Interdigital cell death in the embryonic limb is associated with depletion of Reelin in the extracellular matrix

Abstract

Interdigital cell death is a physiological regression process responsible for sculpturing the digits in the embryonic vertebrate limb. Changes in the intensity of this degenerative process account for the different patterns of interdigital webbing among vertebrate species. Here, we show that Reelin is present in the extracellular matrix of the interdigital mesoderm of chick and mouse embryos during the developmental stages of digit formation. Reelin is a large extracellular glycoprotein which has important functions in the developing nervous system, including neuronal survival; however, the significance of Reelin in other systems has received very little attention. We show that reelin expression becomes intensely downregulated in both the chick and mouse interdigits preceding the establishment of the areas of interdigital cell death. Furthermore, fibroblast growth factors, which are cell survival signals for the interdigital mesoderm, intensely upregulated reelin expression, while BMPs, which are proapototic signals, downregulate its expression in the interdigit. Gene silencing experiments of reelin gene or its intracellular effector Dab-1 confirmed the implication of Reelin signaling as a survival factor for the limb undifferentiated mesoderm. We found that Reelin activates canonical survival pathways in the limb mesoderm involving protein kinase B and focal adhesion kinase. Our findings support that Reelin plays a role in interdigital cell death, and suggests that anoikis (apoptosis secondary to loss of cell adhesion) may be involved in this process.

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“…In all these cases, the embryonic webbing is retained in these species and represents a form of adaptive syndactyly where adjacent digits are joined by interdigital epithelium and mesenchyme, which is typically lost through apoptosis at later stages of embryonic development once digits and phalanges have been patterned (Zuzarte‐Luis and Hurle, ; Zeller et al., ; Hernandez‐Martinez and Covarrubias, ). Fgf8 is generally utilized as a survival factor (retained on axis of the digits) and Bmp signaling is a proapoptotic signal present in the interdigits (Diaz‐Mendoza et al., ; Diaz and Trainor, ). Inhibiting interdigital Bmp signaling in a “webbing free” species like a chicken hindlimb through the introduction of a dominant negative Bmp receptor led to a retention of embryonic interdigital mesenchyme reminiscent of the duck foot and bat hand, for which the latter species inhibit Bmp signaling through heterotopic expression of the Bmp antagonist gremlin in the interdigital mesenchyme (Zou and Niswander, ; Weatherbee et al., ).…”

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

“…In a previous study, we have shown that integrin‐mediated Reelin signaling is a survival factor for the undifferentiated interdigital mesoderm (Díaz‐Mendoza et al, ). In this study, cell death evaluated by flow‐cytometry was not intensified in cultures subjected to knock down of either ApoER2 or VLDLR (Supporting Information Fig.…”

“…However, an increasing number of studies in recent years have provided evidence for a wider distribution of Reelin in the extracellular matrices of tumors and non‐nervous embryonic and adult organs (Maurin et al, ; Sato et al, Khialeeva et al, ; Lutter et al, ; Yuan et al, ; Garcia‐Miranda et al, ). Recognized functions of Reelin unrelated to neural functions include the following: morphogenesis of the lymphatic vessels (Lutter et al, ) inhibition of metastasis in various carcinomas (see Yuan et al, ) homeostasis of the intestinal epithelium (Garcia‐Miranda et al, ) mammary gland morphogenesis (Khialeeva et al, ) and interdigital cell death (Díaz‐Mendoza et al, ). There is also evidence for a greater complexity of the signaling cascade and various transmembrane receptors such as Ephrin B receptor (Bouche et al, ), integrins (Dulabon et al, ), and Amyloid Precursor Protein (Hoe et al, ) appear to be functionally associated with this signaling pathway.…”

“…In addition, ECM store growth factors and regulate their local delivery. Reelin has been detected in the ECM of the developing limb (Khialeeva et al, ) and its depletion precedes interdigital cell death (Díaz‐Mendoza et al, ), but its potential function in the differentiation of the skeletal tissues has not been analyzed.…”

Interdigital tissue remodelling in the embryonic limb involves dynamic regulation of the miRNA profiles

“…However, it is likely that this miRNA was involved in the changes in the extracellular matrix occurring in the course of interdigit remodelling associated with the onset of interdigit regression (Diaz‐Mendoza et al. ). Notably, miR‐30 targets important components of the interdigit such as CTGF, Snail and Tgfβ2 (Roy et al.…”