United States and northern Mexico. We found that this widespread matrilineal lineage is divergent (ca. 2% in mtDNA) from haplotypes that originate from the western United States and western coast of Mexico. Using a dataset that included all five species of Gastrophryne and both species of the closely related genus Hypopachus, we investigated the phylogenetic …

Structure-Function Analysis Of Nel, A Thrombospondin-1-Like Glycoprotein Involved In Neural Development And Functions, Masaru Nakamoto

Biology Faculty Publications

Nel (neural epidermal growth factor (EGF)-like molecule) is a multimeric, multimodular extracellular glycoprotein with heparin-binding activity and structural similarities to thrombospondin-1. Nel is predominantly expressed in the nervous system and has been implicated in neuronal proliferation and differentiation, retinal axon guidance, synaptic functions, and spatial learning. The Nel protein contains an N-terminal thrombospondin-1 (TSP-N) domain, five cysteine-rich domains, and six EGF-like domains. However, little is known about the functions of specific domains of the Nel protein. In this study, we have performed structure-function analysis of Nel, by using a series of expression constructs for different regions of the Nel protein. …

Dynamic Glucoregulation And Mammalian-Like Responses To Metabolic And Developmental Disruption In Zebrafish, Agata Jurczyk, Nicole M. Roy, Rabia Bajwa, Philipp Gut, Kathryn Lipson, Chaoxing Yang, Laurence Covassin, Waldemar J. Racki, Aldo A. Rossini, Nancy Phillips, Didier Y. R. Stainier, Dale L. Greiner, Michael A. Brehm, Rita Bortell, Philip Diiorio

Biology Faculty Publications

Zebrafish embryos are emerging as models of glucose metabolism. However, patterns of endogenous glucose levels, and the role of the islet in glucoregulation, are unknown. We measured absolute glucose levels in zebrafish and mouse embryos, and demonstrate similar, dynamic glucose fluctuations in both species. Further, we show that chemical and genetic perturbations elicit mammalian-like glycemic responses in zebrafish embryos. We show that glucose is undetectable in early zebrafish and mouse embryos, but increases in parallel with pancreatic islet formation in both species. In zebrafish, increasing glucose is associated with activation of gluconeogenic phosphoenolpyruvate carboxykinase1 (pck1) transcription. Non-hepatic Pck1 protein is …

Methods For Karyotyping And For Localization Of Developmentally Relevant Genes On The Chromosomes Of The Purple Sea Urchin Strongylocentrotus Purpuratus, Celeste C. Eno, Stefanie A. Boettger, Charles W. Walker

Biology Faculty Publications

No abstract provided.

Phylogeography Of The Frog Leptodactylus Validus (Amphibia: Anura): Patterns And Timing Of Colonization Events In The Lesser Antilles, Arley Camargo, W. Ronald Heyer, Rafael O. De Sá

Biology Faculty Publications

The frog Leptodactylus validus occurs in northern South America, Trinidad and Tobago, and the southern Lesser Antilles (Grenada and St. Vincent). Mitochondrial DNA sequences were used to perform a nested clade phylogeographic analysis (NCPA), to date colonization events, and to analyze colonization patterns using on a relaxed molecular clock and coalescent simulations. L. validus originated on the mainland and first colonized Trinidad with subsequent independent colonizations of Tobago and the Lesser Antilles from Trinidad. The NCPA suggests a historical vicariant event between populations in Trinidad and Tobago from those in the Lesser Antilles. The colonization of Trinidad occurred 1 million …

In Vitro Guidance Of Retinal Axons By A Tectal Lamina-Specific Glycoprotein Nel, Yulan Jiang, Hiroya Obama, Soh-Leh Kuan, Ritsuko Nakamura, Chizu Nakamoto, Zhufeng Ouyang, Masaru Nakamoto

Biology Faculty Publications

Nel is a glycoprotein containing five chordin-like and six epidermal growth factor-like domains and is strongly expressed in the nervous system. In this study, we have examined expression patterns and in vitro functions of Nel in the chicken retinotectal system. We have found that in the developing tectum, expression of Nel is localized in specific laminae that retinal axons normally do not enter, including the border between the retinorecipient and non-retinorecipient laminae. Nel-binding activity is detected on retinal axons both in vivo and in vitro, suggesting that retinal axons express a receptor for Nel. In vitro, Nel inhibits retinal axon …

Is The Amphibian Tree Of Life Really Fatally Flawed?, Darrel R. Frost, Taran Grant, Julian Faivovich, Raoul H. Bain, Alexander Haas, Celio F.B. Haddad, Rafael O. De Sá, Et Al.

Biology Faculty Publications

Wiens (2007, Q. Rev. Biol. 82, 55–56) recently published a severe critique of Frost et al.'s (2006, Bull. Am. Mus. Nat. Hist. 297, 1–370) monographic study of amphibian systematics, concluding that it is “a disaster” and recommending that readers “simply ignore this study”. Beyond the hyperbole, Wiens raised four general objections that he regarded as “fatal flaws”: (1) the sampling design was insufficient for the generic changes made and taxonomic changes were made without including all type species; (2) the nuclear gene most commonly used in amphibian phylogenetics, RAG-1, was not included, nor were the morphological characters that had justified …

Phylogenetic Analyses Of Mtdna Sequences Reveal Three Cryptic Lineages In The Widespread Neotropical Frog Leptodactylus Fuscus (Schneider, 1799) (Anura, Leptodactylidae), Arley Camargo, Rafael O. De Sá, W. Ronald Heyer

Biology Faculty Publications

Leptodactylus fuscus is a neotropical frog ranging from Panamá to Argentina, to the east of the Andes mountains, and also inhabiting Margarita, Trinidad, and the Tobago islands. We performed phylogenetic analyses of 12S rRNA, 16S rRNA, tRNA-Leu, and ND1 mitochondrial (mt) DNA sequences from specimens collected across the geographic distribution of L. fuscus to examine two alternative hypotheses: (i) L. fuscus is a single, widely distributed species, or (ii) L. fuscus is a species complex. We tested statistically for geographic association and partitioning of genetic variation among mtDNA clades. The mtDNA data supported the hypothesis of several cryptic species within …

Neuronal Pentraxins Mediate Synaptic Refinement In The Developing Visual System, Lisa Bjartmar, Andrew Huberman, Erik Ullian, Rene Renterıa, Xiaoqin Liu, Weifeng Xu, Jennifer Prezioso, Michael Susman, David Stellwagen, Caleb Stokes, Richard Cho, Paul Worley, Robert Malenka, Sherry Ball, Neal Peachey, David Copenhagen, Barbara Chapman, Masaru Nakamoto, Ben Barres, Mark Perin

Biology Faculty Publications

Neuronal pentraxins (NPs) define a family of proteins that are homologous to C-reactive and acute-phase proteins in the immune system and have been hypothesized to be involved in activity-dependent synaptic plasticity. To investigate the role of NPs in vivo , we generated mice that lack one, two, or all three NPs. NP1/2 knock-out mice exhibited defects in the segregation of eye-specific retinal ganglion cell (RGC) projections to the dorsal lateral geniculate nucleus, a process that involves activity-dependent synapse formation and elimination. Retinas from mice lacking NP1 and NP2 had cholinergically driven waves of activity that occurred at a frequency similar …

Loss-Of-Function Analysis Of Epha Receptors In Retinotectal Mapping, David Feldheim, Masaru Nakamoto, Miriam Osterfield, Nicholas Gale, Thomas Dechiara, Rajat Rohatgi, George Yancopoulos, John Flanagan

Biology Faculty Publications

EphA tyrosine kinases are thought to act as topographically specific receptors in the well-characterized projection map from the retina to the tectum. Here, we describe a loss-of-function analysis of EphA receptors in retinotectal mapping. Expressing patches of a cytoplasmically truncated EphA3 receptor in chick retina caused temporal axons to have reduced responsiveness to posterior tectal repellent activity in vitro and to shift more posteriorly within the map in vivo . A gene disruption of mouse EphA5, replacing the intracellular domain with beta-galactosidase, reduced in vitro responsiveness of temporal axons to posterior target membranes. It also caused map abnormalities in vivo …

Skeletal Myosin Heavy Chain Function In Cultured Lung Myofibroblasts, Nancy A. Rice, Leslie A. Leinwand

Biology Faculty Publications

Myofibroblasts are unique contractile cells with both muscle and nonmuscle properties. Typically myofibroblasts are identified by the expression of smooth muscle actin (ASMA); however some myofibroblasts also express sarcomeric proteins. In this study, we show that pulmonary myofibroblasts express three of the eight known sarcomeric myosin heavy chains (MyHCs) (IIa, IId, and embryonic) and that skeletal muscle myosin enzymatic activity is required for pulmonary myofibroblast contractility. Furthermore, inhibition of skeletal myosin activity and myofibroblast contraction results in a decrease in both ASMA and skeletal MyHC promoter activity and ASMA protein expression, suggesting a potential coupling of skeletal myosin activity and …

Regulation Of Zebrafish Hindbrain Development By Fibroblast Growth Factor And Retinoic Acid: A Dissertation, Nicole M. Roy

Biology Faculty Publications

A thesis submitted to the Department of Biochemistry and Molecular Pharmacology and the Graduate School of Biomedical Sciences of the University of Massachusetts, Worcester in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Biochemistry and Molecular Pharmacology.

Fibroblast growth factor (Fgf) and Retinoic acid (RA) are known to be involved in patterning the posterior embryo. Work has shown that Fgf can convert anterior tissue into posterior fates and that embryos deficient in Fgf signaling lack posterior trunk and tail structures. Likewise, studies performed on RA have shown that overexpression of RA posteriorizes anterior tissue, while disrupting …