Whitney A. Bullock

Understanding the geographic distribution of genetic diversity of imperiled species across all life history stages, and identifying the factors that shape those distributions, are key to maintaining long-term genetic diversity and the health of populations. This knowledge is particularly important for highly mobile marine organisms, whose extensive movements can obscure patterns of population structure. We substantially expand the genetic dataset for the critically endangered hawksbill turtle, Eretmochelys imbricata, in the West Atlantic, focusing on the southwest Caribbean. Our dataset comprises nearly 3,000 mtDNA control region sequences (740 bp) assigned to 60 haplotypes: 41 found in rookeries and 47 in foraging grounds, including 17 orphan haplotypes. The Panama metapopulation represents a major center of genetic diversity for hawksbills, with one of the highest recorded diversity values for the species (h = 0.749, π = 0.00782), nine endemic haplotypes, and four additional haplotypes that are endemic to the Southwest Caribbean. Rarefaction analyses indicate that a sample size of at least 100 is necessary to reveal true haplotype richness at most rookeries. Many-to-many mixed stock analyses, which incorporated rookery size and distance priors for 19 rookeries and 15 developmental foraging grounds, suggest that hatchlings from rookeries in the southwest Caribbean are distributed among multiple, widely-spaced foraging grounds across the West Atlantic. These results support a groups-to-soups analogy, in which genetic variability across foraging grounds represents a continuum of genetic diversity that can best be explained by a “current conveyor” model. The dataset shows that philopatry in hawksbills is not absolute, resulting in true biological dispersal and geneflow on local, regional, and ocean-basin scales, likely facilitated by dispersion during the epipelagic stage. The important contribution of oceanographic features to genetic variation at rookeries and foraging grounds is corroborated, as is the concept of oceanographic “dispersal shadows” that limit geneflow between rookeries. This study reinforces the assertion that all range states share responsibility for the recovery of the hawksbill, because foraging grounds, that are often at distant locations, are the source of future generations of reproductive adults. We also document significant movement by hawksbills between regional management units (RMUs) 29 and 30 in the West Atlantic. The Spanish version of the Abstract is available in Supplementary File 1.

The Cre/Lox system has revolutionized the ability of biomedical researchers to ask very specific questions about the function of individual genes in specific cell types at specific times during development and/or disease progression in a variety of animal models. This is true in the skeletal biology field, and numerous Cre driver lines have been created to foster conditional gene manipulation in specific subpopulations of bone cells. However, as our ability to scrutinize these models increases, an increasing number of issues have been identified with most driver lines. All existing skeletal Cre mouse models exhibit problems in one or more of the following three areas: (1) cell type specificity-avoiding Cre expression in unintended cell types; (2) Cre inducibility-improving the dynamic range for Cre in inducible models (negligible Cre activity before induction and high Cre activity after induction); and (3) Cre toxicity-reducing the unwanted biological effects of Cre (beyond loxP recombination) on cellular processes and tissue health. These issues are hampering progress in understanding the biology of skeletal disease and aging, and consequently, identification of reliable therapeutic opportunities. Skeletal Cre models have not advanced technologically in decades despite the availability of improved tools, including multi-promoter-driven expression of permissive or fragmented recombinases, new dimerization systems, and alternative forms of recombinases and DNA sequence targets. We review the current state of skeletal Cre driver lines, and highlight some of the successes, failures, and opportunities to improve fidelity in the skeleton, based on successes pioneered in other areas of biomedical science.

Sclerostin antibody (romosozumab) was recently approved for clinical use in the United States to treat osteoporosis. We and others have explored Wnt-based combination therapy to disproportionately improve the anabolic effects of sclerostin inhibition, including cotreatment with sclerostin antibody (Scl-mAb) and Dkk1 antibody (Dkk1-mAb). To determine the optimal ratio of Scl-mAb and Dkk1-mAb for producing maximal anabolic action, the proportion of Scl-mAb and Dkk1-mAb were systematically varied while holding the total antibody dose constant. A 3:1 mixture of Scl-mAb to Dkk1-mAb produced two to three times as much cancellous bone mass as an equivalent dose of Scl-mAb alone. Further, a 75% reduction in the dose of the 3:1 mixture was equally efficacious to a full dose of Scl-mAb in the distal femur metaphysis. The Scl-mAb/Dkk1-mAb combination approach was highly efficacious in the cancellous bone mass, but the cortical compartment was much more subtly affected. The osteoanabolic effects of Wnt pathway targeting can be made more efficient if multiple antagonists are simultaneously targeted. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Keywords: Akt; osteoporosis; Pten; Wnt; [beta]-catenin Skeletal homeostasis is sensitive to perturbations in Wnt signaling. Beyond its role in the bone, Wnt is a major target for pharmaceutical inhibition in a wide range of diseases, most notably cancers. Numerous clinical trials for Wnt-based candidates are currently underway, and Wnt inhibitors will likely soon be approved for clinical use. Given the bone-suppressive effects accompanying Wnt inhibition, there is a need to expose alternate pathways/molecules that can be targeted to counter the deleterious effects of Wnt inhibition on bone properties. Activation of the Pi3k/Akt pathway via Pten deletion is one possible osteoanabolic pathway to exploit. We investigated whether the osteopenic effects of [beta]-catenin deletion from bone cells could be rescued by Pten deletion in the same cells. Mice carrying floxed alleles for Pten and [beta]-catenin were bred to Dmp1-Cre mice to delete Pten alone, [beta]-catenin alone, or both genes from the late-stage osteoblast/osteocyte population. The mice were assessed for bone mass, density, strength, and formation parameters to evaluate the potential rescue effect of Pten deletion in Wnt-impaired mice. Pten deletion resulted in high bone mass and [beta]-catenin deletion resulted in low bone mass. Compound mutants had bone properties similar to [beta]-catenin mutant mice, or surprisingly in some assays, were further compromised beyond [beta]-catenin mutants. Pten inhibition, or one of its downstream nodes, is unlikely to protect against the bone-wasting effects of Wnt/[beta]cat inhibition. Other avenues for preserving bone mass in the presence of Wnt inhibition should be explored to alleviate the skeletal side effects of Wnt inhibitor-based therapies. CAPTION(S): Supporting information Supporting information Byline: Kyung-Eun Lim, April M. Hoggatt, Whitney A. Bullock, Daniel J. Horan, Hiroki Yokota, Frederick M. Pavalko, Alexander G. Robling

This chapter considers the types of cells and forces likely to impact bone formation/remodeling. It examines the cellular and molecular mechanisms regulating the biological responses of cells to those forces. Cells within bone detect, coordinate, and mediate mechanical loading on the skeleton. The cells most likely to function as the “mechanosensors” in bone are the osteocytes. Chemotransport also affects the response of bone cells to fluid flow independent of mechanical signal transduction. Bone cell mechanoreceptors can be classified into broad categories: integrins; G‐protein coupled receptors/receptor tyrosine kinases; and ion channels/connexin hemichannels. Ion channels sensitive to mechanical stimulation are also found in osteoblastic cells. Detection of a molecular response to mechanical stimulation does not prove that it is important for mechanotransduction in bone. The Wnt pathway has been identified as one of the most important mediators of bone cell mechanotransduction.

The WNT pathway has become an attractive target for skeletal therapies. High-bone-mass phenotypes in patients with loss-of-function mutations in the LRP5/6 inhibitor Sost (sclerosteosis), or in its downstream enhancer region (van Buchem disease), highlight the utility of targeting Sost/sclerostin to improve bone properties. Sclerostin-neutralizing antibody is highly osteoanabolic in animal models and in human clinical trials, but antibody-based inhibition of another potent LRP5/6 antagonist, Dkk1, is largely inefficacious for building bone in the unperturbed adult skeleton. Here, we show that conditional deletion of Dkk1 from bone also has negligible effects on bone mass. Dkk1 inhibition increases Sost expression, suggesting a potential compensatory mechanism that might explain why Dkk1 suppression lacks anabolic action. To test this concept, we deleted Sost from osteocytes in, or administered sclerostin neutralizing antibody to, mice with a Dkk1-deficient skeleton. A robust anabolic response to Dkk1 deletion was manifest only when Sost/sclerostin was impaired. Whole-body DXA scans, μCT measurements of the femur and spine, histomorphometric measures of femoral bone formation rates, and biomechanical properties of whole bones confirmed the anabolic potential of Dkk1 inhibition in the absence of sclerostin. Further, combined administration of sclerostin and Dkk1 antibody in WT mice produced a synergistic effect on bone gain that greatly exceeded individual or additive effects of the therapies, confirming the therapeutic potential of inhibiting multiple WNT antagonists for skeletal health. In conclusion, the osteoanabolic effects of Dkk1 inhibition can be realized if sclerostin upregulation is prevented. Anabolic therapies for patients with low bone mass might benefit from a strategy that accounts for the compensatory milieu of WNT inhibitors in bone tissue. Genetic disruption of a compensatory WNT inhibitor expression reveals a context‐dependent, highly osteoanabolic role for DKK1 inhibition in the skeleton.

The WNT-signaling pathway is involved in cellular and tissue functions that control such diverse processes as body axis patterning, cellular proliferation, differentiation, and life span. The long list of molecules that can participate or modify WNT signaling makes this pathway one of the most complex in cell biology. In bone tissues, WNT signaling is required for proper skeletal development, and human mutations in various components of the cascade revealed insights into pharmacologic targeting that can be harnessed to improve skeletal health. In particular, mutations in genes that code for the WNT-signaling inhibitor sclerostin or the WNT coreceptor lipoprotein receptor-related protein 5 have highlighted the potential therapeutic value of recapitulating those effects in patients with low bone mass. A constant challenge in this area is selectively modifying WNT components in the tissue of interest, as WNT has manifold effects in nearly every tissue.

Sclerostin (Sost) is a negative regulator of bone formation that acts upon the Wnt signaling pathway. Sost is mechanically regulated at both mRNA and protein level such that loading represses and unloading enhances Sost expression, in osteocytes and in circulation. The non-coding evolutionarily conserved enhancer ECR5 has been previously reported as a transcriptional regulatory element required for modulating Sost expression in osteocytes. Here we explored the mechanisms by which ECR5, or several other putative transcriptional enhancers regulate Sost expression, in response to mechanical stimulation. We found that in vivo ulna loading is equally osteoanabolic in wildtype and Sost−/− mice, although Sost is required for proper distribution of load-induced bone formation to regions of high strain. Using Luciferase reporters carrying the ECR5 non-coding enhancer and heterologous or homologous hSOST promoters, we found that ECR5 is mechanosensitive in vitro and that ECR5-driven Luciferase activity decreases in osteoblasts exposed to oscillatory fluid flow. Yet, ECR5−/− mice showed similar magnitude of load-induced bone formation and similar periosteal distribution of bone formation to high-strain regions compared to wildtype mice. Further, we found that in contrast to Sost−/− mice, which are resistant to disuse-induced bone loss, ECR5−/− mice lose bone upon unloading to a degree similar to wildtype control mice. ECR5 deletion did not abrogate positive effects of unloading on Sost, suggesting that additional transcriptional regulators and regulatory elements contribute to load-induced regulation of Sost. •Loading is osteoanabolic in WT and Sost−/− mice; Sost is required for distribution of bone formation to high strain areas•ECR5 is mechanosensitive in vitro and that ECR5-driven Luciferase activity decreases in osteoblasts exposed to oscillatory fluid flow•ECR5−/− mice show similar load-induced bone formation distributed to high-strain regions compared to WT mice•Sost−/− mice, but not ECR5−/− mice, are resistant to disuse-induced bone loss