John J. Rasweiler

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.

Embryonic staging system for the short-tailed fruit bat, Carollia perspicillata, a model organism for the mammalian order Chiroptera, based upon timed pregnancies in captive-bred animals

There are approximately 4,800 extant species of mammals that exhibit tremendous morphological, physiological, and developmental diversity. Yet embryonic development has been studied in only a few mammalian species. Among mammals, bats are second only to rodents with regard to species number and habitat range and are the most abundant mammals in undisturbed tropical regions. Bat development, though, remains relatively unstudied. Here, we describe and illustrate a staging series of embryonic development for the short‐tailed fruit bat, Carollia perspicillata, based on embryos collected at timed intervals after captive matings. As Carollia can be readily maintained and propagated in captivity and is extremely abundant in the wild, it offers an attractive choice as a chiropteran model organism. This staging system provides a framework for studying Carollia embryogenesis and should prove useful as a guide for embryological studies of other bat species and for comparisons with other orders of mammals. Developmental Dynamics 233:721–738, 2005.

Hoxd13 expression in the developing limbs of the short-tailed fruit bat, Carollia perspicillata

Summary Bat forelimbs are highly specialized for sustained flight, providing a unique model to explore the genetic programs that regulate vertebrate limb diversity. Hoxd9–13 genes are important regulators of stylopodium, zeugopodium, and autopodium development and thus evolutionary changes in their expression profiles and biochemical activities may contribute to divergent limb morphologies in vertebrates. We have isolated the genomic region that includes Hoxd12 and Hoxd13 from Carollia perspicillata, the short‐tailed fruit bat. The bat Hoxd13 gene encodes a protein that shares 95% identity with human and mouse HOXD13. The expression pattern of bat Hoxd13 mRNA during limb development was compared with that of mouse. In bat and mouse hindlimbs, the expression patterns of Hoxd13 are relatively similar. However, although the forelimb Hoxd13 expression patterns in both organisms during early limb bud stages are similar, at later stages they diverge; the anterior expression boundary of bat Hoxd13 is posterior‐shifted relative to the mouse. These findings, compared with the Hoxd13 expression profiles of other vertebrates, suggest that divergent Hoxd13 expression patterns may contribute to limb morphological variation.

Evaluation of leuprolide acetate and gonadotropins versus clomiphene citrate and gonadotropins for in vitro fertilization or gamete intrafallopian transfer

A prospective randomized trial was conducted to compare the efficiency of two ovarian stimulation protocols for in vitro fertilization-embryo transfer or gamete intrafallopian transfer. Protocol 1 consisted of clomiphene citrate and human menopausal gonadotropin (hMG) with 55 cycles of 42 patients being evaluated. Protocol 2 had 38 cycles of 34 patients receiving a gonadotropin-releasing hormone agonist (GnRH-a) and hMG. The incidence of a spontaneous luteinizing hormone surge was 38.2% in protocol 1 and 0% in protocol 2. Both protocols had a similar cancellation rate. The total clinical pregnancy rates per oocyte retrieval for patients receiving protocol 1 and protocol 2 were 19.5% and 10.3%, respectively. The difference was not statistically significant. Therefore, as first-line ovulation induction agents, it cannot be concluded that either protocol demonstrates a clear superiority over the other and further trials of the GnRH-a/hMG combination are indicated.

Anatomy and Physiology of the Female Reproductive Tract

Publisher Summary Bats exhibit considerable diversity in their reproductive characteristics. These are of interest and importance for several reasons. Because many bat species are both small and abundant, it is feasible to collect specimens exhibiting major reproductive states and to conduct thorough comparative investigations. The latter can, in turn, provide insights into how and why particular characteristics may have evolved. This would be much more difficult or impossible with many other mammalian groups (for example, primates) because of animal cost and/or availability problems. This can provide a perspective on the evolution of mammalian reproduction that one might have trouble in acquiring through comparative studies of unrelated groups (for example, rodents and primates) or within other common but less diversified orders. A better knowledge of how bats reproduce is important to the proper management or rescue of wild populations that are increasingly threatened by human activities, and to the successful propagation of captive populations in zoological parks for educational purposes. Finally, bats exhibit reproductive specializations which are sometimes either uniquely similar to those of primates or unusual when compared to those operative in more commonly studied mammals.

Improved procedures for maintaining and breeding the short-tailed fruit bat (Carollia perspicillata) in a laboratory setting.

Improved methods have been developed for maintaining and breeding the neotropical short-tailed fruit bat, Carollia perspicillata, in an easily-reproduced, laboratory setting. Bipartite, all-metal cages have been designed which permit efficient, non-injurious handling of the animals. Also, a fruit-based liquid diet, which is simple and inexpensive to prepare, has been formulated from readily-available canned and powdered components. When efforts were initiated to breed wild-caught animals at 3-6 months after capture, this progressed slowly in most (but not all) cages. Breeding took place more rapidly when the bats had been in captivity for 11-24 months. Most females (122/144, 84.7%) then bred within 30 days of the introduction of a stud male, and the overall conception rate was 94.3% in those females which bred. This pattern of breeding activity, evidence that this bat is a spontaneous ovulator, and observed intervals between successive breeding periods (apparently representing much or all of non-pregnant cycles) in some individuals suggest that the cycle length in many of these bats probably falls between 20 and 30 days. Analysis of the breeding data also showed that certain patterns of breeding activity were frequently associated with a failure of females to establish ongoing pregnancies. Most laboratory-bred females (69/81) that were permitted to carry their pregnancies to term did so and successfully reared their young. The laboratory-reared young have generally exhibited excellent body condition, and many have proved to be fertile. The bats were found to exhibit a post-partum oestrus, which in nearly all cases (35/36) was fertile. The interval between the discovery of a new baby and the detection of a sperm-positive vaginal aspirate varied between 3-10 days, but most frequently was 3-6 days. Females which aborted non-term fetuses also had a post-partum oestrus with similar timing. These findings indicate that the short-tailed fruit bat, which is widely abundant in the lowland tropics of the New World, can be successfully maintained under controlled conditions, in cages of modest dimensions, for research purposes.

The evolutionary and developmental basis of parallel reduction in mammalian zeugopod elements.

Understanding the mechanisms by which parallel evolution occurs has the potential to clarify the complex relationship between evolution and development. In this study, we examine the role of development in the repeated reduction of zeugopod elements during mammalian evolution, a functionally important phenomenon enabling locomotor specialization. By completing a morphometric study (incorporating both analyses of variation and phylogenetics) of mammalian limbs, we are able to demonstrate an evolutionary trend toward width reduction in posterior zeugopod elements of the forelimbs and hindlimbs, the ulna and fibula, respectively. We also examine the developmental basis of limb reduction in three test cases, the bat Carollia perspicillata ulna and fibula and the mouse Mus musculus fibula. The most common pattern of reduction, that of reduced element width, was achieved via the same developmental process in both bat and mouse limbs (i.e., by a slower growth rate relative to other skeletal elements), suggesting that the parallel reduction of the posterior zeugopod element within mammals could have occurred primarily by the repeated evolution of the same developmental mechanism. However, our findings also suggest that the developmental mechanisms behind the parallel evolution of other, more taxon‐specific characteristics of limb reduction (i.e., element fusion) are not conserved.

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. Anat Rec, 2009.

Formation of reticulated endoderm, Reichert's membrane, and amniogenesis in blastocysts of captive-bred, short-tailed fruit bats, Carollia perspicillata

As part of an effort to develop the short‐tailed fruit bat (Carollia perspicillata) as a new animal model for the study of interstitial implantation and trophoblast‐uterine interactions, early embryogenesis was examined histologically and ultrastructurally in captive‐bred females at different intervals after the first appearance of spermatozoa in daily vaginal smears (day 1 postcoitum [p.c.]).

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.