Insertional mutation in the Golgb1 gene is associated with osteochondrodysplasia and systemic edema in the OCD rat

Abstract

Homozygous rats (ocd/ocd) of a mutant inbred strain, OCD (osteochondrodysplasia), show osteochondrodysplasia, systemic edema, cleft palate, protruding tongue, disproportionate dwarfism, and lethality immediately after birth. Their epiphyses show decreased levels of glycosaminoglycans and weak staining for extracellular matrix proteins. The epiphyseal chondrocytes have large vesicles and expanded endoplasmic reticulum and Golgi apparatus. These phenotypic features are inherited in an autosomal recessive manner, and the ocd locus responsible for these phenotypes has been mapped close to D11Mgh3 on rat chromosome 11. In the present study, we characterized the embryonic pathogenesis of ocd/ocd rats and identified the mutant gene.

Subcutaneous edema in the dorsal portion was found at embryonic day (E) 16.5, and the other anomalies described above were apparent after E18.5 in ocd/ocd. Whole mount immunohistochemistry for Sox9 revealed that mesenchymal condensation was delayed in limb bud in ocd/ocd, and skeletal preparation showed that the progression of whole-body chondrogenesis was delayed in ocd/ocd. Histological and immunohistological analyses of the femur showed that cell proliferations of resting and proliferative zones of growth plate were significantly reduced in ocd/ocd embryos. Fine linkage mapping localized the ocd locus within 84 kb of positions 65,584–65,668 kb containing a part of Golgb1 gene on chromosome 11. Expression of Golgb1 mRNA was found in limb buds, somite derivatives and calvaria. Sequence analysis identified a 10-bp insertion in exon 13 of the Golgb1 gene in ocd/ocd rats. The Golgb1 gene encodes the COPI vesicle tethering factor, giantin. This insertion mutation causes a frame shift, and introduces a premature termination codon at codon 1082, leading to truncation of the C-terminal two thirds of giantin. By in-gel Western analysis using anti-giantin antibody that recognizes an epitope within 200 aa of the C-terminus, the expression of giantin was not detected in ocd/ocd embryos. As the C-terminal region of giantin is required for localization to the Golgi apparatus, these results strongly suggested that giantin is functionally defective in ocd/ocd rats. Therefore, we concluded that mutation of the Golgb1 gene is responsible for the phenotypic characteristics including osteochondrodysplasia of ocd/ocd, and that giantin plays a pivotal role in multiple aspects of chondrogenesis.

Introduction

The skeletal elements of mammals arise in development through two distinct processes, endochondral ossification and intramembranous ossification. Intramembranous ossification is a process in which undifferentiated mesenchymal cells differentiate into osteoblasts and form bone structures directly. The majority of skull and facial bones and the medial part of the clavicles are formed by intramembranous ossification. Endochondral ossification is a process in which the bone structures are formed via a cartilage template. Most of the skeletal elements, including appendicular skeleton and vertebrae, are formed by endochondral ossification. Endochondral ossification is a multi-step process and is initiated by condensation of undifferentiated mesenchymal cells. While mesenchymal cells in the center of the condensation differentiate into chondrocytes, those localized in the periphery differentiate into perichondrium. Chondrocytes proliferate, differentiate into hypertrophic chondrocytes, and eventually undergo apoptosis, thereby allowing invasion of blood vessels and osteoblasts, and replacement of cartilage template to bone elements and bone marrow [1].

The growth plate of developing bone contains four distinct zones: resting, proliferative, prehypertrophic, and hypertrophic zones. Various extracellular matrix (ECM) components and growth factors are produced by chondrocytes and perichondrium, and are involved in endochondral ossification. Type II collagen is the most abundant ECM protein of cartilage, and mutations or depletion of type II collagen cause several types of chondrodysplasia [2], [3]. Both types X and XI collagens are also indispensable for endochondral ossification [4]. Other non-collagenous ECM proteins and glycosaminoglycans (GAGs) have fundamental roles in bone development in mammals [5], [6], [7], [8], [9]. Some growth factors expressed by chondrocytes interact in complex ways to regulate chondrocyte proliferation and differentiation and osteoblast development [10], [11], [12].

The Golgi apparatus is a central part of the intracellular vesicle transport system and the main compartment for protein modification and GAG synthesis. As synthesis and secretion of growth factors and ECM components are required for proper chondrogenesis, chondrocytes possess well-developed endoplasmic reticulum (ER) and Golgi apparatus [13]. Chondrocytes seem to have specialized molecules and/or mechanisms for intracellular vesicle transport [14]. Although mutations of several genes encoding intracellular vesicle transport components have been identified as the causes of skeletal disorders in humans, mice, and zebrafish [15], [16], [17], [18], the molecules and mechanisms involved in intracellular vesicle transport required for chondrogenesis are not fully understood [14].

Osteochondrodysplasia (ocd) is a mutation that arose spontaneously in a closed colony of Wistar–Imamichi rats in our laboratory, and homozygous rats (ocd/ocd) are characterized by osteochondrodysplasia, cleft palate, protruding tongue, and systemic edema. The abnormal skeletal system at birth includes shortenings of long bones, deformation of cranial bones and vertebrae, and fusion of ribs [19]. Homozygous ocd/ocd rats die soon after birth from respiratory insufficiency, due to both morphological and functional anomalies of the respiratory system [20]. The growth plates of ocd/ocd femurs contain reduced amounts of ECM components and have swollen chondrocytes [21], [22]. Chondrocyte degeneration is observed in the central portion of epiphyseal cartilage in the neonatal ocd/ocd femur [23]. Electron microscopic analysis showed that the chondrocytes in the femur growth plate of ocd/ocd neonates possess large vesicles containing ruthenium red granules and expanded ER and Golgi apparatus [21]. These phenotypic features are inherited in an autosomal recessive manner by ocd, and the ocd locus has been mapped close to D11Mgh3 on rat chromosome 11 [24].

In the present study, to characterize the embryonic ocd/ocd phenotype and identify the gene responsible for ocd, we performed anatomical, histological, and immunohistological analyses during the late embryonic period, further linkage analysis to make a fine linkage map around the ocd locus, and expression and sequence analyses of candidate genes located within the critical region. Our results clearly indicated that an insertion mutation in Golgb1 gene encoding the COPI vesicle tethering protein giantin is associated with osteochondrodysplasia and systemic edema in OCD rats. This insertion causes a frame shift and results in a deduced amino acid sequence lacking the C-terminal two thirds of giantin. This is the first description of the phenotype induced by a giantin loss-of-function mutation.