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1. Differential Impact of Hoxa11 and Hoxd11 on Pisiform Ossification and Epiphysis Formation

   Graziano, Timothy; Sweeney, Brendan; Kjosness, Kelsey M; Kistler, Sabrina; Schuetz, Emily; Thompson, Ryan; Reno, Philip (May 2021, The FASEB journal)

   null (Ed.)

   Hox genes are key developmental patterning genes that impact segmental identity and skeletal patterning. Hox11 genes are known to impact wrist and ankle development and are expressed around the developing pisiform and calcaneus. These paralogous bones in the wrist and ankle are the only carpal and tarsal to form a growth plate in mammals, although humans have lost this growth plate and the associated primary ossification center in the pisiform. Loss-of-function mutations to Hoxa11 and Hoxd11 result in pisiform truncation and appear to also cause at least some disorganization of the growth plate cartilage; however, little is known about the nature of this disorganization or if ossification timing is impacted by Hox11 genes. The present study investigates the role of Hoxa11 and Hoxd11 in pisiform growth plate organization and ossification timing. We conducted histological analysis of the pisiform growth plate in juvenile mice with Hoxa11 and Hoxd11 loss-of-function mutations and compared them to ossification patterns observed in age- and genotype-matched whole-mount specimens that were cleared and stained with Alizarin red and Alcian blue to visualize bone and cartilage, respectively. Histological analysis reveals a dosage-dependent impact of Hox11 mutations on pisiform ossification to both the primary and secondary ossification center. As the number of Hox11 mutation alleles increase, less bone is present in the early primary ossification center compared to age-matched specimens. In specimens with three loss-of-function alleles, no trabeculae or growth plate organization are visible at P9, when both are well established in wild type specimens. Cleared and stained specimens indicate a possible pseudo epiphysis forming with Hoxd11 mutation, while Hoxa11 knockout specimens have not formed any visible epiphysis or calcification by P9. These results indicate that ossification timing and patterns, along with growth plate organization, are affected by Hox11 mutations during early pisiform ossification. Furthermore, Hoxa11 and Hoxd11 alter the pisiform epiphysis differently, suggesting that each plays a specific role in formation of the ossification front and epiphysis ossification either by influencing timing, ossification progression, or both. Further work is needed to understand the mechanisms by which Hox genes impact ossification patterns and timing, as well as the differential roles of Hoxa11 and Hoxd11 in growth plate organization and epiphysis formation. 

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2. Morphological Changes in Calcanei of Mice with Hoxa11 and Hoxd11 loss-of-function mutations

   Goldman, Samuel; Bhavsar, Avani; Kjosness, Kelsey M; Kistler, Sabrina; Schuetz, Emily; Stephens, Nicholas; Ryan, Timothy M; Reno, Philip L (May 2021, The FASEB journal)

   null (Ed.)

   Hox11 genes are expressed around the developing wrist and ankle and are known to substantially impact pisiform shape and length in mice. The calcaneus is a tarsal bone that is paralogous to the pisiform in the wrist, but previous descriptions of mice with Hox11 mutations have suggested that little morphological change takes place unless Hoxa11 and Hoxd11 are both knocked out, at which point the calcaneus fails to form. However, these studies primarily relied on cleared and stained whole-mount specimens which limit resolution of morphological features. This study seeks to determine if calcaneus morphology is altered by three or fewer loss-of-function Hoxa11 and Hoxd11 alleles. We obtained microCT scans of 8 week old mice and compared calcaneus morphology in wild type mice and mice with one, two, and three Hoxa11 and Hoxd11 loss-of-function alleles. We used auto3dgm to conduct a 3D geometric morphometric analysis of shape variation using surface semi-landmarks. Principle components (PC) analysis indicates that calcaneus morphology is altered in mice with Hoxa11 and Hoxd11 loss-of-function mutations. PC1 accounts for 35.4% of shape variation and results from changes to the width and height of the calcaneal neck and shape of peroneal tubercle/process. PC2 accounts for 11.9% of shape variation and results from changes to the width of the calcaneal tuberosity and height of the posterior talar facet. Most specimens with either combination of three out of four Hoxa11 and Hoxd11 loss-of-function alleles cluster together. The other genotypes form a gradient of morphological change with WT, Hoxd11 heterozygotes, and Hoxd11 knockouts being most similar to each other and Hoxa11 heterozygotes, Hoxa11 knockouts, and heterozygotes for both genes being most similar to each other. These results suggest that Hox11 loss-of-function mutations result in altered calcaneus morphology and Hoxa11 and Hoxd11 loss-of-function mutations alter the shape of the calcaneus in different ways when fewer than three alleles are knocked out. 

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3. Ossification pattern of the unusual pisiform in two‐toed ( Choloepus ) and three‐toed sloths ( Bradypus )

   https://doi.org/10.1002/ar.24832  

   Gavazzi, Lia_M; Kjosness, Kelsey_M; Reno, Philip_L (November 2021, The Anatomical Record)

   Abstract Two‐toed (Choloepus sp.) and three‐toed (Bradypus sp.) sloths possess short, rounded pisiforms that are rare among mammals and differ from other members of Xenarthra like the giant anteater (Myrmecophaga tridactyla) which retain elongated, rod‐like pisiforms in common with most mammals. Using photographs, radiographs, and μCT, we assessed ossification patterns in the pisiform and the paralogous tarsal, the calcaneus, for two‐toed sloths, three‐toed sloths, and giant anteaters to determine the process by which pisiform reduction occurs in sloths and compare it to other previously studied examples of pisiform reduction in humans and orangutans. Both extant sloth genera achieve pisiform reduction through the loss of a secondary ossification center and the likely disruption of the associated growth plate based on an unusually porous subchondral surface. This represents a third unique mechanism of pisiform reduction among mammals, along with primary ossification center loss in humans and retention of two ossification centers with likely reduced growth periods in orangutans. Given the remarkable similarities between two‐toed and three‐toed sloth pisiform ossification patterns and the presence of pisiform reduction in fossil sloths, extant sloth pisiform morphology does not appear to represent a recent convergent adaptation to suspensory locomotion, but instead is likely to be an ancestral trait of Folivora that emerged early in the radiation of extant and fossil sloths. 

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4. The non-canonical Wnt receptor Ror2 is required for cartilage cell polarity and morphogenesis of the craniofacial skeleton in zebrafish

   https://doi.org/10.1242/dev.201273  

   Dranow, Daniel B.; Le Pabic, Pierre; Schilling, Thomas F. (April 2023, Development)

   ABSTRACT Non-canonical/β-catenin-independent Wnt signaling plays crucial roles in tissue/cell polarity in epithelia, but its functions have been less well studied in mesenchymal tissues, such as the skeleton. Mutations in non-canonical Wnt signaling pathway genes cause human skeletal diseases such as Robinow syndrome and Brachydactyly Type B1, which disrupt bone growth throughout the endochondral skeleton. Ror2 is one of several non-canonical Wnt receptor/co-receptors. Here, we show that ror2−/− mutant zebrafish have craniofacial skeletal defects, including disruptions of chondrocyte polarity. ror1−/− mutants appear to be phenotypically wild type, but loss of both ror1 and ror2 leads to more severe cartilage defects, indicating partial redundancy. Skeletal defects in ror1/2 double mutants resemble those of wnt5b−/− mutants, suggesting that Wnt5b is the primary Ror ligand in zebrafish. Surprisingly, the proline-rich domain of Ror2, but not its kinase domain, is required to rescue its function in mosaic transgenic experiments in ror2−/− mutants. These results suggest that endochondral bone defects in ROR-related human syndromes reflect defects in cartilage polarity and morphogenesis. 

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5. Zebrafish endochondral growth zones as they relate to human bone size, shape and disease

   https://doi.org/10.3389/fendo.2022.1060187  

   Le Pabic, Pierre; Dranow, Daniel B.; Hoyle, Diego J.; Schilling, Thomas F. (December 2022, Frontiers in Endocrinology)

   Research on the genetic mechanisms underlying human skeletal development and disease have largely relied on studies in mice. However, recently the zebrafish has emerged as a popular model for skeletal research. Despite anatomical differences such as a lack of long bones in their limbs and no hematopoietic bone marrow, both the cell types in cartilage and bone as well as the genetic pathways that regulate their development are remarkably conserved between teleost fish and humans. Here we review recent studies that highlight this conservation, focusing specifically on the cartilaginous growth zones (GZs) of endochondral bones. GZs can be unidirectional such as the growth plates (GPs) of long bones in tetrapod limbs or bidirectional, such as in the synchondroses of the mammalian skull base. In addition to endochondral growth, GZs play key roles in cartilage maturation and replacement by bone. Recent studies in zebrafish suggest key roles for cartilage polarity in GZ function, surprisingly early establishment of signaling systems that regulate cartilage during embryonic development, and important roles for cartilage proliferation rather than hypertrophy in bone size. Despite anatomical differences, there are now many zebrafish models for human skeletal disorders including mutations in genes that cause defects in cartilage associated with endochondral GZs. These point to conserved developmental mechanisms, some of which operate both in cranial GZs and limb GPs, as well as others that act earlier or in parallel to known GP regulators. Experimental advantages of zebrafish for genetic screens, high resolution live imaging and drug screens, set the stage for many novel insights into causes and potential therapies for human endochondral bone diseases. 

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