Cryptic Variation in Morphological Evolution: HSP90 as a Capacitor for Loss of Eyes in Cavefish

In the process of morphological evolution, the extent to which cryptic, preexisting variation provides a substrate for natural selection has been controversial. We provide evidence that heat shock protein 90 (HSP90) phenotypically masks standing eye-size variation in surface populations of the cavefish Astyanax mexicanus. This variation is exposed by HSP90 inhibition and can be selected for, ultimately yielding a reduced-eye phenotype even in the presence of full HSP90 activity. Raising surface fish under conditions found in caves taxes the HSP90 system, unmasking the same phenotypic variation as does direct inhibition of HSP90. These results suggest that cryptic variation played a role in the evolution of eye loss in cavefish and provide the first evidence for HSP90 as a capacitor for morphological evolution in a natural setting

Distinct Compartments of the Proepicardial Organ Give Rise to Coronary Vascular Endothelial Cells

SummaryThe proepicardial organ is an important transient structure that contributes cells to various cardiac lineages. However, its contribution to the coronary endothelium has been disputed, with conflicting data arising in chick and mouse. Here we resolve this conflict by identifying two proepicardial markers, Scleraxis (Scx) and Semaphorin3D (Sema3D), that genetically delineate heretofore uncharacterized proepicardial subcompartments. In contrast to previously fate-mapped Tbx18/WT-1-expressing cells that give rise to vascular smooth muscle, Scx- and Sema3D-expressing proepicardial cells give rise to coronary vascular endothelium both in vivo and in vitro. Furthermore, Sema3D+ and Scx+ proepicardial cells contribute to the early sinus venosus and cardiac endocardium, respectively, two tissues linked to vascular endothelial formation at later stages. Taken together, our studies demonstrate that the proepicardial organ is a molecularly compartmentalized structure, reconciling prior chick and mouse data and providing a more complete understanding of the progenitor populations that establish the coronary vascular endothelium

Expression of Distal-less, dachshund, and optomotor blind in Neanthes arenaceodentata (Annelida, Nereididae) does not support homology of appendage-forming mechanisms across the Bilateria

The similarity in the genetic regulation of arthropod and vertebrate appendage formation has been interpreted as the product of a plesiomorphic gene network that was primitively involved in bilaterian appendage development and co-opted to build appendages (in modern phyla) that are not historically related as structures. Data from lophotrochozoans are needed to clarify the pervasiveness of plesiomorphic appendage forming mechanisms. We assayed the expression of three arthropod and vertebrate limb gene orthologs, Distal-less (Dll), dachshund (dac), and optomotor blind (omb), in direct-developing juveniles of the polychaete Neanthes arenaceodentata. Parapodial Dll expression marks premorphogenetic notopodia and neuropodia, becoming restricted to the bases of notopodial cirri and to ventral portions of neuropodia. In outgrowing cephalic appendages, Dll activity is primarily restricted to proximal domains. Dll expression is also prominent in the brain. dac expression occurs in the brain, nerve cord ganglia, a pair of pharyngeal ganglia, presumed interneurons linking a pair of segmental nerves, and in newly differentiating mesoderm. Domains of omb expression include the brain, nerve cord ganglia, one pair of anterior cirri, presumed precursors of dorsal musculature, and the same pharyngeal ganglia and presumed interneurons that express dac. Contrary to their roles in outgrowing arthropod and vertebrate appendages, Dll, dac, and omb lack comparable expression in Neanthes appendages, implying independent evolution of annelid appendage development. We infer that parapodia and arthropodia are not structurally or mechanistically homologous (but their primordia might be), that Dll’s ancestral bilaterian function was in sensory and central nervous system differentiation, and that locomotory appendages possibly evolved from sensory outgrowths

A Computational Clonal Analysis of the Developing Mouse Limb Bud

A comprehensive spatio-temporal description of the tissue movements underlying organogenesis would be an extremely useful resource to developmental biology. Clonal analysis and fate mappings are popular experiments to study tissue movement during morphogenesis. Such experiments allow cell populations to be labeled at an early stage of development and to follow their spatial evolution over time. However, disentangling the cumulative effects of the multiple events responsible for the expansion of the labeled cell population is not always straightforward. To overcome this problem, we develop a novel computational method that combines accurate quantification of 2D limb bud morphologies and growth modeling to analyze mouse clonal data of early limb development. Firstly, we explore various tissue movements that match experimental limb bud shape changes. Secondly, by comparing computational clones with newly generated mouse clonal data we are able to choose and characterize the tissue movement map that better matches experimental data. Our computational analysis produces for the first time a two dimensional model of limb growth based on experimental data that can be used to better characterize limb tissue movement in space and time. The model shows that the distribution and shapes of clones can be described as a combination of anisotropic growth with isotropic cell mixing, without the need for lineage compartmentalization along the AP and PD axis. Lastly, we show that this comprehensive description can be used to reassess spatio-temporal gene regulations taking tissue movement into account and to investigate PD patterning hypothesis

Sequential and Coordinated Actions of c-Myc and N-Myc Control Appendicular Skeletal Development

BACKGROUND: During limb development, chondrocytes and osteoblasts emerge from condensations of limb bud mesenchyme. These cells then proliferate and differentiate in separate but adjacent compartments and function cooperatively to promote bone growth through the process of endochondral ossification. While many aspects of limb skeletal formation are understood, little is known about the mechanisms that link the development of undifferentiated limb bud mesenchyme with formation of the precartilaginous condensation and subsequent proliferative expansion of chondrocyte and osteoblast lineages. The aim of this study was to gain insight into these processes by examining the roles of c-Myc and N-Myc in morphogenesis of the limb skeleton. METHODOLOGY/PRINCIPAL FINDINGS: To investigate c-Myc function in skeletal development, we characterized mice in which floxed c-Myc alleles were deleted in undifferentiated limb bud mesenchyme with Prx1-Cre, in chondro-osteoprogenitors with Sox9-Cre and in osteoblasts with Osx1-Cre. We show that c-Myc promotes the proliferative expansion of both chondrocytes and osteoblasts and as a consequence controls the process of endochondral growth and ossification and determines bone size. The control of proliferation by c-Myc was related to its effects on global gene transcription, as phosphorylation of the C-Terminal Domain (pCTD) of RNA Polymerase II, a marker of general transcription initiation, was tightly coupled to cell proliferation of growth plate chondrocytes where c-Myc is expressed and severely downregulated in the absence of c-Myc. Finally, we show that combined deletion of N-Myc and c-Myc in early limb bud mesenchyme gives rise to a severely hypoplastic limb skeleton that exhibits features characteristic of individual c-Myc and N-Myc mutants. CONCLUSIONS/SIGNIFICANCE: Our results show that N-Myc and c-Myc act sequentially during limb development to coordinate the expansion of key progenitor populations responsible for forming the limb skeleton

No Pathogenic Mutations Identified in the COL8A2 Gene or Four Positional Candidate Genes in Patients with Posterior Polymorphous Corneal Dystrophy

PURPOSE. To identify the genetic basis of posterior polymorphous corneal dystrophy (PPCD) through screening of four positional candidate genes and the COL8A2 gene, in which a presumed pathogenic mutation has previously been identified in affected patients. METHODS. DNA extraction, PCR amplification, and direct sequencing of the COL8A2, BFSP1, CST3, MMP9, and SLPI genes were performed in 14 unrelated, affected patients and in unaffected family members. RESULTS. In the COL8A2 gene, the previously identified, presumed pathogenic mutation (Gln455Lys) was not discovered in any of the affected patients. A missense mutation, Thr502Met, was identified in 2 of the 14 affected probands, although it was not considered to be pathogenic, as it has been identified in unaffected individuals. Although several novel and previously identified single nucleotide polymorphisms producing synonymous and missense amino acid substitutions were identified in the COL8A2, BFSP1, CST3, MMP9, and SLPI genes, no presumed pathogenic sequence variants were found. CONCLUSIONS. No pathogenic mutations were identified in the COL8A2 gene or in several positional candidate genes in a series of patients with PPCD, indicating that other genetic factors are involved in the development of this autosomal dominant corneal dystrophy. (Invest Ophthalmol Vis Sci

Left-Right Function of dmrt2 Genes Is Not Conserved between Zebrafish and Mouse

Background: Members of the Dmrt family, generally associated with sex determination, were shown to be involved in several other functions during embryonic development. Dmrt2 has been studied in the context of zebrafish development where, due to a duplication event, two paralog genes dmrt2a and dmrt2b are present. Both zebrafish dmrt2a/terra and dmrt2b are important to regulate left-right patterning in the lateral plate mesoderm. In addition, dmrt2a/terra is necessary for symmetric somite formation while dmrt2b regulates somite differentiation impacting on slow muscle development. One dmrt2 gene is also expressed in the mouse embryo, where it is necessary for somite differentiation but with an impact on axial skeleton development. However, nothing was known about its role during left-right patterning in the lateral plate mesoderm or in the symmetric synchronization of somite formation. Methodology/Principal Findings: Using a dmrt2 mutant mouse line, we show that this gene is not involved in symmetric somite formation and does not regulate the laterality pathway that controls left-right asymmetric organ positioning. We reveal that dmrt2a/terra is present in the zebrafish laterality organ, the Kupffer’s vesicle, while its homologue is excluded from the mouse equivalent structure, the node. On the basis of evolutionary sub-functionalization and neo-functionalization theories we discuss this absence of functional conservation. Conclusions/Significance: Our results show that the role of dmrt2 gene is not conserved during zebrafish and mous

Non-Redundant Selector and Growth-Promoting Functions of Two Sister Genes, buttonhead and Sp1, in Drosophila Leg Development

The radically distinct morphologies of arthropod and tetrapod legs argue that these appendages do not share a common evolutionary origin. Yet, despite dramatic differences in morphology, it has been known for some time that transcription factors encoded by the Distalless (Dll)/Dlx gene family play a critical role in the development of both structures. Here we show that a second transcription factor family encoded by the Sp8 gene family, previously implicated in vertebrate limb development, also plays an early and fundamental role in arthropod leg development. By simultaneously removing the function of two Sp8 orthologs, buttonhead (btd) and Sp1, during Drosophila embryogenesis, we find that adult leg development is completely abolished. Remarkably, in the absence of these factors, transformations from ventral to dorsal appendage identities are observed, suggesting that adult dorsal fates become derepressed when ventral fates are eliminated. Further, we show that Sp1 plays a much more important role in ventral appendage specification than btd and that Sp1 lies genetically upstream of Dll. In addition to these selector-like gene functions, Sp1 and btd are also required during larval stages for the growth of the leg. Vertebrate Sp8 can rescue many of the functions of the Drosophila genes, arguing that these activities have been conserved, despite more than 500 million years of independent evolution. These observations suggest that an ancient Sp8/Dlx gene cassette was used in an early metazoan for primitive limb-like outgrowths and that this cassette was co-opted multiple times for appendage formation in multiple animal phyla

Role of the Gut Endoderm in Relaying Left-Right Patterning in Mice

Analysis of Sox17 mutant mice reveals that gap junction coupling across the gut endoderm of the embryo transmits the left-right asymmetric signal from the node to the site of asymmetric organogenesis in mice

Tbx6 Regulates Left/Right Patterning in Mouse Embryos through Effects on Nodal Cilia and Perinodal Signaling

Background: The determination of left/right body axis during early embryogenesis sets up a developmental cascade that coordinates the development of the viscera and is essential to the correct placement and alignment of organ systems and vasculature. Defective left-right patterning can lead to congenital cardiac malformations, vascular anomalies and other serious health problems. Here we describe a novel role for the T-box transcription factor gene Tbx6 in left/right body axis determination in the mouse. Results: Embryos lacking Tbx6 show randomized embryo turning and heart looping. Our results point to multiple mechanisms for this effect. First, Dll1, a direct target of Tbx6, is down regulated around the node in Tbx6 mutants and there is a subsequent decrease in nodal signaling, which is required for laterality determination. Secondly, in spite of a lack of expression of Tbx6 in the node, we document a profound effect of the Tbx6 mutation on the morphology and motility of nodal cilia. This results in the loss of asymmetric calcium signaling at the periphery of the node, suggesting that unidirectional nodal flow is disrupted. To carry out these studies, we devised a novel method for direct labeling and live imaging cilia in vivo using a genetically-encoded fluorescent protein fusion that labels tubulin, combined with laser point scanning confocal microscopy for direct visualization of cilia movement. Conclusions: We conclude that the transcription factor gene Tbx6 is essential for correct left/right axis determination in th