Reelin/DAB‐1 Signaling in the Embryonic Limb Regulates the Chondrogenic Differentiation of Digit Mesodermal Progenitors

Abstract

Reelin is a bioactive component of some extracellular matrices. Most studies on this signaling glycoprotein have been performed in the developing nervous system, where Reelin binds to the very-low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) of target cells. This induces phosphorylation of the intracellular adaptor protein Disabled-1 (Dab-1), which subsequently activates downstream effectors to regulate important aspects of neuroblast biology. Here, we show that the components of the Reelin signaling pathway exhibit a dynamic expression pattern during the development of the digits in chick and mouse embryonic limbs. Reelin and Dab-1 are highly expressed in the differentiating digit cartilages and tendinous blastemas. Immunolabeling of phospho-Dab-1 indicates that the pattern of gene expression correlates with zones of active signaling. Intense signaling is also present in the early stages of cartilage differentiation in micromass cultures of digit mesodermal progenitors. In this in vitro assay, disruption of the Reelin signaling pathway by gene silencing causes cystoskeletal and cell shape modifications accompanied by reduced chondrogenesis and down-regulation of specific cartilage molecular markers. Of note, Scleraxis and Six2, which are master genes of tendinous blastemas, become up-regulated in these experiments. We further show that the receptors ApoER2 and VLDLR are differentially expressed in cartilage and tendons and that these receptors show temporal expression differences in the micromass cultures. Sox9 and other chondrogenic markers were downregulated in micromass cultures after ApoER2 gene silencing, while gene silencing of VLDLR up-regulates Scleraxis. In summary, our findings provide evidence of a role for Reelin signaling in skeletogenesis that promotes chondrogenesis through ApoER2 and inhibits tenogenic differentiation through VLDLR.

Nonneuronal roles for the reelin signaling pathway

“…Reelin and Dab1 are expressed in the undifferentiated limb mesoderm, and their levels decrease before interdigital cell death, which is necessary for digit formation (Díaz‐Mendoza et al, ). The reelin receptors ApoER2 and Vldlr are also expressed in the developing limb cartilage and tendons (Díaz‐Mendoza et al, ).…”

“…Analysis of the reelin signaling pathway components in micromass cultures of limb skeletogenic progenitors by quantitative PCR showed that levels of reelin, Dab1 and ApoER2 decrease as the cartilage undergoes differentiation, while expression of Vldlr follows the opposite pattern (Díaz‐Mendoza et al, ). Reelin expression in the limb is up‐regulated in response to FGF2, a cell survival factor, and down‐regulated in response to bone morphogenetic protein 7 (BMP7), which induces apoptosis (Díaz‐Mendoza et al, ). Dab1 silencing in micromass cultures reduced chondrogenesis and induced cell death, with decreased phosphorylation of Akt and lower levels of focal adhesion kinase (Díaz‐Mendoza et al, ).…”

“…Reelin expression in the limb is up‐regulated in response to FGF2, a cell survival factor, and down‐regulated in response to bone morphogenetic protein 7 (BMP7), which induces apoptosis (Díaz‐Mendoza et al, ). Dab1 silencing in micromass cultures reduced chondrogenesis and induced cell death, with decreased phosphorylation of Akt and lower levels of focal adhesion kinase (Díaz‐Mendoza et al, ). In addition, silencing of Dab1 in limb ectoderm‐mesoderm recombinants resulted in the absence of cartilage and tendon differentiation (Díaz‐Mendoza et al, ).…”

Reelin Deficiency Delays Mammary Tumor Growth and Metastatic Progression

“…Recent studies have identified a number of non-neuronal functions for reelin [8–12]. For example, reelin signaling was found to regulate the homeostasis of the intestinal crypt-villus unit [11], formation of thrombin clots [10], and to be important for establishment of the lymphatic vasculature [12].…”

Assessment of promoter methylation and expression of SIX2 as a diagnostic and prognostic biomarker in Wilms’ tumor

“…A member of the sine oculis homeobox (SIX) transcription factor family, SIX2, acts as a marker of nephronic progenitor cell self-renewal and plays a pivotal role in the growth process of cartilage, pyloric sphincter, eyes, tendon germ cells, craniofacial system, and cerebrum [4][5][6][7][8][9]. SIX2 is ubiquitously present throughout the development of the genitourinary system, including nephrogenic cord and metanephros [10][11][12].…”