Insights into the formation and diversification of a novel chiropteran wing membrane from embryonic development.

BACKGROUND: Through the evolution of novel wing structures, bats (Order Chiroptera) became the only mammalian group to achieve powered flight. This achievement preceded the massive adaptive radiation of bats into diverse ecological niches. We investigate some of the developmental processes that underlie the origin and subsequent diversification of one of the novel membranes of the bat wing: the plagiopatagium, which connects the fore- and hind limb in all bat species. RESULTS: Our results suggest that the plagiopatagium initially arises through novel outgrowths from the body flank that subsequently merge with the limbs to generate the wing airfoil. Our findings further suggest that this merging process, which is highly conserved across bats, occurs through modulation of the programs controlling the development of the periderm of the epidermal epithelium. Finally, our results suggest that the shape of the plagiopatagium begins to diversify in bats only after this merging has occurred. CONCLUSIONS: This study demonstrates how focusing on the evolution of cellular processes can inform an understanding of the developmental factors shaping the evolution of novel, highly adaptive structures.

Transcriptomic insights into the genetic basis of mammalian limb diversity.

BACKGROUND: From bat wings to whale flippers, limb diversification has been crucial to the evolutionary success of mammals. We performed the first transcriptome-wide study of limb development in multiple species to explore the hypothesis that mammalian limb diversification has proceeded through the differential expression of conserved shared genes, rather than by major changes to limb patterning. Specifically, we investigated the manner in which the expression of shared genes has evolved within and among mammalian species. RESULTS: We assembled and compared transcriptomes of bat, mouse, opossum, and pig fore- and hind limbs at the ridge, bud, and paddle stages of development. Results suggest that gene expression patterns exhibit larger variation among species during later than earlier stages of limb development, while within species results are more mixed. Consistent with the former, results also suggest that genes expressed at later developmental stages tend to have a younger evolutionary age than genes expressed at earlier stages. A suite of key limb-patterning genes was identified as being differentially expressed among the homologous limbs of all species. However, only a small subset of shared genes is differentially expressed in the fore- and hind limbs of all examined species. Similarly, a small subset of shared genes is differentially expressed within the fore- and hind limb of a single species and among the forelimbs of different species. CONCLUSIONS: Taken together, results of this study do not support the existence of a phylotypic period of limb development ending at chondrogenesis, but do support the hypothesis that the hierarchical nature of development translates into increasing variation among species as development progresses.

The Relationship between Gene Network Structure and Expression Variation among Individuals and Species.

Variation among individuals is a prerequisite of evolution by natural selection. As such, identifying the origins of variation is a fundamental goal of biology. We investigated the link between gene interactions and variation in gene expression among individuals and species using the mammalian limb as a model system. We first built interaction networks for key genes regulating early (outgrowth; E9.5-11) and late (expansion and elongation; E11-13) limb development in mouse. This resulted in an Early (ESN) and Late (LSN) Stage Network. Computational perturbations of these networks suggest that the ESN is more robust. We then quantified levels of the same key genes among mouse individuals and found that they vary less at earlier limb stages and that variation in gene expression is heritable. Finally, we quantified variation in gene expression levels among four mammals with divergent limbs (bat, opossum, mouse and pig) and found that levels vary less among species at earlier limb stages. We also found that variation in gene expression levels among individuals and species are correlated for earlier and later limb development. In conclusion, results are consistent with the robustness of the ESN buffering among-individual variation in gene expression levels early in mammalian limb development, and constraining the evolution of early limb development among mammalian species.

Early oogenesis in the short-tailed fruit bat Carollia perspicillata: transient germ cell cysts and noncanonical intercellular bridges.

The ovaries of early embryos (40 days post coitum/p.c.) of the bat Carollia perspicillata contain numerous germ-line cysts, which are composed of 10 to 12 sister germ cells (cystocytes). Variability in the number of cystocytes within the cyst and between the cysts (defying the Giardina rule) indicates that the mitotic divisions of the cystoblast are asynchronous in this bat species. Serial section analysis showed that the cystocytes are interconnected via intercellular bridges that are atypical, strongly elongated, short-lived, and rich in microtubule bundles and microfilaments. During slightly later stages of embryonic development (44-46 days p.c.), somatic cells penetrate the cyst, and their cytoplasmic projections separate individual oocytes. Separated oocytes surrounded by a single layer of somatic cells constitute the primordial ovarian follicles. The oocytes of C. perspicillata are similar to mouse oocytes and are asymmetric. In both species, this asymmetry is clearly recognizable in the localization of the Golgi complexes. The presence of germ-line cysts and intercellular bridges (although noncanonical) in the fetal ovaries of C. perspicillata suggest that the formation of germ-line cysts is an evolutionarily conserved phase in the development of the female gametes in a substantial part of the animal kingdom.

The evolution and development of mammalian flight.

Mammals have evolved a stunning diversity of limb morphologies (e.g., wings, flippers, hands, and paws) that allowed access to a wide range of habitats. Over 50 million years ago, bats (Order Chiroptera) evolved a wing (composed of a thin membrane encasing long digits) and thereby achieved powered flight. Unfortunately, the fossil record currently lacks any transitional fossils between a rodent-like ancestor and a winged bat. To reconstruct how this important evolutionary transition occurred, researchers have begun to employ an evolutionary developmental approach. This approach has revealed some of the embryological and molecular changes that have contributed to the evolution of the bat wing. For example, bat and mouse forelimb morphologies are similar during earliest limb development. Despite this, some key signaling centers for limb development are already divergent in bat and mouse at these early stages. Bat and mouse limb development continues to diverge, such that at later stages many differences are apparent. For example, at these later stages bats redeploy expression of toolkit genes (i.e., Fgf, Shh, Bmp, Grem) in a novel expression domain to inhibit apoptosis of the interdigital tissues. When results are taken together, a broad picture of the developmental changes that drove the transition from a hand to a wing over 50 million years ago is beginning to take shape. Moreover, studies seem to suggest that small changes in gene regulation during organogenesis can generate large evolutionary changes in phenotype.

Embryonic staging system for the Black Mastiff Bat, Molossus rufus (Molossidae), correlated with structure-function relationships in the adult.

An embryonic staging system for Molossus rufus (also widely known as Molossus ater) was devised using 17 reference specimens obtained during the postimplantation period of pregnancy from wild-caught, captive-bred females. This was done in part by comparing the embryos to a developmental staging system that had been created for another, relatively unrelated bat, Carollia perspicillata (family Phyllostomidae). Particular attention was paid to the development of species-specific features, such as wing and ear morphology, and these are discussed in light of the adaptive significance of these structures in the adult. M. rufus can be maintained and bred in captivity and is relatively abundant in the wild. This embryonic staging system will facilitate further developmental studies of M. rufus, a model species for one of the largest and most successful chiropteran families, the Molossidae.

A second wave of Sonic hedgehog expression during the development of the bat limb.

Sonic hedgehog (Shh) plays an integral role in both the anterior-posterior (A-P) patterning and expansion of developing vertebrate limbs through a feedback loop involving Fgfs, Bmps, and Gremlin. In bat limbs A-P patterning and the size of the digital field are unique. The posterior digits of the forelimb are elongated and joined by tissue, whereas the thumb is short. The hindlimb digits often are uniform in length. Here, we reveal novel expression patterns for Shh and its target, Patched 1 (Ptc1), during limb development in two bat species. Early Shh expression in the zone of polarizing activity is wider in the bat forelimb than in the mouse forelimb, correlating with the reported expansion of Fgf8 expression in the apical ectodermal ridge and the early loss of symmetry in the bat forelimb. Later in limb development, Shh and Ptc1 expression is reinitiated in the interdigital tissue. Shh is graded along the A-P axis in forelimb and is expressed uniformly at a lower level across the hindlimb interdigital tissue. We also show that the reported Fgf8 expression in the interdigital tissue precedes the expression of Shh. We propose that the reinitiation of Shh and Fgf8 expression in bat limbs reactivates the Shh-Fgf feedback loop in the interdigital tissue of stage 16 bat embryos. The cell survival and proliferation signals provided by the Shh-Fgf signaling loop probably contribute to the lengthening of the posterior forelimb digits, the survival of the forelimb interdigital webbing, and the extension of the hindlimb digits to a uniform length.

Regulatory divergence modifies limb length between mammals.

Natural selection acts on variation within populations, resulting in modified organ morphology, physiology, and ultimately the formation of new species. Although variation in orthologous proteins can contribute to these modifications, differences in DNA sequences regulating gene expression may be a primary source of variation. We replaced a limb-specific transcriptional enhancer of the mouse Prx1 locus with the orthologous sequence from a bat. Prx1 expression directed by the bat enhancer results in elevated transcript levels in developing forelimb bones and forelimbs that are significantly longer than controls because of endochondral bone formation alterations. Surprisingly, deletion of the mouse Prx1 limb enhancer results in normal forelimb length and Prx1 expression, revealing regulatory redundancy. These findings suggest that mutations accumulating in pre-existing noncoding regulatory sequences within a population are a source of variation for the evolution of morphological differences between species and that cis-regulatory redundancy may facilitate accumulation of such mutations.

Polarized ovaries of the long-tongued bat, Glossophaga soricina: a novel model for studying ovarian development, folliculogenesis, and ovulation.

Glossophaga soricina is a spontaneously ovulating, monovular, polyestrous bat with a simplex uterus, exhibiting true menstruation. Studies conducted on reproductively active, captive-maintained animals established that G. soricina also has polarized ovaries, with the ovarian surface epithelium (OSE) restricted to the medial side of the ovary, and primordial follicles limited to an immediately adjacent zone. Follicles selected for further development are recruited from the medullary side of this zone, and ovulation is restricted to the portion of the ovary covered by the OSE. To further develop G. soricina as a model for studying ovarian development and physiology, ovaries were collected from fetal, neonatal, and adult females and processed for morphological and immunohistochemical analyses. The latter included staining for factor VIII-related antigen (von Willebrand factor) to assess regional differences in ovarian vascularity. The ovarian structure in fetal and neonatal animals was very similar to that in other species that do not have polarized ovaries at comparable stages of development. This indicated that polarization of the ovary does not occur during fetal development, but rather sometime between the neonatal period and adulthood. Vascular elements were abundant in areas of the ovary surrounding early growing follicles, but sparse in the zone of the ovary containing primordial follicles. The polarized nature of the ovaries in G. soricina suggests that this species might be used as a model to investigate the formation, long-term maintenance, and activation of primordial follicles, and the role of the OSE in ovulation and folliculogenesis.

Isolation, genomic structure and developmental expression of Fgf8 in the short-tailed fruit bat, Carollia perspicillata.

Fibroblast growth factor-8 (Fgf8) encodes a secreted protein which was initially identified as the factor responsible for androgen-dependant growth of mouse mammary carcinoma cells (Tanaka et al., 1992). Fgf8 has been subsequently implicated in the patterning and growth of the gastrulating embryo, paraxial mesoderm (somites), limbs, craniofacial tissues, central nervous system and other organ systems during the development of several vertebrate model animals. Consistent with these findings, Fgf8 is expressed in a complex and dynamic pattern during vertebrate embryogenesis. Here we report the isolation and characterization of a bat (Carollia perspicillata) Fgf8 orthologue. Compared with those of other model vertebrates, Carollia Fgf8 is conserved with respect to genomic structure, sequence and many domains of developmental expression pattern. Interestingly, the expression domain marking the apical ectodermal ridge of the developing limb shows a striking difference compared to that of mouse, consistent with evolutionary diversification of bat limb morphology.