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

Date Published: August 26, 2019

Publisher: Public Library of Science

Author(s): Nadine Schroeder, Manuela Wuelling, Daniel Hoffmann, Beate Brand-Saberi, Andrea Vortkamp, Andre van Wijnen.


Atonal homolog 8 (Atoh8) is a transcription factor of the basic helix-loop-helix (bHLH) protein family, which is expressed in the cartilaginous elements of endochondral bones. To analyze its function during chondrogenesis we deleted Atoh8 in mice using a chondrocyte- (Atoh8flox/flox;Col2a1-Cre) and a germline- (Atoh8flox/flox;Prx1-Crefemale) specific Cre allele. In both strains, Atoh8 deletion leads to a reduced skeletal size of the axial and appendicular bones, but the stages of phenotypic manifestations differ. While we observed obviously shortened bones in Atoh8flox/flox;Col2a1-Cre mice only postnatally, the bones of Atoh8flox/flox;Prx1-Crefemale mice are characterized by a reduced bone length already at prenatal stages. Detailed histological and molecular investigations revealed reduced zones of proliferating and hypertrophic chondrocytes. In addition, Atoh8 deletion identified Atoh8 as a positive regulator of chondrocyte proliferation. As increased Atoh8 expression is found in the region of prehypertrophic chondrocytes where the expression of Ihh, a main regulator of chondrocyte proliferation and differentiation, is induced, we investigated a potential interaction of Atoh8 function and Ihh signaling. By activating Ihh signaling with Purmorphamine we demonstrate that Atoh8 regulates chondrocyte proliferation in parallel or downstream of Ihh signaling while it acts on the onset of hypertrophy upstream of Ihh likely by modulating Ihh expression levels.

Partial Text

During endochondral ossification, bones are formed by a multistep process, which includes the formation of a cartilage template of the later skeletal element and its subsequent replacement by bone. The cartilage anlagen originate in mesenchymal cells, which condense and differentiate into chondrocytes. These chondrocytes proliferate and express the extracellular matrix protein Collagen type 2 (Col2) [1]. Two subtypes of proliferating chondrocytes can be distinguished: round, slow proliferating cells at the end of the cartilage elements (round/resting chondrocytes) and flat, highly proliferating cells organized in columns towards the hypertrophic region (columnar chondrocytes). When the cartilage anlagen reach a critical size, proliferating chondrocytes in their center exit the cell cycle and differentiate into Indian hedgehog (Ihh) producing, prehypertrophic [2, 3] and, subsequently, Collagen type 10 (Col10) expressing, hypertrophic chondrocytes [4]. Eventually, blood vessels invade the zone of hypertrophic chondrocytes and the hypertrophic cells are replaced by bone and bone marrow. Postnatally, secondary ossification centers (SOC) are formed at the ends of the endochondral long bones. Between the two regions of ossification, parts of the embryonic cartilage, the so-called growth plates, remain to organize longitudinal growth after birth [5]. As longitudinal bone growth depends on the balance between chondrocyte proliferation and hypertrophic differentiation, both processes are tightly regulated. Although many regulators have been identified, clarifying the detailed molecular mechanisms is still in progress.

Members of the Atonal gene family, e.g. Atoh1 and Atoh7, have been associated with the development of sensory neurons and are typically classified as proneuronal genes [35–37]. Here we have investigated the role of Atoh8, a less well characterized member of the protein family, which is widely expressed in the developing embryo [8, 9, 14, 38, 39].