RESEARCH ARTICLE Vascular patterning regulates interdigital cell death by a ROS-mediated mechanism Idit Eshkar-Oren 1, *, Sharon Krief 1, *, Napoleone Ferrara 2 , Alison M. Elliott 3 and Elazar Zelzer 1, ‡ ABSTRACT Blood vessels serve as key regulators of organogenesis by providing oxygen, nutrients and molecular signals. During limb development, programmed cell death (PCD) contributes to separation of the digits. Interestingly, prior to the onset of PCD, the autopod vasculature undergoes extensive patterning that results in high interdigital vascularity. Here, we show that in mice, the limb vasculature positively regulates interdigital PCD. In vivo, reduction in interdigital vessel number inhibited PCD, resulting in syndactyly, whereas an increment in vessel number and distribution resulted in elevation and expansion of PCD. Production of reactive oxygen species (ROS), toxic compounds that have been implicated in PCD, also depended on interdigital vascular patterning. Finally, ex vivo incubation of limbs in gradually decreasing oxygen levels led to a correlated reduction in both ROS production and interdigital PCD. The results support a role for oxygen in these processes and provide a mechanistic explanation for the counterintuitive positive role of the vasculature in PCD. In conclusion, we suggest a new role for vascular patterning during limb development in regulating interdigital PCD by ROS production. More broadly, we propose a double safety mechanism that restricts PCD to interdigital areas, as the genetic program of PCD provides the first layer and vascular patterning serves as the second. KEY WORDS: Programmed cell death, Vascular patterning, Reactive oxygen species, Interdigit, Oxygen, Limb development, Syndactyly INTRODUCTION In the mouse embryo, the limbs emerge at embryonic day (E) 9.5, consisting of a mesenchymal core covered by ectoderm. The distal tip of the ectoderm forms a specialized epithelial structure known as the apical ectodermal ridge (AER), which acts as a major signaling center for limb patterning. Soon afterwards, mesenchymal cells form precartilaginous condensations that will serve as templates for the various skeletal elements, including the digits (Cooper et al., 2011; Johnson and Tabin, 1997; Zeller et al., 2009). The developing digits are initially connected by soft tissue. During limb morphogenesis, the digits are separated by coordinated regression of the soft tissue through programmed cell death (PCD) and outgrowth of the digits (Fallon and Cameron, 1977; Fernandez- Teran et al., 2006; Zuzarte-Luis and Hurle, 2002). Interdigital PCD commences at E12.5 in the mesenchyme underlying the AER. It then spreads proximally and by E14.0 it extends throughout the interdigital spaces, forming well-defined regions of cell death. By E14.5, most of the distal interdigital tissue has regressed and the digits are almost completely separated, as PCD is still active in the remaining proximal interdigital soft tissue (Fernandez-Teran et al., 2006; Salas-Vidal et al., 2001). Extensive work aimed at exposing the regulatory signals of PCD has concentrated mostly on the AER and interdigital mesenchyme as potential sources of these signals. Fibroblast growth factors (FGFs) produced by the AER were shown to act as negative regulators of PCD (Fernandez-Teran and Ros, 2008; Hernández- Martínez and Covarrubias, 2011; Montero et al., 2001). Bone morphogenetic proteins (BMP) 2, 4 and 7 are expressed in the AER and interdigital mesenchyme prior to the onset of PCD. BMPs were suggested to promote PCD by acting directly on interdigital tissue (Guha et al., 2002; Macias et al., 1997; Zou and Niswander, 1996) or indirectly, by downregulating the expression of FGFs in the AER (Maatouk et al., 2009; Pajni-Underwood et al., 2007). Similarly, the transcription factors MSX1 and MSX2 have been shown to act downstream of the BMP signaling pathway in the regulation of interdigital PCD (Lallemand et al., 2005). Retinoic acid (RA) is another molecule that has been implicated in PCD. RA was shown to affect the interdigital mesenchyme by upregulation of Bmp genes in that region (Dupé et al., 1999; Rodriguez-Leon et al., 1999). In the AER, RA was suggested to antagonize the survival effect of FGFs (Hernandez-Martinez et al., 2009). Reactive oxygen species (ROS) are chemically reactive molecules that are generated through the partial reduction of molecular oxygen (O 2 ). ROS are produced mostly in the mitochondria, as a byproduct of the respiratory chain. These toxic compounds might damage cells by oxidizing constituents such as DNA, proteins and lipids (Bokov et al., 2004). ROS were shown to participate in the regulation of interdigital PCD in mouse embryos (Covarrubias et al., 2008). During mouse development, high levels of ROS were detected at interdigital regions and coincided with areas of PCD. Treatment with antioxidants caused reduction of PCD and interdigital regression, supporting a role for ROS in the activation of PCD (Salas-Vidal et al., 1998). Moreover, expression patterns of specific antioxidant enzymes determined ROS production and PCD at interdigital areas (Schnabel et al., 2006). During organ development, blood vessels not only supply oxygen and nutrients but also provide vital regulatory signals (Cleaver and Melton, 2003; Coultas et al., 2005; Nikolova and Lammert, 2003; Tirziu and Simons, 2009). Vascular impairments during embryogenesis can lead to aberrant organ formation and lethality (Carmeliet, 2005; Ferrara et al., 1996; Gao et al., 2005; Lammert et al., 2003; Matsumoto et al., 2001). During the initial stages of mouse limb development, the mesenchyme core is vascularized by a primary unpatterned vascular plexus, which is formed by sprouts from the dorsal aorta and by somite-derived angioblasts that invade the limb bud (Coffin and Poole, 1988; Drake et al., 1998; Folkman, 2003; Risau and Flamme, 1995; Sabin, 1920; Received 25 November 2014; Accepted 11 December 2014 1 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel. 2 Genentech, Inc., 1 DNA Way, S. San Francisco, CA 94080, USA. 3 Departments of Pediatrics and Child Health and Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB R3A 1S1, Manitoba, Canada. *These authors contributed equally to this work ‡ Author for correspondence (eli.zelzer@weizmann.ac.il) 672 © 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 672-680 doi:10.1242/dev.120279 DEVELOPMENT