Cellular Stiffness as a Novel Stemness Marker in the Corneal Limbus.

Healthy eyes contain a population of limbal stem cells (LSCs) that continuously renew the corneal epithelium. However, each year, 1 million Americans are afflicted with severely reduced visual acuity caused by corneal damage or disease, including LSC deficiency (LSCD). Recent advances in corneal transplant technology promise to repair the cornea by implanting healthy LSCs to encourage regeneration; however, success is limited to transplanted tissues that contain a sufficiently high percentage of LSCs. Attempts to screen limbal tissues for suitable implants using molecular stemness markers are confounded by the poorly understood signature of the LSC phenotype. For cells derived from the corneal limbus, we show that the performance of cell stiffness as a stemness indicator is on par with the performance of ΔNP63α, a common molecular marker. In combination with recent methods for sorting cells on a biophysical basis, the biomechanical stemness markers presented here may enable the rapid purification of LSCs from a heterogeneous population of corneal cells, thus potentially enabling clinicians and researchers to generate corneal transplants with sufficiently high fractions of LSCs, regardless of the LSC percentage in the donor tissue.

Temporal and spatial requirements for Hoxa3 in mouse embryonic development.

Hoxa3(null) mice have severe defects in the development of pharyngeal organs including athymia, aparathyroidism, thyroid hypoplasia, and ultimobranchial body persistence, in addition to defects of the throat cartilages and cranial nerves. Some of the structures altered in the Hoxa3(null) mutant embryos are anterior to the described Hoxa3 gene expression boundary: the thyroid, soft palate, and lesser hyoid horn. All of these structures develop over time and through the interactions of multiple cell types. To investigate the specific cellular targets for HOXA3 function in these structures across developmental time, we performed a comprehensive analysis of the temporal and tissue-specific requirements for Hoxa3, including a lineage analysis using Hoxa3(Cre). The combination of these approaches showed that HOXA3 functions in both a cell autonomous and non-cell autonomous manner during development of the 3rd and 4th arch derivatives, and functions in a neural crest cell (NCC)-specific, non-cell autonomous manner for structures that were Hoxa3-negative by lineage tracing. Our data indicate that HOXA3 is required for tissue organization and organ differentiation in endodermal cells (in the tracheal epithelium, thymus, and parathyroid), and contributes to organ migration and morphogenesis in NCCs. These data provide a detailed picture of where and when HOXA3 acts to promote the development of the diverse structures that are altered in the Hoxa3(null) mutant. Data presented here, combined with our previous studies, indicate that the regionally restricted defects in Hoxa3 mutants do not reflect a role in positional identity (establishment of cell or tissue fate), but instead indicate a wider variety of functions including controlling distinct genetic programs for differentiation and morphogenesis in different cell types during development.

Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.

Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans.

Multiple roles for HOXA3 in regulating thymus and parathyroid differentiation and morphogenesis in mouse.

Hoxa3 was the first Hox gene to be mutated by gene targeting in mice and is required for the development of multiple endoderm and neural crest cell (NCC)-derived structures in the pharyngeal region. Previous studies have shown that the Hoxa3 null mutant lacks third pharyngeal pouch derivatives, the thymus and parathyroids by E18.5, and organ-specific markers are absent or downregulated during initial organogenesis. Our current analysis of the Hoxa3 null mutant shows that organ-specific domains did undergo initial patterning, but the location and timing of key regional markers within the pouch, including Tbx1, Bmp4 and Fgf8, were altered. Expression of the parathyroid marker Gcm2 was initiated but was quickly downregulated and differentiation failed; by contrast, thymus markers were delayed but achieved normal levels, concurrent with complete loss through apoptosis. To determine the cell type-specific roles of Hoxa3 in third pharyngeal pouch development, we analyzed tissue-specific mutants using endoderm and/or NCC-specific Cre drivers. Simultaneous deletion with both drivers resulted in athymia at E18.5, similar to the null. By contrast, the individual tissue-specific Hoxa3 deletions resulted in small, ectopic thymi, although each had a unique phenotype. Hoxa3 was primarily required in NCCs for morphogenesis. In endoderm, Hoxa3 temporally regulated initiation of the thymus program and was required in a cell-autonomous manner for parathyroid differentiation. Furthermore, Hoxa3 was required for survival of third pharyngeal pouch-derived organs, but expression in either tissue was sufficient for this function. These data show that Hoxa3 has multiple complex and tissue-specific functions during patterning, differentiation and morphogenesis of the thymus and parathyroids.

Parsimony and model-based analyses of indels in avian nuclear genes reveal congruent and incongruent phylogenetic signals.

Insertion/deletion (indel) mutations, which are represented by gaps in multiple sequence alignments, have been used to examine phylogenetic hypotheses for some time. However, most analyses combine gap data with the nucleotide sequences in which they are embedded, probably because most phylogenetic datasets include few gap characters. Here, we report analyses of 12,030 gap characters from an alignment of avian nuclear genes using maximum parsimony (MP) and a simple maximum likelihood (ML) framework. Both trees were similar, and they exhibited almost all of the strongly supported relationships in the nucleotide tree, although neither gap tree supported many relationships that have proven difficult to recover in previous studies. Moreover, independent lines of evidence typically corroborated the nucleotide topology instead of the gap topology when they disagreed, although the number of conflicting nodes with high bootstrap support was limited. Filtering to remove short indels did not substantially reduce homoplasy or reduce conflict. Combined analyses of nucleotides and gaps resulted in the nucleotide topology, but with increased support, suggesting that gap data may prove most useful when analyzed in combination with nucleotide substitutions.

An unbiased approach to identify genes involved in development in a turtle with temperature-dependent sex determination.

BACKGROUND: Many reptiles exhibit temperature-dependent sex determination (TSD). The initial cue in TSD is incubation temperature, unlike genotypic sex determination (GSD) where it is determined by the presence of specific alleles (or genetic loci). We used patterns of gene expression to identify candidates for genes with a role in TSD and other developmental processes without making a priori assumptions about the identity of these genes (ortholog-based approach). We identified genes with sexually dimorphic mRNA accumulation during the temperature sensitive period of development in the Red-eared slider turtle (Trachemys scripta), a turtle with TSD. Genes with differential mRNA accumulation in response to estrogen (estradiol-17ß; E(2)) exposure and developmental stages were also identified. RESULTS: Sequencing 767 clones from three suppression-subtractive hybridization libraries yielded a total of 581 unique sequences. Screening a macroarray with a subset of those sequences revealed a total of 26 genes that exhibited differential mRNA accumulation: 16 female biased and 10 male biased. Additional analyses revealed that C16ORF62 (an unknown gene) and MALAT1 (a long noncoding RNA) exhibited increased mRNA accumulation at the male producing temperature relative to the female producing temperature during embryonic sexual development. Finally, we identified four genes (C16ORF62, CCT3, MMP2, and NFIB) that exhibited a stage effect and five genes (C16ORF62, CCT3, MMP2, NFIB and NOTCH2) showed a response to E(2) exposure. CONCLUSIONS: Here we report a survey of genes identified using patterns of mRNA accumulation during embryonic development in a turtle with TSD. Many previous studies have focused on examining the turtle orthologs of genes involved in mammalian development. Although valuable, the limitations of this approach are exemplified by our identification of two genes (MALAT1 and C16ORF62) that are sexually dimorphic during embryonic development. MALAT1 is a noncoding RNA that has not been implicated in sexual differentiation in other vertebrates and C16ORF62 has an unknown function. Our results revealed genes that are candidates for having roles in turtle embryonic development, including TSD, and highlight the need to expand our search parameters beyond protein-coding genes.

Homoplastic microinversions and the avian tree of life.

BACKGROUND: Microinversions are cytologically undetectable inversions of DNA sequences that accumulate slowly in genomes. Like many other rare genomic changes (RGCs), microinversions are thought to be virtually homoplasy-free evolutionary characters, suggesting that they may be very useful for difficult phylogenetic problems such as the avian tree of life. However, few detailed surveys of these genomic rearrangements have been conducted, making it difficult to assess this hypothesis or understand the impact of microinversions upon genome evolution. RESULTS: We surveyed non-coding sequence data from a recent avian phylogenetic study and found substantially more microinversions than expected based upon prior information about vertebrate inversion rates, although this is likely due to underestimation of these rates in previous studies. Most microinversions were lineage-specific or united well-accepted groups. However, some homoplastic microinversions were evident among the informative characters. Hemiplasy, which reflects differences between gene trees and the species tree, did not explain the observed homoplasy. Two specific loci were microinversion hotspots, with high numbers of inversions that included both the homoplastic as well as some overlapping microinversions. Neither stem-loop structures nor detectable sequence motifs were associated with microinversions in the hotspots. CONCLUSIONS: Microinversions can provide valuable phylogenetic information, although power analysis indicates that large amounts of sequence data will be necessary to identify enough inversions (and similar RGCs) to resolve short branches in the tree of life. Moreover, microinversions are not perfect characters and should be interpreted with caution, just as with any other character type. Independent of their use for phylogenetic analyses, microinversions are important because they have the potential to complicate alignment of non-coding sequences. Despite their low rate of accumulation, they have clearly contributed to genome evolution, suggesting that active identification of microinversions will prove useful in future phylogenomic studies.

Phylogenomic evidence for multiple losses of flight in ratite birds.

Ratites (ostriches, emus, rheas, cassowaries, and kiwis) are large, flightless birds that have long fascinated biologists. Their current distribution on isolated southern land masses is believed to reflect the breakup of the paleocontinent of Gondwana. The prevailing view is that ratites are monophyletic, with the flighted tinamous as their sister group, suggesting a single loss of flight in the common ancestry of ratites. However, phylogenetic analyses of 20 unlinked nuclear genes reveal a genome-wide signal that unequivocally places tinamous within ratites, making ratites polyphyletic and suggesting multiple losses of flight. Phenomena that can mislead phylogenetic analyses, including long branch attraction, base compositional bias, discordance between gene trees and species trees, and sequence alignment errors, have been eliminated as explanations for this result. The most plausible hypothesis requires at least three losses of flight and explains the many morphological and behavioral similarities among ratites by parallel or convergent evolution. Finally, this phylogeny demands fundamental reconsideration of proposals that relate ratite evolution to continental drift.

A phylogenomic study of birds reveals their evolutionary history.

Deep avian evolutionary relationships have been difficult to resolve as a result of a putative explosive radiation. Our study examined approximately 32 kilobases of aligned nuclear DNA sequences from 19 independent loci for 169 species, representing all major extant groups, and recovered a robust phylogeny from a genome-wide signal supported by multiple analytical methods. We documented well-supported, previously unrecognized interordinal relationships (such as a sister relationship between passerines and parrots) and corroborated previously contentious groupings (such as flamingos and grebes). Our conclusions challenge current classifications and alter our understanding of trait evolution; for example, some diurnal birds evolved from nocturnal ancestors. Our results provide a valuable resource for phylogenetic and comparative studies in birds.

Introns outperform exons in analyses of basal avian phylogeny using clathrin heavy chain genes.

Neoaves is the most diverse major avian clade, containing ~95% of avian species, and it underwent an ancient but rapid diversification that has made resolution of relationships at the base of the clade difficult. In fact, Neoaves has been suggested to be a "hard" polytomy that cannot be resolved with any amount of data. However, this conclusion was based on slowly evolving coding sequences and ribosomal RNAs and some recent studies using more rapidly evolving intron sequences have suggested some resolution at the base of Neoaves. To further examine the utility of introns and exons for phylogenetics, we sequenced parts of two unlinked clathrin heavy chain genes (CLTC and CLTCL1). Comparisons of phylogenetic trees based upon individual partitions (i.e. introns and exons), the combined dataset, and published phylogenies using Robinson-Foulds distances (a metric of topological differences) revealed more similarity than expected by chance, suggesting there is structure at the base of Neoaves. We found that introns provided more informative sites, were subject to less homoplasy, and provided better support for well-accepted clades, suggesting that intron evolution is better suited to determining closely-spaced branching events like the base of Neoaves. Furthermore, phylogenetic power analyses indicated that existing molecular datasets for birds are unlikely to provide sufficient phylogenetic information to resolve relationships at the base of Neoaves, especially when comprised of exon or other slowly evolving regions. Although relationships among the orders in Neoaves cannot be definitively established using available data, the base of Neoaves does not appear to represent a hard polytomy. Our analyses suggest that large intron datasets have the best potential to resolve relationships among avian orders and indicate that the utility of intron data for other phylogenetic questions should be examined.

Turtle isochore structure is intermediate between amphibians and other amniotes.

Vertebrate genomes are comprised of isochores that are relatively long (>100 kb) regions with a relatively homogenous (either GC-rich or AT-rich) base composition and with rather sharp boundaries with neighboring isochores. Mammals and living archosaurs (birds and crocodilians) have heterogeneous genomes that include very GC-rich isochores. In sharp contrast, the genomes of amphibians and fishes are more homogeneous and they have a lower overall GC content. Because DNA with higher GC content is more thermostable, the elevated GC content of mammalian and archosaurian DNA has been hypothesized to be an adaptation to higher body temperatures. This hypothesis can be tested by examining structure of isochores across the reptilian clade, which includes the archosaurs, testudines (turtles), and lepidosaurs (lizards and snakes), because reptiles exhibit diverse body sizes, metabolic rates, and patterns of thermoregulation. This study focuses on a comparative analysis of a new set of expressed genes of the red-eared slider turtle and orthologs of the turtle genes in mammalian (human, mouse, dog, and opossum), archosaurian (chicken and alligator), and amphibian (western clawed frog) genomes. EST (expressed sequence tag) data from a turtle cDNA library enriched for genes that have specialized functions (developmental genes) revealed using the GC content of the third-codon-position to examine isochore structure requires careful consideration of the types of genes examined. The more highly expressed genes (e.g., housekeeping genes) are more likely to be GC-rich than are genes with specialized functions. However, the set of highly expressed turtle genes demonstrated that the turtle genome has a GC content that is intermediate between the GC-poor amphibians and the GC-rich mammals and archosaurs. There was a strong correlation between the GC content of all turtle genes and the GC content of other vertebrate genes, with the slope of the line describing this relationship also indicating that the isochore structure of turtles is intermediate between that of amphibians and other amniotes. These data are consistent with some thermal hypotheses of isochore evolution, but we believe that the credible set of models for isochore evolution still includes a variety of models. These data expand the amount of genomic data available from reptiles upon which future studies of reptilian genomics can build.

Patterns of vertebrate isochore evolution revealed by comparison of expressed mammalian, avian, and crocodilian genes.

Vertebrate genomes are mosaics of isochores, defined as long (>100 kb) regions with relatively homogeneous within-region base composition. Birds and mammals have more GC-rich isochores than amphibians and fish, and the GC-rich isochores of birds and mammals have been suggested to be an adaptation to homeothermy. If this hypothesis is correct, all poikilothermic (cold-blooded) vertebrates, including the nonavian reptiles, are expected to lack a GC-rich isochore structure. Previous studies using various methods to examine isochore structure in crocodilians, turtles, and squamates have led to different conclusions. We collected more than 6000 expressed sequence tags (ESTs) from the American alligator to overcome sample size limitations suggested to be the fundamental problem in the previous reptilian studies. The alligator ESTs were assembled and aligned with their human, mouse, chicken, and western clawed frog orthologs, resulting in 366 alignments. Analyses of third-codon-position GC content provided conclusive evidence that the poikilothermic alligator has GC-rich isochores, like homeothermic birds and mammals. We placed these results in a theoretical framework able to unify available models of isochore evolution. The data collected for this study allowed us to reject the models that explain the evolution of GC content using changes in body temperature associated with the transition from poikilothermy to homeothermy. Falsification of these models places fundamental constraints upon the plausible pathways for the evolution of isochores.