Persistent expression of Twist1 in chondrocytes causes growth plate abnormalities and dwarfism in mice

Abstract

Evidence from various in vitro gain and loss of function studies indicate that the bHLH transcription factor Twist1 negatively regulates chondrocyte differentiation; however limited information regarding Twist1 function in postnatal cartilage development and maintenance is available. Twist1 expression within the postnatal growth plate is restricted to immature, proliferating chondrocytes, and is significantly decreased or absent in hypertrophic chondrocytes. In order to examine the effect of maintaining the expression of Twist1 at later stages of chondocyte differentiation, we used type II collagen Cre (Col2-Cre) mice to activate a Cre-inducible Twist1 transgene specifically in chondrocytes (Col2-Twist1). At two weeks, postnatal growth was inhibited in Col2-Twist1 mice, as evidenced by limb shortening. Histological examination revealed abnormal growth plate structure, characterized by poor columnar organization of proliferating cartilaginous cells, decreased cellularity, and expansion of the hypertrophic zone. Moreover, structural defects within the growth plates of Col2-Twist1 transgenic mice included abnormal vascular invasion and focal regions of bony formation. Quantitative analysis of endochondral bone formation via micro-computed topography revealed impaired trabecular bone formation in the hindlimbs of Col2-Twist1 transgenic mice at various timepoints of postnatal development. Taken together, these findings indicate that regulated Twist1 expression contributes to growth plate organization and endochondral ossification to modulate postnatal longitudinal bone growth. KEY WORDS: twist1, chondrocyte, growth plateTransition of chondrocytes from proliferation to terminal maturation within the growth plate is vital for longitudinal bone growth. Proliferating, immature chondrocytes synthesize an extracellular matrix (ECM) abundant in type II collagen and aggrecan. As cells differentiate, they mature into hypertrophic, postmitotic chondrocytes that enrich the ECM in type X collagen. Following hypertrophy, chondrocytes terminally mature and the calcified cartilage matrix is degraded by proteases and is infiltrated by blood vessels. Terminally mature chondrocytes then undergo apoptosis, thus facilitating the remodeling of the vascularized calcified matrix and its invasion by osteoblast precursors. Various families of transcription factors and signaling molecules, including transforming growth factor beta (TGF-b) and Wnts mediate this fine balance of chondrocyte proliferation, hypertrophy and terminal maturation necessary for longitudinal bone growth (Wuelling and Vortkamp, 2010). Further elucidation of the downstream effectors of these pathways may Int. J. Dev. Biol. 55: [641][642][643][644][645][646][647]

Craniofacial Shape Variation in Twist1+/− Mutant Mice

“…Twist1 may alter chondrocranial development as it plays a role in chondrocyte differentiation (Miraoui and Marie, ). Twist1 indirectly regulates chondrocyte maturation and the persistent expression of Twist1 in chondrocytes impedes endochondral bone growth (Hinoi et al, ; Guzzo et al, ). As for the face, the loss of Twist1 in cranial neural crest cells results in compromised frontonasal development and it likely mediates interactions between the cranial mesoderm and neural crest (Bildsoe et al, , ).…”

Aberrant expression of Twist1 in diseased articular cartilage and a potential role in the modulation of osteoarthritis severity

“…Human osteochondral specimens were fixed in 10% formalin and decalcified in EDTA prior to paraffin embedding as we previously reported. 23 , 26 Microtome sections (5um) were deparaffinized in xylene, rehydrated through graded ethanol, and stained with 1% Alcian Blue or Safranin O/Fast Green (SO/FG). Immunohistochemical staining was performed following routine methods for antibodies against Twist1, Sox9, PCNA and VEGF.…”

“…Immunohistochemical staining was performed following routine methods for antibodies against Twist1, Sox9, PCNA and VEGF. 23 Image analysis was performed using NIS-Elements BR 3.0 (Nikon, Melville, NY).…”

“… 22 We previously determined that Twist1 expression is maintained in the proliferating, immature chondrocytes of the postnatal growth plate, whereas its expression is repressed in hypertrophic chondrocytes both in vitro and in vivo. 4 , 23 Bialek et al had previously demonstrated that the direct interaction between Twist box with the DNA-binding domain of the transcriptional factor Runx2 led to inhibition of Runx2 transcriptional activity. 6 Using in vitro models of chondrocyte maturation, we demonstrated that Twist1 acts as a potent repressor of hypertrophy in growth plate chondrocytes downstream of TGF-beta and canonical Wnt signaling 4 and speculated that the inhibition of chondrocyte maturation is mediated through its repressive effects on Runx2, the master regulator of chondrocyte hypertrophy.…”

“…When we generated Twist1 transgenic (TG) mice with persistent expression in growth plate chondrocytes (Col2a1 expressing cells and progeny), we observed abnormal growth plate organization and postnatal longitudinal growth retardation attributed to impaired endochondral ossification. 23 While these collective studies point to the importance of Twist1 for normal formation and growth of cartilage structures, the role of Twist1 in normal maintenance and homeostasis of adult cartilage has remained elusive.…”