Endothelin-2 signaling in the neural retina promotes PNAS PLUS the endothelial tip cell state and inhibits angiogenesis Amir Rattnera,1, Huimin Yua, John Williamsa,b, Philip M. Smallwooda,b, and Jeremy Nathansa,b,c,d,1 Departments of aMolecular Biology and Genetics, cNeuroscience, and dOphthalmology and bHoward Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205 Contributed by Jeremy Nathans, August 20, 2013 (sent for review February 19, 2013) Endothelin signaling is required for neural crest migration and time lapse imaging studies of vascular development in zebrafish homeostatic regulation of blood pressure. Here, we report that and mammalian EC dynamics in explant culture show that the tip constitutive overexpression of Endothelin-2 (Edn2) in the mouse cell and stalk cell states are highly plastic, with frequent retina perturbs vascular development by inhibiting endothelial cell exchanges between the two cell states (8, 9). migration across the retinal surface and subsequent endothelial Several other signaling pathways are also essential for retinal cell invasion into the retina. Developing endothelial cells exist in vascular development. Norrin, a Muller-glia–derived ligand, and one of two states: tip cells at the growing front and stalk cells in its EC receptor Frizzled4 (Fz4), coreceptor Lrp5, and receptor the vascular plexus behind the front. This division of endothelial chaperone Tspan12 activate canonical Wnt signaling in de- cell states is one of the central organizing principles of angiogen- veloping ECs (10). In humans and mice, defects in any of these esis. In the developing retina, Edn2 overexpression leads to components lead to retinal hypovascularization. Similar pheno- overproduction of endothelial tip cells by both morphologic and types are observed in mice lacking Angiopoietin2 (Ang2), an molecular criteria. Spatially localized overexpression of Edn2 pro- antagonist of the Tie2 receptor tyrosine kinase, or overexpressing duces a correspondingly localized endothelial response. Edn2 over- leukemia inhibitory factor (11–13). expression in the early embryo inhibits vascular development at Interest in retinal vascular development is motivated, in part, midgestation, but Edn2 overexpression in developing skin and by the central role that neovascularization plays in age-related brain has no discernible effect on vascular structure. Inhibition of macular degeneration and diabetic retinopathy, two of the most BIOLOGY retinal angiogenesis by Edn2 requires expression of Endothelin prevalent adult-onset eye diseases (14). The success of anti-Vegf DEVELOPMENTAL receptor A but not Endothelin receptor B in the neural retina. Taken therapies in treating these disorders has motivated the search for together, these observations imply that the neural retina responds additional regulators of retinal vascular growth (15). to Edn2 by synthesizing one or more factors that promote the The experiments reported here focus on the effects of endo- endothelial tip cell state and inhibit angiogenesis. The response to thelin signaling on retinal vascular development. Endothelins Edn2 is sufficiently potent that it overrides the activities of other were initially discovered as endogenous vasoconstricting pep- homeostatic regulators of angiogenesis, such as Vegf. tides (16). In mammals, there are three closely related peptides [Endothelin-1 (Edn1), Edn2, and Edn3], each of which is enco- he architecture of the vertebrate vasculature has long been ded by a distinct gene and released by proteolysis from a large Tan object of fascination for biologists. At a macroscopic scale, polypeptide precursor. Two G protein-coupled receptors, Ednra the trajectories and branch points of major arteries and veins are and Ednrb, mediate the effects of the endothelins. In addition highly stereotyped, and at a microscopic scale, capillary density to their roles in homeostatic regulation of blood pressure— within each tissue is governed by characteristic statistical param- including regulation of arterial smooth muscle tone, renal fluid eters. These attributes are particularly accessible to observation and salt retention, and cardiac contractility—endothelins and and manipulation in the mammalian retina (1, 2). In many endothelin receptors play critical roles in neural crest migration mammals, including mice and humans, the retinal vasculature (17). In mice and humans, defects in Edn3 or Ednrb lead to develops by radial migration of endothelial cells (ECs) along the inner (vitreal) face of the retina starting from their point of entry Significance at the optic disk. In a second phase of development, branches from the primary vascular plexus penetrate into the retina to Two distinct and interconvertible types of endothelial cells are fl form two parallel tiers of capillaries that ank a central layer present during blood vessel growth: tip cells at the growing of retinal neurons, the inner nuclear layer (INL). During both front of the vascular network and stalk cells behind the front. phases, EC proliferation and migration are driven, at least in In the present study, overexpression of Endothelin-2, a peptide part, by tissue-derived Vegf (3, 4). previously implicated in the control of blood pressure, is shown During radial growth, there is a clear morphologic distinction to promote the tip cell fate and arrest vascular growth within between ECs at the growing front of the vascular plexus, referred the mouse retina. Genetic experiments show that this effect to as tip cells, and the bulk of the ECs that follow behind the requires Endothelin receptor A expression in the neural retina, front, referred to as stalk cells (5). In particular, tip cells possess implying the existence of a retina-derived regulator of vascular numerous filopodia and are highly motile, acting like the vascular growth and development that is under Endothelin control. equivalent of axonal growth cones. Stalk cells proliferate, but they lack filopodia. The balance between tip and stalk cell states Author contributions: A.R. and J.N. designed research; A.R., H.Y., J.W., P.M.S., and J.N. is orchestrated by asymmetric signaling through the Notch performed research; A.R. and J.N. analyzed data; and J.N. wrote the paper. pathway: tip cells express the Notch ligand Δ-like4 (Dll4), which The authors declare no conflict of interest. acts on stalk cell Notch receptors to decrease stalk-to-tip con- Data deposition: The sequence reported in this paper has been deposited in the Gene version, whereas stalk cells express the Notch ligand Jagged1, Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE50059) and the NCBI sequence read archive, www.ncbi.nlm.nih.gov/sra (accession no. which antagonizes Dll4 activity (6, 7). This pathway is intimately SRP029473). linked to Vegf signaling; Vegfa promotes Dll4 expression in tip 1To whom correspondence may be addressed. E-mail: [email protected] or arattner@ cells, and Notch signaling in stalk cells suppresses the response jhmi.edu. to Vegfa. Additionally, Vegfa, acting through Vegfr2, directly This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. promotes the tip cell fate, including filopodia formation. Recent 1073/pnas.1315509110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1315509110 PNAS Early Edition | 1of10 Downloaded by guest on October 1, 2021 deficiencies in skin melanocytes and enteric neurons in the co- In Z/Edn2;Six3-Cre mice, Edn2 is produced throughout the lon, both of which are neural crest derivatives. In mice, defects in developing and mature retina beginning at approximately em- Edn1 or Ednra lead to deficiencies in cranial and cardiac neural bryonic day 11 (E11). An examination of adult Z/Edn2;Six3-Cre crest derivatives, with resulting deficiencies in craniofacial and retinas revealed diverse morphologic defects (Fig. 1). Some cardiac outflow tract development, respectively. Mutations in retinal regions are very thin, especially the ganglion cell layer and Edn2 have not been reported. INL (compare Fig. 1A with Fig. 1B) or less commonly, the outer In earlier work, we and others identified Edn2 transcripts nuclear layer (ONL) (Fig. 1C). [Throughout this paper, control among a small set of transcripts that increase in abundance in retinas are derived from age-matched and phenotypically WT injured or diseased photoreceptors (18–20). This induction is mice—typically littermates—that carry a subset of the genetic observed in a wide variety of experimental paradigms, including markers of the experimental mice.] Other retinal regions exhibit retinal detachment, light damage, and inherited photoreceptor extensive detachment, with numerous macrophages occupying degeneration. Here we report the surprising discovery that Edn2 the subretinal space, severe folding of the retina, accumulation fl fi overexpression in the developing mouse retina potently and of interstitial uid, and/or development of interstitial brosis – specifically inhibits retinal angiogenesis and promotes the tip (Fig. 1 D F). In some regions, few or no blood vessels were cell state. observed on the vitreal face of the retina (Fig. 1 B and C), whereas in other regions, clusters of blood vessels occupy the Results vitreal face (Fig. 1D). It is unclear whether these retina-associ- fi Edn2 Overexpression in the Retina Inhibits Vascular Development. ated vessels were initially derived from bona de retinal vascu- Our point of departure for the experiments described below lature or recruited from the hyaloid