Douglas B. Menke

The vertebrate forelimb initiates as a localized swelling in the somatic lateral plate mesoderm (somatic LPM) in response to TBX5-dependent transcription. The molecular pathways driving limb morphogenesis have been extensively studied but the steps directly preceding limb bud formation remain poorly characterized. To address this, we defined the temporal onset of forelimb initiation in mouse embryos using sequencing based high-throughput approaches (RNA-seq, scRNA-seq, ChIP-seq, and Ribo-ITP) benchmarked to known features in forelimb development, identifying four distinct stages. Using scRNA-seq at the onset of forelimb-specific transcription, we determined the transcriptional profile of the somatic LPM and identified signature genes that distinguish the nascent forelimb from other cell types. This group includes multiple genes involved in neural projection as well as cell adhesion. Interestingly, these genes are highly enriched for TBX5 binding sites, suggesting they are candidate early transcriptional targets of TBX5. As TBX5 is essential for forelimb outgrowth, the identification of these genes suggests new mechanistic models for TBX5-driven limb initiation.

The vertebrate forelimb initiates as a localized swelling in the somatic lateral plate mesoderm (somatic LPM) in response to TBX5-dependent transcription. The molecular pathways driving limb morphogenesis have been extensively studied but the steps directly preceding limb bud formation remain poorly characterized. To address this, we defined the temporal onset of forelimb initiation in mouse embryos using sequencing based high-throughput approaches (RNA-seq, scRNA-seq, ChIP-seq, and Ribo-ITP) benchmarked to known features in forelimb development, identifying four distinct stages. Using scRNA-seq at the onset of forelimb-specific transcription, we determined the transcriptional profile of the somatic LPM and identified signature genes that distinguish the nascent forelimb from other cell types. This group includes multiple genes involved in neural projection as well as cell adhesion. Interestingly, these genes are highly enriched for TBX5 binding sites, suggesting they are candidate early transcriptional targets of TBX5. As TBX5 is essential for forelimb outgrowth, the identification of these genes suggests new mechanistic models for TBX5-driven limb initiation.

Squamates, the taxon that comprises lizards and snakes, are a diverse assemblage of reptiles represented by more than 11,000 described species. Studies of gene function in squamates, however, have remained very limited, largely due to the lack of established genetic tools and suitable experimental systems. A major challenge for the development of CRISPR-based gene editing in these reptiles is that the isolation of fertilized oocytes or single-celled embryos is impractical for most species, given that fertilization occurs internally, the females of many species can store sperm, and simple methods for detecting ovulation are lacking. To overcome these challenges, we have developed a unique surgical approach in the brown anole lizard Anolis sagrei . The procedure enables users to access and microinject unfertilized oocytes while they are still maturing within the lizard ovary. We describe here the methods to anesthetize adult female anoles, access the ovary through a surgical incision into the coelomic cavity, and microinject unfertilized oocytes with CRISPR–Cas9 ribonucleoprotein complexes to generate targeted mutations, enabling the routine production of gene-edited lizards.

Anolis lizards are an ecologically diverse group that includes more than 400 described species. These reptiles have been the subject of wide-ranging studies, from speciation and convergent evolution to climate adaptation and tail regeneration. While CRISPR-based gene editing has tremendous potential to reveal new insights into these and other aspects of Anolis biology, the reproductive biology of these reptiles has presented significant barriers to gene editing. Here, we briefly summarize gene editing approaches in vertebrates and discuss some of the major challenges associated with the performance of gene editing in anoles. We then introduce a recently established surgical procedure that enables the injection of CRISPR–Cas into the developing oocytes of female lizards. This approach circumvents the need to manipulate early-stage embryos and permits the production of gene-edited anoles. This method has recently been successfully adapted for use in other reptiles, suggesting that it may be effective in a wide range of species and will broadly enable studies of gene function in reptiles.

The baculum, a bone in the penis of many mammal species, shows an astonishing level of morphological divergence between species. Despite hundreds of years of interest, biologists have been unable to directly test its function. The goal of the current study is to uncover molecular details that could allow selective disruption of the baculum while allowing normal sexual differentiation and skeletal development. We compare patterns of androgen receptor binding and single cell gene expression in the developing penis, forelimbs and hindlimbs of mice. We identified chondrocytes in all three tissue types, but those from the developing penis show several unique features, including a population of chondrocytes that express both Runt-related transcription factor 2 (Runx2) and Androgen receptor (Ar). By combining a Runx2-Cre allele with a floxed Ar allele in mice, we selectively knocked out androgen signaling in late chondrocytes, resulting in a range of defects in baculum morphology. Males with the most disrupted bacula were unable to copulate, and their bacula appears to be disconnected from the corpus cavernosum muscle. Our study provides insights into the diversity of molecular mechanisms leading to bone and offers the first opportunity to directly test the function of the baculum.

The fovea, a pit in the retina, is crucial for high‐acuity vision in humans and is found in the eyes of other vertebrates, including certain primates, birds, lizards, and fish. Despite its importance for vision, our understanding of the mechanisms involved in fovea development remains limited. Widely used ocular research models lack a foveated retina, and studies on fovea development are mostly limited to histological and molecular studies in primates. As a first step toward elucidating fovea development in nonprimate vertebrates, we present a detailed histological atlas of retina and fovea development in the bifoveated Anolis sagrei lizard, a novel reptile model for fovea research. We test the hypothesis that retinal remodeling, leading to fovea formation and photoreceptor cell packing, is related to asymmetric changes in eye shape. Our findings show that anole retina development follows the typical spatiotemporal patterning observed in most vertebrates: retinal neurogenesis starts in the central retina, progresses through the temporal retina, and finishes in the nasal retina. However, the areas destined to become the central or temporal fovea differentiate earlier than the rest of the retina. We observe dynamic changes in retinal thickness during ocular elongation and retraction—thinning during elongation and thickening during retraction. Additionally, a transient localized thickening of the ganglion cell layer occurs in the temporal fovea region just before pit formation. Our data indicate that anole retina development is similar to that of humans, including the onset and progression of retinal neurogenesis, followed by changes in ocular shape and retinal remodeling leading to pit formation. We propose that anoles are an excellent model system for fovea development research.

The establishment of CRISPR/Cas9 gene editing in Anolis sagrei has positioned this species as a powerful model for studies of reptilian gene function. To enhance this model, we developed an immortalized lizard fibroblast cell line (ASEC-1) for the exploration of reptilian gene function in cellular processes. We demonstrate the use of this cell line by scrutinizing the role of primary cilia in lizard Hedgehog (Hh) signaling. Using CRISPR/Cas9 mutagenesis, we disrupted the ift88 gene, which is required for ciliogenesis in diverse organisms. We determined that loss of itf88 from lizard cells leads to an absence of primary cilia, a partial derepression of gli1 transcription, and an inability of the cells to respond to the Smoothened agonist, SAG. Through a cross-species analysis of SAG-induced transcriptional responses in cultured limb bud cells, we further determined that ∼46% of genes induced as a response to Hh pathway activation in A. sagrei are also SAG responsive in Mus musculus limb bud cells. Our results highlight conserved and diverged aspects of Hh signaling in anoles and establish a new resource for investigations of reptilian gene function.

The recent establishment of CRISPR/Cas9 gene editing in A. sagrei lizards makes it a powerful model system for studies of reptilian gene function. To enhance the versatility of this model, we developed an immortalized lizard fibroblast cell line (ASEC-1) for the exploration of reptilian gene function in cellular processes. We demonstrate the use of this in vitro system by scrutinizing the role of primary cilia in lizard Hedgehog (Hh) signaling. Through CRISPR/Cas9 mutagenesis we disrupted the ift88 gene, which is required for ciliogenesis in diverse organisms. We find that the loss of itf88 from lizard cells results in an absence of primary cilia, a partial derepression of gli1 transcription, and an inability of the cells to respond to the Smoothened agonist, SAG. Through a cross-species analysis of SAG-induced transcriptional responses in cultured limb bud cells, we further determined that ∼46% of genes induced as a response to Hh pathway activation in A. sagrei, are also SAG-responsive in M. musculus limb bud cells. Our results highlight conserved and diverged aspects of Hh signaling in anoles and establish a new resource for investigations of reptilian gene function.

In primates, the fovea is a pit in the center of the macula, which is a region of the retina with a high concentration of cone photoreceptor cells that 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 packing of photoreceptor cells more densely into this area of the retina, which occurs sometime after birth. Maturation occurs concurrently with progressing age and development, which further correlates with increasing eye size, retinal length, and retinal 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 from five different age groups – 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 the macular borders and peripheral retina areas. Highlights - Foveal and retinal refinement occurs post-hatching into adulthood in anoles. - The temporal pit shallows and widens, whereas central pit morphology remains relatively constant with age. - Retina cell number within a section decreases as the eye grows larger with age. - Anole macular diameter increases from hatching to adult but does not increase proportionately to overall eye growth.