The teleost intervertebral region acts as a growth center of the centrum: In vivo visualization of osteoblasts and their progenitors in transgenic fish

Abstract

The vertebral column is a defined feature of vertebrates. In birds and mammals, the sclerotome yields cartilaginous material for the vertebral column. In teleosts, however, it remains uncertain whether the sclerotome participates in vertebral column formation. To investigate osteoblast development in the teleost, we established transgenic systems that allow in vivo observation of osteoblasts and their progenitors marked by fluorescence of DsRed and enhanced green fluorescent protein (EGFP), respectively. In twist-EGFP transgenic medaka, EGFP-positive cells first appeared in the ventromedial portion of respective somites corresponding to the sclerotome, migrated dorsally around the notochord, and concentrated in the intervertebral regions. Ultrastructural analysis of the intervertebral regions revealed that some of these cells were directly located on the osteoidal surface of the perichordal centrum, and enriched with rough endoplasmic reticulum in their cytoplasm. By using the double transgenic medaka of twist-EGFP and osteocalcin-DsRed, we clarified that the EGFP-positive cells in the intervertebral region differentiated into mature osteoblasts expressing the DsRed. In vivo bone labeling in fact confirmed active matrix formation and mineralization of the perichordal centrum exclusively in the intervertebral region of zebrafish larvae as well as medaka larvae. These findings strongly suggest that the teleost intervertebral region acts as a growth center of the perichordal centrum, where the sclerotome-derived cells differentiate into osteoblasts. Developmental Dynamics 236:3031-3046, 2007.

Osterix‐mCherry transgenic medaka for in vivo imaging of bone formation

“…These two cell types most likely correspond to the osx-mCherry-positive cells in our transgenic lines. We did not observe mCherry expression between two centrae, as was described for the two additional twist-positive cell populations (II, III), which do not become osteocalcin-positive (Inohaya et al, 2007). This shows that osx-mCherry expression enables to distinguish early between twist-positive cells that differentiate into osteoblasts and those that do not.…”

“…We have chosen the medaka for this analysis, as bone formation is more rapid in this model compared with zebrafish. Appearance of first alizarin red-positive bone matrix in the vertebrae, for example, is detected 5 days after fertilization in medaka (Inohaya et al, 2007), while the same structures become visible only at 9 days in zebrafish (Gavaia et al, 2006). Osterix is a key regulator driving the differentiation of mesenchymal progenitor cells into the osteoblast lineage in mammals (Nakashima et al, 2002).…”

“…Two transgenic twist-eGFP and osteocalcin-DsRed medaka lines have recently been reported for the analysis of bone formation (Inohaya et al, 2007). Unfortunately, these lines do not cover the intermediate stages when twist-positive mesenchymal progenitors convert into osteocalcin-expressing mature osteoblasts.…”

“…mCherry fluorescence is first detectable in the neural arches, as shown by life imaging, and only later at the edges of the centrae with additional single cells also appearing in the middle of the centrae. Inohaya et al (2007) suggested that twist-eGFP-expressing cells differentiate into four distinct cell types (I-IV). Two of these cell types (I, IV) are located at the outer surface of the edges and the middle part of the centrae and later become osteocalcin-positive.…”

“…For bone research, twist-eGFP and osteocalcin-DsRed transgenic medaka lines have recently been established. These lines contributed significantly to our understanding of the dynamic processes during early vertebrae formation (Inohaya et al, 2007). Twist expression is a marker for mesenchymal cells, which differentiate into several cell types, including adipocytes, chondrocytes, and osteoblasts (Komori, 2006).…”