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(Foxn4) Activates Dll4-Notch Signaling to Suppress Photoreceptor Cell

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(Foxn4) Activates Dll4-Notch Signaling to Suppress Photoreceptor Cell Forkhead box N4 (Foxn4) activates Dll4-Notch PNAS PLUS signaling to suppress photoreceptor cell fates of early retinal progenitors Huijun Luoa,1, Kangxin Jina, Zhenhui Xiea, Feng Qiua, Shengguo Lia, Min Zoua, Li Caib, Katsuto Hozumic, David T. Shimad, and Mengqing Xianga,2 aCenter for Advanced Biotechnology and Medicine and Department of Pediatrics, University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, Piscataway, NJ 08854; bDepartment of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854; cDepartment of Immunology and Research Center for Embryogenesis and Organogenesis, Tokai University School of Medicine, Kanagawa 259-1193, Japan; and dLaboratory for Translational Vision Research, University College London Institute of Ophthalmology, London EC1V 9EL, United Kingdom Edited* by Aaron J. Shatkin, Center for Advanced Biotechnology and Medicine, Piscataway, NJ, and approved January 17, 2012 (received for review September 26, 2011) The generation of diverse neuronal types and subtypes from multi- stages responsible for generating early-born cell types (9). We potent progenitors during development is crucial for assembling have shown previously that the forkhead/winged helix TF Foxn4 functional neural circuits in the adult central nervous system. During is required by early retinal progenitors to establish the compe- mouse retinogenesis, early retinal progenitors give rise to several tence for the generation of amacrine and horizontal cells. Loss of cell types, including ganglion, amacrine, horizontal, cone, and rod Foxn4 function eliminates most amacrine cells and all horizontal cells. It is unknown at present how each of these fates is selected cells and causes a progenitor fate switch to photoreceptors (10). from the multiple neuronal fates available to the early progenitor. Foxn4 appears to regulate progenitor competence in part by By using a combination of bioinformatic, genetic, and biochemical activating the expression of proneural bHLH TF genes Ptf1a, approaches, we investigated the mechanism by which Foxn4 selects Neurod1, and Neurod4/Math3 (10), which are required for the the amacrine and horizontal cell fates from multipotential retinal specification of amacrine and/or horizontal cells (11–13). progenitors. These studies indicate that Foxn4 has an intrinsic activity It has long been recognized that retinogenic TFs often play dual NEUROSCIENCE to suppress the alternative photoreceptor cell fates of early retinal roles to promote certain cell fate(s) as well as suppress other fates progenitors by selectively activating Dll4-Notch signaling. Gene available to a progenitor. For instance, Otx2 is required for expression and conditional ablation analyses reveal that Dll4 is di- photoreceptor generation while suppressing the amacrine cell rectly activated by Foxn4 via phylogenetically conserved enhancers fate (14), and Nr2e3 in association with Crx promotes the rod fate and that Dll4 can partly mediate the Foxn4 function by serving as while inhibiting cone differentiation (15–17). Similarly, Atoh7 a major Notch ligand to expand the progenitor pool and limit pho- and Pou4f2 are essential for specifying RGCs while suppressing toreceptor production. Our data together define a Foxn4-mediated the cone, amacrine, and horizontal fates (18–20). Foxn4 appears molecular and signaling pathway that underlies the suppression of also to confer early retinal progenitors with amacrine and hori- alternative cell fates of early retinal progenitors. zontal competence not only by activating TF genes involved in their specification and differentiation but also by limiting the al- forkhead/winged-helix transcription factor | proneural bHLH gene | Crx | ternative fates of early progenitors. The increased photoreceptor Otx2 production and Crx up-regulation in Foxn4-null retinas suggest that Foxn4 normally represses Crx expression, which in turn uring vertebrate development, neurons are usually specified suppresses photoreceptor differentiation (10). How Foxn4 Dand differentiated from multipotent progenitor cells that are represses the expression of photoreceptor TFs is unknown—it can capable of producing two or more types of neurons and glial cells. be direct or indirect, such as through activating Notch signaling, For instance, during mouse retinogenesis, embryonic retinal which has been shown to inhibit photoreceptor fates (21, 22). progenitors are able to generate several early-born cell types in- In this study, we set out to explore the Foxn4-mediated molec- cluding ganglion, amacrine, horizontal, cone, and rod cells; and ular pathways and signaling events that lead to the suppression of postnatal progenitors can generate several late-born cell types the alternative photoreceptor fates of early retinal progenitors. fi including rod, bipolar, and Müller glial cells (1, 2). Production of Microarray pro ling and misexpression analyses demonstrated that these diverse retinal cell types is essential for assembling a func- Foxn4 selectively activates Dll4 expression among various Notch tional retinal circuitry consisting of rods and cones as photo- ligand and receptor genes. It colocalizes with Dll4 in a subset of receptors; amacrine, horizontal, and bipolar cells as interneurons; retinal progenitors and directly activates its gene expression and retinal ganglion cells (RGCs) as output neurons. It has been through phylogenetically conserved enhancers. Similar to Foxn4 deletion, inactivating Dll4 in retinal progenitors resulted in pho- proposed that intrinsic and extrinsic factors together determine toreceptor overproduction and reduced progenitor proliferation. the choice of retinal cell fates and that the progenitors may pass through successive and distinct states of competence for the or- dered generation of different cell types (3–5). At present, how- ever, there is little known about the molecular basis of com- Author contributions: H.L., K.J., Z.X., F.Q., M.Z., L.C., and M.X. designed research; H.L., K.J., Z.X., petence states and how a particular cell fate is selected from the F.Q., S.L., M.Z., and L.C. performed research; K.H. and D.T.S. contributed new reagents/analytic multiple fates available to a retinal progenitor. tools; H.L., K.J., Z.X., F.Q., M.Z., L.C., and M.X. analyzed data; and H.L. and M.X. wrote the paper. During retinogenesis, neuroepithelial cells in the optic vesicle The authors declare no conflict of interest. must acquire multipotency and establish competence for the *This Direct Submission article had a prearranged editor. generation of the full range of retinal cell types. Genetic ablation 1Present address: Department of Biochemistry and Molecular Biology, Mayo Clinic Ari- studies have demonstrated that the Pax6 and Sox2 transcription zona, Scottsdale, AZ 85259. factors (TFs) are required to coordinately confer multipotency to 2To whom correspondence should be addressed. E-mail: [email protected]. – fi retinal progenitors (6 8), and that the Ikzf1/Ikaros zinc nger TF This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. plays a key role in establishing the early temporal competence 1073/pnas.1115767109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1115767109 PNAS Early Edition | 1of10 Downloaded by guest on September 24, 2021 These data together thus suggest that Foxn4 limits alternative cell addition, we noted that several genes in the Notch signaling fates of early retinal progenitors in part by directly activating Dll4- pathway are also down-regulated in the mutant (Fig. 1C). Notch signaling to suppress photoreceptor production. Regulation of Dll4 Notch Ligand Gene Expression by Foxn4. We car- Results ried out RNA in situ hybridization analysis for a number of Notch Down-Regulation of Notch Signaling Genes in Foxn4-Null Mutant signaling component genes in Foxn4 mutant retinas to verify di- Retinas. To understand at the molecular level how Foxn4 may minished Notch signaling revealed by microarray profiling (Fig. 1). regulate the competence of embryonic retinal progenitors and The Dll4 ligand and Hes5 effector genes were expressed within the bias them toward amacrine and horizontal cell fates, we carried outer neuroblastic layer of E14.5 WT retinas, but their expression out microarray analysis by using the Affymetrix Mouse Genome was dramatically reduced in Foxn4lacZ/lacZ retinas (Fig. 2 C, D, G, 430A arrays. Array hybridization was carried out in quad- and H), in agreement with the microarray data (Fig. 1C). Similarly, ruplicates by using probes derived from embryonic day (E) 14.5 the expression of the Notch signaling integrator gene Rbpj was retinas of Foxn4+/+ and Foxn4lacZ/lacZ embryos (10). The moderately decreased (Fig. S1 A and B). However, there was obtained data were analyzed by using the Affymetrix Microarray barely any change in the expression of Dll1, Notch1, Hes1,and Suite and dChip software (23) to calculate fold changes of Hey1 genes in the null retina (Fig. 2 A, B, E,andF and Fig. S1 C, D, transcripts between the control and mutant. We found that 176 G,andH). The same was true for the expression of the neurogenic transcripts displayed a decrease or increase of 1.7-fold or higher gene Hes6 (Fig. S1 E and F). in their expression levels in Foxn4 mutant retinas. A total of 134 Our microarray and RNA in situ hybridization analyses sug- of them are down-regulated and 42 are up-regulated (Fig. 1 A gested that Foxn4 may selectively modulate Dll4 expression and B; and Dataset S1). The differentially expressed genes were among
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