Identification and Tissue-Specific Characterization of Novel SHOX-Regulated Genes in Zebrafish Highlights SOX Family Members Among Other Genes.

Sandra Hoffmann,Jasmin Gogel,Ralph Roeth,Gudrun A Rappold,Steffen Just,Sabrina Diebold,David Hassel

doi:10.3389/fgene.2021.688808

Abstract

SHOX deficiency causes a spectrum of clinical phenotypes related to skeletal dysplasia and short stature, including Léri-Weill dyschondrosteosis, Langer mesomelic dysplasia, Turner syndrome, and idiopathic short stature. SHOX controls chondrocyte proliferation and differentiation, bone maturation, and cellular growth arrest and apoptosis via transcriptional regulation of its direct target genes NPPB, FGFR3, and CTGF. However, our understanding of SHOX-related pathways is still incomplete. To elucidate the underlying molecular mechanisms and to better understand the broad phenotypic spectrum of SHOX deficiency, we aimed to identify novel SHOX targets. We analyzed differentially expressed genes in SHOX-overexpressing human fibroblasts (NHDF), and confirmed the known SHOX target genes NPPB and FGFR among the most strongly regulated genes, together with 143 novel candidates. Altogether, 23 genes were selected for further validation, first by whole-body characterization in developing shox-deficient zebrafish embryos, followed by tissue-specific expression analysis in three shox-expressing zebrafish tissues: head (including brain, pharyngeal arches, eye, and olfactory epithelium), heart, and pectoral fins. Most genes were physiologically relevant in the pectoral fins, while only few genes were also significantly regulated in head and heart tissue. Interestingly, multiple sox family members (sox5, sox6, sox8, and sox18) were significantly dysregulated in shox-deficient pectoral fins together with other genes (nppa, nppc, cdkn1a, cdkn1ca, cyp26b1, and cy26c1), highlighting an important role for these genes in shox-related growth disorders. Network-based analysis integrating data from the Ingenuity pathways revealed that most of these genes act in a common network. Our results provide novel insights into the genetic pathways and molecular events leading to the clinical manifestation of SHOX deficiency.

Highlights

Linear growth and skeletal development are highly dynamic processes that depend on transcription factors, signaling molecules, and extracellular matrix proteins
The highest endogenous expression of SHOX so far was found in primary human fibroblasts (NHDF; Durand et al, 2011) and zebrafish embryos (Sawada et al, 2015; Montalbano et al, 2016)
SHOX-homeodomain mutant (HM) was previously detected in a patient with short stature carrying a c.421T>G missense mutation that led to an amino acid exchange (p.Y141D) in the homeodomain, which impairs SHOX function by reducing DNA-binding and transactivation capacity (Schneider et al, 2005)

Summary

Introduction

Linear growth and skeletal development are highly dynamic processes that depend on transcription factors, signaling molecules, and extracellular matrix proteins. The growth plate, located near the ends of the long bones, provides a continuous supply of chondrocytes for endochondral ossification, which is initiated during early fetal development and continues until adolescence (Kronenberg, 2003; Lui et al, 2018). Any failure in this process causes a wide range of skeletal disorders. The penetrance of SHOX deficiency is high, its clinical expression is variable, even among family members (Schiller et al, 2000; Binder, 2011). CYP26C1 has been identified as a modifier of severity in patients with SHOX deficiency, but certainly others will follow that contribute to the variable expressivity (Montalbano et al, 2016)

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