Anatomy, ontogeny, and evolution of the archosaurian respiratory system: A case study on Alligator mississippiensis and Struthio camelus.

The avian lung is highly specialized and is both functionally and morphologically distinct from that of their closest extant relatives, the crocodilians. It is highly partitioned, with a unidirectionally ventilated and immobilized gas-exchanging lung, and functionally decoupled, compliant, poorly vascularized ventilatory air-sacs. To understand the evolutionary history of the archosaurian respiratory system, it is essential to determine which anatomical characteristics are shared between birds and crocodilians and the role these shared traits play in their respective respiratory biology. To begin to address this larger question, we examined the anatomy of the lung and bronchial tree of 10 American alligators (Alligator mississippiensis) and 11 ostriches (Struthio camelus) across an ontogenetic series using traditional and micro-computed tomography (µCT), three-dimensional (3D) digital models, and morphometry. Intraspecific variation and left to right asymmetry were present in certain aspects of the bronchial tree of both taxa but was particularly evident in the cardiac (medial) region of the lungs of alligators and the caudal aspect of the bronchial tree in both species. The cross-sectional area of the primary bronchus at the level of the major secondary airways and cross-sectional area of ostia scaled either isometrically or negatively allometrically in alligators and isometrically or positively allometrically in ostriches with respect to body mass. Of 15 lung metrics, five were significantly different between the alligator and ostrich, suggesting that these aspects of the lung are more interspecifically plastic in archosaurs. One metric, the distances between the carina and each of the major secondary airways, had minimal intraspecific or ontogenetic variation in both alligators and ostriches, and thus may be a conserved trait in both taxa. In contrast to previous descriptions, the 3D digital models and CT scan data demonstrate that the pulmonary diverticula pneumatize the axial skeleton of the ostrich directly from the gas-exchanging pulmonary tissues instead of the air sacs. Global and specific comparisons between the bronchial topography of the alligator and ostrich reveal multiple possible homologies, suggesting that certain structural aspects of the bronchial tree are likely conserved across Archosauria, and may have been present in the ancestral archosaurian lung.

miR-199 family contributes to regulation of sonic hedgehog expression during craniofacial development.

BACKGROUND: The frontonasal ectodermal zone (FEZ) is a signaling center that regulates patterned development of the upper jaw, and Sonic hedgehog (SHH) mediates FEZ activity. Induction of SHH expression in the FEZ results from SHH-dependent signals from the brain and neural crest cells. Given the role of miRNAs in modulating gene expression, we investigated the extent to which miRNAs regulate SHH expression and FEZ signaling. RESULTS: In the FEZ, the miR-199 family appears to be regulated by SHH-dependent signals from the brain; expression of this family increased from HH18 to HH22, and upon activation of SHH signaling in the brain. However, the miR-199 family is more broadly expressed in the mesenchyme of the frontonasal process and adjacent neuroepithelium. Downregulating the miR-199 genes expanded SHH expression in the FEZ, resulting in wider faces, while upregulating miR-199 genes resulted in decreased SHH expression and narrow faces. Hypoxia inducible factor 1 alpha (HIF1A) and mitogen-activated protein kinase kinase kinase 4 (MAP3K4) appear to be potential targets of miR-199b. Reduction of MAP3K4 altered beak development but increased apoptosis, while reducing HIF1A reduced expression of SHH in the FEZ and produced malformations independent of apoptosis. CONCLUSIONS: Our results demonstrate that this miRNA family appears to participate in regulating SHH expression in the FEZ; however, specific molecular mechanisms remain unknown.

Tiludronate and clodronate do not affect bone structure or remodeling kinetics over a 60 day randomized trial.

BACKGROUND: Tiludronate and clodronate are FDA-approved bisphosphonate drug therapies for navicular disease in horses. Although clinical studies have determined their ability to reduce lameness associated with skeletal disorders in horses, data regarding the effect on bone structure and remodeling is lacking. Additionally, due to off-label use of these drugs in young performance horses, effects on bone in young horses need to be investigated. Therefore, the purpose of this randomized, experimental pilot study was to determine the effect of tiludronate and clodronate on normal bone cells, structure and remodeling after 60 days in clinically normal, young horses. Additionally, the effect of clodronate on bone healing 60 days after an induced defect was investigated. RESULTS: All horses tolerated surgery well, with no post-surgery lameness and all acquired biopsies being adequate for analyses. Overall, tiludronate and clodronate did not significantly alter any bone structure or remodeling parameters, as evaluated by microCT and dynamic histomorphometry. Tiludronate did not extensively impact bone formation or resorption parameters as evaluated by static histomorphometry. Similarly, clodronate did not affect bone formation or resorption after 60 days. Sixty days post-defect, healing was minimally affected by clodronate. CONCLUSIONS: Tiludronate and clodronate do not appear to significantly impact bone tissue on a structural or cellular level using standard dose and administration schedules.

Assessment of tuber coxae bone biopsy in the standing horse.

OBJECTIVE: To describe a biopsy technique in standing horses with minimal morbidity that consistently provides a substantial bone biopsy with intact, undamaged architecture. STUDY DESIGN: Experimental, prospective study. ANIMALS: Ten Thoroughbred horses. METHODS: Biopsies were obtained from the tuber coxae of 10 sedated, standing horses using an oscillating saw. Bilateral biopsies, separated by 60 days, were evaluated with micro-computed tomography (microCT). The first biopsy was prepared for decalcified histology; the second for undecalcified histology. Both biopsies were evaluated qualitatively for histologic quality. RESULTS: The biopsy technique did not result in any significant complications, was well tolerated and all biopsies were of good histologic quality. CONCLUSION: Cortical and trabecular bone biopsies can be successfully collected from the tuber coxa using a simple technique that creates minimal morbidity and allows sequential samples to be collected. The biopsies were larger than those described previously, provided adequate bone for multiple histologic sections, and had intact, undamaged architecture on examination with microCT and light microscopy.

Dosage-dependent effects of FGFR2W290R mutation on craniofacial shape and cellular dynamics of the basicranial synchondroses.

Craniosynostosis is a common yet complex birth defect, characterized by premature fusion of the cranial sutures that can be syndromic or nonsyndromic. With over 180 syndromic associations, reaching genetic diagnoses and understanding variations in underlying cellular mechanisms remains a challenge. Variants of FGFR2 are highly associated with craniosynostosis and warrant further investigation. Using the missense mutation FGFR2W290R , an effective mouse model of Crouzon syndrome, craniofacial features were analyzed using geometric morphometrics across developmental time (E10.5-adulthood, n = 665 total). Given the interrelationship between the cranial vault and basicranium in craniosynostosis patients, the basicranium and synchondroses were analyzed in perinates. Embryonic time points showed minimal significant shape differences. However, hetero- and homozygous mutant perinates and adults showed significant differences in shape and size of the cranial vault, face, and basicranium, which were associated with cranial doming and shortening of the basicranium and skull. Although there were also significant shape and size differences associated with the basicranial bones and clear reductions in basicranial ossification in cleared whole-mount samples, there were no significant alterations in chondrocyte cell shape, size, or orientation along the spheno-occipital synchondrosis. Finally, shape differences in the cranial vault and basicranium were interrelated at perinatal stages. These results point toward the possibility that facial shape phenotypes in craniosynostosis may result in part from pleiotropic effects of the causative mutations rather than only from the secondary consequences of the sutural defects, indicating a novel direction of research that may shed light on the etiology of the broad changes in craniofacial morphology observed in craniosynostosis syndromes.

MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses.

Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N = 10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase ( www.facebase.org , https://doi.org/10.25550/3-HXMC ) and GitHub ( https://github.com/jaydevine/MusMorph ).

Anatomical Variation of the Tarsus in Common Inbred Mouse Strains.

Rodent models are used for a variety of orthopedic research applications; however, anatomy references include mostly artistic representations. Advanced imaging techniques, including micro-computed tomography (microCT), can provide more accurate representations of subtle anatomical characteristics. A recent microCT atlas of laboratory mouse (Mus musculus) anatomy depicts the central and tarsal bone III (T3) as a single bone, differing from previous references. Fusion of tarsal bones is generally characterized as pathological secondary to mutations associated with growth factors, and normal variation has not been documented in the mouse tarsus. Therefore, it is unclear if this fusion is a normal or a pathological characteristic. The aim of this study is to characterize the tarsus of the laboratory mouse and compare it to the rat and selected outgroup species (i.e., white-footed mouse) via microCT and histology to determine if the central and T3 are separate or fused into a single bone. Laboratory mice (C57/Bl6 [n = 17] and BalbC [n = 2]) and rats (n = 5) were scanned with microCT. A representative laboratory mouse from each strain was evaluated histologically via serial sagittal sections through the mid-tarsus. General pedal anatomy was similar between all species; however, the central and T3 bones were fused in all laboratory mice but not the rat or white-footed mouse. A band of hyaline cartilage was identified within the fused bone of the laboratory mice. We conclude that the fusion found is a normal characteristic in laboratory mice, but timing of the fusion remains ambiguous. Anat Rec, 300:450-459, 2017. © 2016 Wiley Periodicals, Inc.

Gait Changes Vary among Horses with Naturally Occurring Osteoarthritis Following Intra-articular Administration of Autologous Platelet-Rich Plasma.

Mechanisms to reduce lameness associated with osteoarthritis (OA) are vital to equine health and performance. This study was designed to quantify response to autologous, intra-articular platelet-rich plasma (PRP) in horses with OA. Kinetic gait analysis was performed on 12 horses with unilateral forelimb lameness and OA in the same limb before and after intra-articular anesthesia (IAA). Radiographs and kinetic data were obtained before and 6 and 16 weeks after PRP administration to same joint, 4 weeks after IAA. Statistical evaluations included filtration effect on platelet concentration, relationship between kinetic variable changes after IAA versus PRP in the affected limb, and associations between response to PRP and response to IAA, platelet concentration, and radiographic OA. A positive response to IAA or PRP was defined as ≥5% improvement in peak vertical force, vertical impulse, or breaking impulse of the affected limb. Out of 10 horses that responded to IAA, 3 responded to PRP at both time points and 4 responded at one. Of the two horses that did not respond to IAA, one responded to PRP at both time points. Filtration increased platelet concentration significantly. The relationship between kinetic variable alterations of the affected limb after IAA and PRP was not significant, and response to PRP was not associated with response to IAA, platelet concentration, or radiographic OA. Changes in kinetic variables following IAA in joints with naturally occurring OA provide a custom standard to assess intra-articular therapy. Kinetic gait changes after intra-articular PRP are variable in horses with moderate to severe forelimb OA.