Chris J. Cretekos

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.

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

Abstract 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.

Genetic Regulation of Mammalian Diversity

Mammals have evolved a variety of morphological adaptations that have allowed them to compete in their natural environments. The developmental genetic basis of this morphological diversity remains largely unknown. Bats are mammals that have the unique ability of powered flight. We have examined the molecular embryology of bats and investigated the developmental genetic basis for their highly derived limbs used for flight. Initially, we developed an embryo staging system for a model chiropteran, Carollia perspicillata, the short-tailed fruit bat that has subsequently been used for staging other bat species. Expression studies focusing on genes that regulate limb development indicate that there are similarities and differences between bats and mice. To determine the consequences of these expression differences, we have conducted an enhancer switch assay by gene targeting in mouse embryonic stem cells to create mice whose genes are regulated by bat sequences. Our studies indicate that cis-regulatory elements contribute to the morphological differences that have evolved among mammalian species.

Whole-mount in situ hybridization of short-tailed fruit bat (Carollia perspicillata) embryos with RNA probes.

This protocol describes whole-mount in situ hybridization of short-tailed fruit bat (Carollia perspicillata) embryos with RNA probes. This technique allows direct visualization of the mRNA expression pattern of a gene of interest within embryos fixed at a wide range of developmental stages. Due to limitations in probe penetration, the size of the specimen must be considered when using whole-mount procedures. In addition, the preparation of probe is an important step in whole-mount in situ hybridization. Two primary considerations for making a good probe are (1) a good template sequence and (2) an efficient labeling reaction. In our experience, the ideal template sequence for a whole-mount in situ probe should be 300-1000 bp in length. We have had little success using shorter probes, although they can be made to work in a case-by-case (and presumably sequence-dependent) manner. Typically, one begins with a cDNA or coding genomic fragment cloned into an in vitro transcription vector. The linearized vector is purified and used as the template for in vitro transcription. In this protocol, the in situ hybridization procedure is presented as a three-day schedule: Day 1, Pre-treatment and hybridization; Day 2, Post-hybridization washes and antibody incubation; and Day 3, Post-antibody washes and color reaction.

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. Genesis 50:18‐27, 2012.

Palate Variation and Evolution in New World Leaf-Nosed and Old World Fruit Bats (Order Chiroptera)

Two bat families, the leaf-nosed (Phyllostomidae) and fruit bats (Pteropodidae), have independently evolved the ability to consume plant resources. However, despite their similar ages, species richness and the strong selective pressures placed on the evolution of skull shape by plant-based foods, phyllostomids display more craniofacial diversity than pteropodids. In this study, we used morphometrics to investigate the distribution of palate variation and the evolution of palate diversity in these groups. We focused on the palate because evolutionary alterations in palate morphology are thought to underlie much feeding specialization in bats. We hypothesize that the distribution of palate variation differs in phyllostomids and pteropodids, and that the rate of palate evolution is higher in phyllostomids than pteropodids. The results suggest that the overall level of palate integration is higher in adult populations of pteropodids than phyllostomids but that the distribution of palate variation is otherwise generally conserved among phyllostomids and pteropodids. Furthermore, the results are consistent with these differences in palate integration likely having a developmental basis. The results also suggest that palate evolution has occurred significantly more rapidly in phyllostomids than pteropodids. These findings are consistent with a scenario in which the greater integration of the pteropodid palate has limited its evolvability.

The Short-Tailed Fruit Bat Carollia perspicillata: A Model for Studies in Reproduction and Development

The bat (Chiroptera) order, its distribution, and various developmental studies are described at http://embryology.med.unsw.edu.au/OtherEmb/bat.htm. The first release of the low-coverage 1.7X assembly of the genome of the microbat or little brown bat (Myotis lucifugus) is described at http://www.ensembl.org/Myotis_lucifugus/index.html. M. lucifugus is one of 16 mammals that will be sequenced as part of the Mammalian Genome Project.

Whole-mount immunohistochemistry of short-tailed fruit bat (Carollia perspicillata)

Cold Spring Harb Protoc John J. Rasweiler IV, Chris J. Cretekos and Richard R. Behringer ) perspicillata Carollia Whole-Mount Immunohistochemistry of Short-Tailed Fruit Bat ( Service Email Alerting click here. Receive free email alerts when new articles cite this article Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (89 articles) Visualization of Proteins (458 articles) Visualization (480 articles) Proteins and Proteomics, general (159 articles) Protein Identification and Analysis (873 articles) Laboratory Organisms, general (64 articles) Immunostaining, general (85 articles) Immunostaining (71 articles) Immunohistochemistry (318 articles) Genetics, general (283 articles) Emerging Model Organisms (563 articles) Developmental Biology (988 articles) Cell Biology, general (214 articles) Antibodies, general

Transcriptomic insights into the genetic basis of mammalian limb diversity

BackgroundFrom 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.ResultsWe 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.ConclusionsTaken 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.

Alcian blue staining of cartilage of short-tailed fruit bat (Carollia perspicillata).

This protocol is used to visualize the cartilaginous elements of the developing skeleton using alcian blue. It has been used with excellent results on the fruit bat Carollia perspicillata, as well as several other bat species at stages from CS 14 through CS 22. Staining has been shown to last at least 2 yr in benzyl alcohol:benzyl benzoate (BABB). However, staining has been observed to weaken during the course of months. It is therefore advisable to document results immediately.