Dynamic changes in ocular shape during human development and its implications for retina fovea formation.

The human fovea is known for its distinctive pit-like appearance, which results from the displacement of retinal layers superficial to the photoreceptors cells. The photoreceptors are found at high density within the foveal region but not the surrounding retina. Efforts to elucidate the mechanisms responsible for these unique features have ruled out cell death as an explanation for pit formation and changes in cell proliferation as the cause of increased photoreceptor density. These findings have led to speculation that mechanical forces acting within and on the retina during development underly the formation of foveal architecture. Here we review eye morphogenesis and retinal remodeling in human embryonic development. Our meta-analysis of the literature suggests that fovea formation is a protracted process involving dynamic changes in ocular shape that start early and continue throughout most of human embryonic development. From these observations, we propose a new model for fovea development.

Changes in selection pressure can facilitate hybridization during biological invasion in a Cuban lizard.

Hybridization is among the evolutionary mechanisms most frequently hypothesized to drive the success of invasive species, in part because hybrids are common in invasive populations. One explanation for this pattern is that biological invasions coincide with a change in selection pressures that limit hybridization in the native range. To investigate this possibility, we studied the introduction of the brown anole (Anolis sagrei) in the southeastern United States. We find that native populations are highly genetically structured. In contrast, all invasive populations show evidence of hybridization among native-range lineages. Temporal sampling in the invasive range spanning 15 y showed that invasive genetic structure has stabilized, indicating that large-scale contemporary gene flow is limited among invasive populations and that hybrid ancestry is maintained. Additionally, our results are consistent with hybrid persistence in invasive populations resulting from changes in natural selection that occurred during invasion. Specifically, we identify a large-effect X chromosome locus associated with variation in limb length, a well-known adaptive trait in anoles, and show that this locus is often under selection in the native range, but rarely so in the invasive range. Moreover, we find that the effect size of alleles at this locus on limb length is much reduced in hybrids among divergent lineages, consistent with epistatic interactions. Thus, in the native range, epistasis manifested in hybrids can strengthen extrinsic postmating isolation. Together, our findings show how a change in natural selection can contribute to an increase in hybridization in invasive populations.

Maturation and refinement of the maculae and foveae in the Anolis sagrei lizard.

The fovea is a pit in the center of the macula, which is a region of the retina with a high concentration of photoreceptor cells, which accounts for a large degree of visual acuity in primates. The maturation of this primate visual acuity area is characterized by the shallowing and widening of the foveal pit, a decrease in the diameter of the rod-free zone, and an increase in photoreceptor cells packing after birth. Maturation occurs concurrently with progressing age, increasing eye size, and retinal length/area. These observations have led to the hypothesis that the maturation of the fovea might be a function of mechanical variables that remodel the retina. However, this has never been explored outside of primates. Here, we take advantage of the Anolis sagrei lizard, which has a bifoveated retina, to study maturation of the fovea and macula. Eyes were collected from male and female lizards-hatchling, 2-month, 4-month, 6-month, and adult. We found that Anolis maculae undergo a maturation process somewhat different than what has been observed in primates. Anole macular diameters actually increase in size and undergo minimal photoreceptor cell packing, possessing a near complete complement of these cells at the time of hatching. As the anole eye expands, foveal centers experience little change in overall retina cell density with most cell redistribution occurring at macular borders and peripheral retina areas. Gene editing technology has recently been developed in lizards; this study provides a baseline of normal retina maturation for future genetic manipulation studies in anoles.

Ocular elongation and retraction in foveated reptiles.

BACKGROUND: Pronounced asymmetric changes in ocular globe size during eye development have been observed in a number of species ranging from humans to lizards. In contrast, largely symmetric changes in globe size have been described for other species like rodents. We propose that asymmetric changes in the three-dimensional structure of the developing eye correlate with the types of retinal remodeling needed to produce areas of high photoreceptor density. To test this idea, we systematically examined three-dimensional aspects of globe size as a function of eye development in the bifoveated brown anole, Anolis sagrei. RESULTS: During embryonic development, the anole eye undergoes dynamic changes in ocular shape. Initially spherical, the eye elongates in the presumptive foveal regions of the retina and then proceeds through a period of retraction that returns the eye to its spherical shape. During this period of retraction, pit formation and photoreceptor cell packing are observed. We found a similar pattern of elongation and retraction associated with the single fovea of the veiled chameleon, Chamaeleo calyptratus. CONCLUSIONS: These results, together with those reported for other foveated species, support the idea that areas of high photoreceptor packing occur in regions where the ocular globe asymmetrically elongates and retracts during development.

Pigeon foot feathering reveals conserved limb identity networks.

The tetrapod limb is a stunning example of evolutionary diversity, with dramatic variation not only among distantly related species, but also between the serially homologous forelimbs (FLs) and hindlimbs (HLs) within species. Despite this variation, highly conserved genetic and developmental programs underlie limb development and identity in all tetrapods, raising the question of how limb diversification is generated from a conserved toolkit. In some breeds of domestic pigeon, shifts in the expression of two conserved limb identity transcription factors, PITX1 and TBX5, are associated with the formation of feathered HLs with partial FL identity. To determine how modulation of PITX1 and TBX5 expression affects downstream gene expression, we compared the transcriptomes of embryonic limb buds from pigeons with scaled and feathered HLs. We identified a set of differentially expressed genes enriched for genes encoding transcription factors, extracellular matrix proteins, and components of developmental signaling pathways with important roles in limb development. A subset of the genes that distinguish scaled and feathered HLs are also differentially expressed between FL and scaled HL buds in pigeons, pinpointing a set of gene expression changes downstream of PITX1 and TBX5 in the partial transformation from HL to FL identity. We extended our analyses by comparing pigeon limb bud transcriptomes to chicken, anole lizard, and mammalian datasets to identify deeply conserved PITX1- and TBX5-responsive components of the limb identity program. Our analyses reveal a suite of predominantly low-level gene expression changes that are conserved across amniotes to regulate the identity of morphologically distinct limbs.

Isl1 mediates mesenchymal expansion in the developing external genitalia via regulation of Bmp4, Fgf10 and Wnt5a.

Genital malformations are among the most common human birth defects, and both genetic and environmental factors can contribute to these malformations. Development of the external genitalia in mammals relies on complex signaling networks, and disruption of these signaling pathways can lead to genital defects. Islet-1 (ISL1), a member of the LIM/Homeobox family of transcription factors, has been identified as a major susceptibility gene for classic bladder exstrophy in humans, a common form of the bladder exstrophy-epispadias complex (BEEC), and is implicated in a role in urinary tract development. We report that deletion of Isl1 from the genital mesenchyme in mice led to hypoplasia of the genital tubercle and prepuce, with an ectopic urethral opening and epispadias-like phenotype. These mice also developed hydroureter and hydronephrosis. Identification of ISL1 transcriptional targets via ChIP-Seq and expression analyses revealed that Isl1 regulates several important signaling pathways during embryonic genital development, including the BMP, WNT, and FGF cascades. An essential function of Isl1 during development of the external genitalia is to induce Bmp4-mediated apoptosis in the genital mesenchyme. Together, these studies demonstrate that Isl1 plays a critical role during development of the external genitalia and forms the basis for a greater understanding of the molecular mechanisms underlying the pathogenesis of BEEC and urinary tract defects in humans.

PITX1 promotes chondrogenesis and myogenesis in mouse hindlimbs through conserved regulatory targets.

The PITX1 transcription factor is expressed during hindlimb development, where it plays a critical role in directing hindlimb growth and the specification of hindlimb morphology. While it is known that PITX1 regulates hindlimb formation, in part, through activation of the Tbx4 gene, other transcriptional targets remain to be elucidated. We have used a combination of ChIP-seq and RNA-seq to investigate enhancer regions and target genes that are directly regulated by PITX1 in embryonic mouse hindlimbs. In addition, we have analyzed PITX1 binding sites in hindlimbs of Anolis lizards to identify ancient PITX1 regulatory targets. We find that PITX1-bound regions in both mouse and Anolis hindlimbs are strongly associated with genes implicated in limb and skeletal system development. Gene expression analyses reveal a large number of misexpressed genes in the hindlimbs of Pitx1-/- mouse embryos. By intersecting misexpressed genes with genes that have neighboring mouse PITX1 binding sites, we identified 440 candidate targets of PITX1. Of these candidates, 68 exhibit ultra-conserved PITX1 binding events that are shared between mouse and Anolis hindlimbs. Among the ancient targets of PITX1 are important regulators of cartilage and skeletal muscle development, including Sox9 and Six1. Our data suggest that PITX1 promotes chondrogenesis and myogenesis in the hindlimb by direct regulation of several key members of the cartilage and muscle transcriptional networks.

Appendages and gene regulatory networks: Lessons from the limbless.

Among squamate reptiles, dozens of lineages have independently evolved complete or partial limb reduction. This remarkable convergence of limbless and limb-reduced phenotypes provides multiple natural replicates of different ages to explore the evolution and development of the vertebrate limb and the gene regulatory network that controls its formation. The most successful and best known of the limb-reduced squamates are snakes, which evolved a limb-reduced body form more than 100 million years ago. Recent studies have revealed the unexpected finding that many ancient limb enhancers are conserved in the genomes of snakes. Analyses in limbed animals show that many of these limb enhancers are also active during development of the phallus, suggesting that these enhancers may have been retained in snakes due their importance in regulating transcription in the external genitalia. This hypothesis is substantiated by functional tests of snake enhancers, which demonstrate that snake enhancer elements have lost limb function while retaining genital enhancer function. The large degree of overlap in the gene regulatory networks deployed during limb and phallus development may act to constrain the divergence of shared gene network components and the evolution of appendage morphology. Future studies will reveal whether limb regulatory elements have undergone similar functional changes in other lineages of limb-reduced squamates.

Human-specific loss of regulatory DNA and the evolution of human-specific traits.

Humans differ from other animals in many aspects of anatomy, physiology, and behaviour; however, the genotypic basis of most human-specific traits remains unknown. Recent whole-genome comparisons have made it possible to identify genes with elevated rates of amino acid change or divergent expression in humans, and non-coding sequences with accelerated base pair changes. Regulatory alterations may be particularly likely to produce phenotypic effects while preserving viability, and are known to underlie interesting evolutionary differences in other species. Here we identify molecular events particularly likely to produce significant regulatory changes in humans: complete deletion of sequences otherwise highly conserved between chimpanzees and other mammals. We confirm 510 such deletions in humans, which fall almost exclusively in non-coding regions and are enriched near genes involved in steroid hormone signalling and neural function. One deletion removes a sensory vibrissae and penile spine enhancer from the human androgen receptor (AR) gene, a molecular change correlated with anatomical loss of androgen-dependent sensory vibrissae and penile spines in the human lineage. Another deletion removes a forebrain subventricular zone enhancer near the tumour suppressor gene growth arrest and DNA-damage-inducible, gamma (GADD45G), a loss correlated with expansion of specific brain regions in humans. Deletions of tissue-specific enhancers may thus accompany both loss and gain traits in the human lineage, and provide specific examples of the kinds of regulatory alterations and inactivation events long proposed to have an important role in human evolutionary divergence.

Mesenchymal adenomatous polyposis coli plays critical and diverse roles in regulating lung development.

BACKGROUND: Adenomatous polyposis coli (Apc) is a tumor suppressor that inhibits Wnt/Ctnnb1. Mutations of Apc will not only lead to familial adenomatous polyposis with associated epithelial lesions, but will also cause aggressive fibromatosis in mesenchymal cells. However, the roles of Apc in regulating mesenchymal cell biology and organogenesis during development are unknown. RESULTS: We have specifically deleted the Apc gene in lung mesenchymal cells during early lung development in mice. Loss of Apc function resulted in immediate mesenchymal cell hyperproliferation through abnormal activation of Wnt/Ctnnb1, followed by a subsequent inhibition of cell proliferation due to cell cycle arrest at G0/G1, which was caused by a mechanism independent of Wnt/Ctnnb1. Meanwhile, abrogation of Apc also disrupted lung mesenchymal cell differentiation, including decreased airway and vascular smooth muscle cells, the presence of Sox9-positive mesenchymal cells in the peripheral lung, and excessive versican production. Moreover, lung epithelial branching morphogenesis was drastically inhibited due to disrupted Bmp4-Fgf10 morphogen production and regulation in surrounding lung mesenchyme. Lastly, lung mesenchyme-specific Apc conditional knockout also resulted in altered lung vasculogenesis and disrupted pulmonary vascular continuity through a paracrine mechanism, leading to massive pulmonary hemorrhage and lethality at mid-gestation when the pulmonary circulation should have started. CONCLUSIONS: Our study suggests that Apc in lung mesenchyme plays central roles in coordinating the proper development of several quite different cellular compartments including lung epithelial branching and pulmonary vascular circulation during lung organogenesis.

In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication.

The transition from mitosis to meiosis is a defining juncture in the life cycle of sexually reproducing organisms. In yeast, the decision to enter meiosis is made before the single round of DNA replication that precedes the two meiotic divisions. We present genetic evidence of an analogous decision point in the germ line of a multicellular organism. The mouse Stra8 gene is expressed in germ cells of embryonic ovaries, where meiosis is initiated, but not in those of embryonic testes, where meiosis does not begin until after birth. Here we report that in female embryos lacking Stra8 gene function, the early, mitotic development of germ cells is normal, but these cells then fail to undergo premeiotic DNA replication, meiotic chromosome condensation, cohesion, synapsis and recombination. Combined with previous findings, these genetic data suggest that active differentiation of ovarian germ cells commences at a regulatory point upstream of premeiotic DNA replication.

Pitx1 broadly associates with limb enhancers and is enriched on hindlimb cis-regulatory elements.

Extensive functional analyses have demonstrated that the pituitary homeodomain transcription factor Pitx1 plays a critical role in specifying hindlimb morphology in vertebrates. However, much less is known regarding the target genes and cis-regulatory elements through which Pitx1 acts. Earlier studies suggested that the hindlimb transcription factors Tbx4, HoxC10, and HoxC11 might be transcriptional targets of Pitx1, but definitive evidence for direct regulatory interactions has been lacking. Using ChIP-Seq on embryonic mouse hindlimbs, we have pinpointed the genome-wide location of Pitx1 binding sites during mouse hindlimb development and identified potential gene targets for Pitx1. We determined that Pitx1 binding is significantly enriched near genes involved in limb morphogenesis, including Tbx4, HoxC10, and HoxC11. Notably, Pitx1 is bound to the previously identified HLEA and HLEB hindlimb enhancers of the Tbx4 gene and to a newly identified Tbx2 hindlimb enhancer. Moreover, Pitx1 binding is significantly enriched on hindlimb relative to forelimb-specific cis-regulatory features that are differentially marked by H3K27ac. However, our analysis revealed that Pitx1 also strongly associates with many functionally verified limb enhancers that exhibit similar levels of activity in the embryonic mesenchyme of forelimbs and hindlimbs. We speculate that Pitx1 influences hindlimb morphology both through the activation of hindlimb-specific enhancers as well as through the hindlimb-specific modulation of enhancers that are active in both sets of limbs.

Conserved regulation of hoxc11 by pitx1 in Anolis lizards.

Anolis lizards are an emerging model system for the study of limb development and evolution, but very little is known concerning the regulatory interactions that control limb patterning differences among Anolis species or what regulatory interactions are deeply conserved between Anolis and other tetrapod groups. Here we report the establishment of an embryonic limb micromass culture system that enables functional studies of forelimb and hindlimb gene regulatory networks in Anolis. Characterization of this culture system demonstrated that embryonic forelimb and hindlimb micromasses from different Anolis species are easy to sustain in culture for weeks, and the expression of forelimb and hindlimb-specific gene expression patterns are maintained for at least 8 days in culture. We tested the ability of this system to explore regulatory linkages between transcription factors and their putative target genes through the ectopic expression of a hindlimb-specific transcription factor, pitx1, in forelimb micromasses. We found that pitx1 expression in forelimb cells is sufficient to strongly induce the expression of hoxc11, a gene that normally exhibits hindlimb-restricted expression.

Lung mesenchymal expression of Sox9 plays a critical role in tracheal development.

BACKGROUND: Embryonic lung development is instructed by crosstalk between mesenchyme and epithelia, which results in activation of transcriptional factors, such as Sox9, in a temporospatial manner. Sox9 is expressed in both distal lung epithelium and proximal lung mesenchyme. Here, we investigated the effect of lung mesenchyme-specific inducible deletion of Sox9 during murine lung development. RESULTS: Transgenic mice lacking Sox9 expression were unable to breathe and died at birth, with noticeable tracheal defects. Cartilage rings were missing, and the tracheal lumen was collapsed in the mutant trachea. In situ hybridization showed an altered expression pattern of Tbx4, Tbx5 and Fgf10 genes and marked reduction of Collagen2 expression in the tracheal mesenchyme. The tracheal phenotype was increasingly severe, with longer duration of deletion. Lymphatic vasculature was underdeveloped in the mutant trachea: Prox1, Lyve1, and Vegfr3 were decreased after Sox9 knockout. We also found that compared with normal tracheal epithelium, the mutant tracheal epithelium had an altered morphology with fewer P63-positive cells and more CC10-positive cells, fewer goblet cells, and downregulation of surfactant proteins A and C. CONCLUSION: The appropriate temporospatial expression of Sox9 in lung mesenchyme is necessary for appropriate tracheal cartilage formation, lymphatic vasculature system development, and epithelial differentiation. We uncovered a novel mechanism of lung epithelium differentiation: tracheal cartilage rings instruct the tracheal epithelium to differentiate properly during embryonic development. Thus, besides having a mechanical function, tracheal cartilage also appears to be a local signaling structure in the embryonic lung.

Spatial-temporal targeting of lung-specific mesenchyme by a Tbx4 enhancer.

BACKGROUND: Reciprocal interactions between lung mesenchymal and epithelial cells play essential roles in lung organogenesis and homeostasis. Although the molecular markers and related animal models that target lung epithelial cells are relatively well studied, molecular markers of lung mesenchymal cells and the genetic tools to target and/or manipulate gene expression in a lung mesenchyme-specific manner are not available, which becomes a critical barrier to the study of lung mesenchymal biology and the related pulmonary diseases. RESULTS: We have identified a mouse Tbx4 gene enhancer that contains conserved DNA sequences across many vertebrate species with lung or lung-like gas exchange organ. We then generate a mouse line to express rtTA/LacZ under the control of the Tbx4 lung enhancer, and therefore a Tet-On inducible transgenic system to target lung mesenchymal cells at different developmental stages. By combining a Tbx4-rtTA driven Tet-On inducible Cre expression mouse line with a Cre reporter mouse line, the spatial-temporal patterns of Tbx4 lung enhancer targeted lung mesenchymal cells were defined. Pulmonary endothelial cells and vascular smooth muscle cells were targeted by the Tbx4-rtTA driver line prior to E11.5 and E15.5, respectively, while other subtypes of lung mesenchymal cells including airway smooth muscle cells, fibroblasts, pericytes could be targeted during the entire developmental stage. CONCLUSIONS: Developmental lung mesenchymal cells can be specifically marked by Tbx4 lung enhancer activity. With our newly created Tbx4 lung enhancer-driven Tet-On inducible system, lung mesenchymal cells can be specifically and differentially targeted in vivo for the first time by controlling the doxycycline induction time window. This novel system provides a unique tool to study lung mesenchymal cell lineages and gene functions in lung mesenchymal development, injury repair, and regeneration in mice.

Engineering subtle targeted mutations into the mouse genome.

Homologous recombination in embryonic stem (ES) cells offers an exquisitely precise mechanism to introduce targeted modifications to the mouse genome. This ability to produce specific alterations to the mouse genome has become an essential tool for the analysis of gene function and the development of mouse models of human disease. Of the many thousands of mouse alleles that have been generated by gene targeting, the majority are designed to completely ablate gene function, to create conditional alleles that are inactivated in the presence of Cre recombinase, or to produce reporter alleles that label-specific tissues or cell populations (Eppig et al., 2012, Nucleic Acids Res 40:D881-D886). However, there is a variety of powerful motivations for the introduction of subtle targeted mutations (STMs) such as point mutations, small deletions, or small insertions into the mouse genome. The introduction of STMs allows the ablation of specific transcript isoforms, permits the functional investigation of particular domains or amino acids within a protein, provides the ability to study the role of specific sites with in cis-regulatory elements, and can result in better mouse models of human genetic disorders. In this review, I examine the current strategies that are commonly used to introduce STMs into the mouse genome and highlight new gene targeting technologies, including TALENs and CRISPR/Cas, which are likely to influence the future of gene targeting in mice.

Piebaldism and chromatophore development in reptiles are linked to the tfec gene.

Reptiles display great diversity in color and pattern, yet much of what we know about vertebrate coloration comes from classic model species such as the mouse and zebrafish.1,2,3,4 Captive-bred ball pythons (Python regius) exhibit a remarkable degree of color and pattern variation. Despite the wide range of Mendelian color phenotypes available in the pet trade, ball pythons remain an overlooked species in pigmentation research. Here, we investigate the genetic basis of the recessive piebald phenotype, a pattern defect characterized by patches of unpigmented skin (leucoderma). We performed whole-genome sequencing and used a case-control approach to discover a nonsense mutation in the gene encoding the transcription factor tfec, implicating this gene in the leucodermic patches in ball pythons. We functionally validated tfec in a lizard model (Anolis sagrei) using the gene editing CRISPR/Cas9 system and TEM imaging of skin. Our findings show that reading frame mutations in tfec affect coloration and lead to a loss of iridophores in Anolis, indicating that tfec is required for chromatophore development. This study highlights the value of captive-bred ball pythons as a model species for accelerating discoveries on the genetic basis of vertebrate coloration.

A single locus regulates a female-limited color pattern polymorphism in a reptile.

Animal coloration is often expressed in periodic patterns that can arise from differential cell migration, yet how these processes are regulated remains elusive. We show that a female-limited polymorphism in dorsal patterning (diamond/chevron) in the brown anole is controlled by a single Mendelian locus. This locus contains the gene CCDC170 that is adjacent to, and coexpressed with, the Estrogen receptor-1 gene, explaining why the polymorphism is female limited. CCDC170 is an organizer of the Golgi-microtubule network underlying a cell's ability to migrate, and the two segregating alleles encode structurally different proteins. Our agent-based modeling of skin development demonstrates that, in principle, a change in cell migratory behaviors is sufficient to switch between the two morphs. These results suggest that CCDC170 might have been co-opted as a switch between color patterning morphs, likely by modulating cell migratory behaviors.

Chromosome-scale genome assembly of the brown anole (Anolis sagrei), an emerging model species.

Rapid technological improvements are democratizing access to high quality, chromosome-scale genome assemblies. No longer the domain of only the most highly studied model organisms, now non-traditional and emerging model species can be genome-enabled using a combination of sequencing technologies and assembly software. Consequently, old ideas built on sparse sampling across the tree of life have recently been amended in the face of genomic data drawn from a growing number of high-quality reference genomes. Arguably the most valuable are those long-studied species for which much is already known about their biology; what many term emerging model species. Here, we report a highly complete chromosome-scale genome assembly for the brown anole, Anolis sagrei - a lizard species widely studied across a variety of disciplines and for which a high-quality reference genome was long overdue. This assembly exceeds the vast majority of existing reptile and snake genomes in contiguity (N50 = 253.6 Mb) and annotation completeness. Through the analysis of this genome and population resequence data, we examine the history of repetitive element accumulation, identify the X chromosome, and propose a hypothesis for the evolutionary history of fusions between autosomes and the X that led to the sex chromosomes of A. sagrei.

Evolution of a chordate-specific mechanism for myoblast fusion.

Vertebrate myoblast fusion allows for multinucleated muscle fibers to compound the size and strength of mononucleated cells, but the evolution of this important process is unknown. We investigated the evolutionary origins and function of membrane-coalescing agents Myomaker and Myomixer in various groups of chordates. Here, we report that Myomaker likely arose through gene duplication in the last common ancestor of tunicates and vertebrates, while Myomixer appears to have evolved de novo in early vertebrates. Functional tests revealed a complex evolutionary history of myoblast fusion. A prevertebrate phase of muscle multinucleation driven by Myomaker was followed by the later emergence of Myomixer that enables the highly efficient fusion system of vertebrates. Evolutionary comparisons between vertebrate and nonvertebrate Myomaker revealed key structural and mechanistic insights into myoblast fusion. Thus, our findings suggest an evolutionary model of chordate fusogens and illustrate how new genes shape the emergence of novel morphogenetic traits and mechanisms.