Atoh8 acts as a regulator of chondrocyte proliferation and differentiation in endochondral bones

Abstract

24 Atonal homolog 8 (Atoh8) is a transcription factor of the basic helix-loop-helix (bHLH) protein 25 family, which is expressed in the cartilaginous elements of endochondral bones. To analyze 26 its function during chondrogenesis we deleted Atoh8 in mice using a chondrocyte-27 (Atoh8 flox/flox ;Col2a1-Cre) and a germline-(Atoh8 flox/flox ;Prx1-Cre female ) specific Cre allele. In 28 both strains, Atoh8 deletion leads to a reduced skeletal size of the axial and appendicular 29 bones, but the stages of phenotypic manifestations differ. While we observed obviously 30 shortened bones in Atoh8 flox/flox ;Col2a1-Cre mice only postnatally, the bones of 31 Atoh8 flox/flox ;Prx1-Cre female mice are characterized by a reduced bone length already at 32 prenatal stages. Detailed histological and molecular investigations revealed reduced zones 33 of proliferating and hypertrophic chondrocytes. In addition, Atoh8 deletion identified Atoh8 as 34 a positive regulator of chondrocyte proliferation. As increased Atoh8 expression is found in 35 the region of prehypertrophic chondrocytes where the expression of Ihh, a main regulator of 36 chondrocyte proliferation and differentiation, is induced, we investigated a potential 37 interaction of Atoh8 function and Ihh signaling. By activating Ihh signaling with 38 Purmorphamine we demonstrate that Atoh8 regulates chondrocyte proliferation in parallel or 39 downstream of Ihh signaling while it acts on the onset of hypertrophy upstream of Ihh likely 40 by modulating Ihh expression levels.41 42 Introduction 43 During endochondral ossification, bones are formed by a multistep process, which includes 44 the formation of a cartilage template of the later skeletal element and its subsequent 45 replacement by bone. The cartilage anlagen originate in mesenchymal cells, which condense 46 and differentiate into chondrocytes. These chondrocytes proliferate and express the 47 extracellular matrix protein Collagen type 2 (Col2) [1]. Two subtypes of proliferating 48 chondrocytes can be distinguished: round, slow proliferating cells at the end of the cartilage 49 elements (round/resting chondrocytes) and flat, highly proliferating cells organized in 3 50 columns towards the hypertrophic region (columnar chondrocytes). When the cartilage 51 anlagen reach a critical size, proliferating chondrocytes in their center exit the cell cycle and 52 differentiate into Indian hedgehog (Ihh) producing, prehypertrophic [2, 3] and, subsequently, 53 Collagen type 10 (Col10) expressing, hypertrophic chondrocytes [4]. Eventually, blood 54 vessels invade the zone of hypertrophic chondrocytes and the hypertrophic cells are 55 replaced by bone and bone marrow. Postnatally, secondary ossification centers (SOC) are 56 formed at the ends of the endochondral long bones. Between the two regions of ossification, 57 parts of the embryonic cartilage, the so-called growth plates, remain to organize longitudinal 58 growth after birth [5]. As longitudinal bone growth depends on the balance between 59 chondrocyte proliferation and hyp...

BMP-induced Atoh8 attenuates osteoclastogenesis by suppressing Runx2 transcriptional activity and reducing the Rankl/Opg expression ratio in osteoblasts

“…However, no signals were observed in osteoblasts ( Fig. 2a, b, asterisks), and prehypertrophic chondrocytes exhibited substantial signal spots of Atoh8, as reported previously 34 (Fig. 2b, arrowhead).…”

“…33,34,[44][45][46] These Atoh8-KO mice are viable and healthy and obtained at the expected Mendelian ratio. 33,34,45,46 However, there is no report on the bone phenotype of adult Atoh8-deficient mice. In this study, we found ubiquitous expression of Atoh8 in tissues from adult mice, with high levels in the lung, stomach, liver, spleen, and skin (Fig.…”

“…Recently, Schroeder et al reported that Atoh8 is expressed in proliferating and hypertrophic chondrocytes, especially prehypertrophic chondrocytes, but not in osteoblasts of embryonic and developing mouse bone. 34 Cartilage-specific Atoh8-KO mice showed delayed chondrocyte proliferation and differentiation in the short bones. 34 Therefore, the decreased bone length in our Atoh8-KO adult mice might be the result of cartilage defects.…”

“…34 Cartilage-specific Atoh8-KO mice showed delayed chondrocyte proliferation and differentiation in the short bones. 34 Therefore, the decreased bone length in our Atoh8-KO adult mice might be the result of cartilage defects. However, the adult bone density appeared to be unaffected in cartilage-specific mutant mice.…”

“…However, the adult bone density appeared to be unaffected in cartilage-specific mutant mice. 34 Atoh8 is expressed in a subset of embryonic muscle cells in the dermomyotome and myotome of chicken embryos 47 and in human skeletal muscle during regeneration, 48 and it plays roles in muscle differentiation and regeneration, whereas Atoh8 is downregulated during myogenic maturation of C2C12 cells in vitro. 47 We also detected Atoh8 expression in embryonic muscle ( Fig.…”