Rod and cone photoreceptors detect light and relay this information through a multisynaptic pathway to the brain via retinal ganglion cells (RGCs) 1 . These retinal outputs support not only pattern vision, but also non-image forming (NIF) functions, which include circadian photoentrainment and pupillary light reflex (PLR). In mammals, NIF functions are mediated by rods, cones and the melanopsincontaining intrinsically photosensitive retinal ganglion cells (ipRGCs) 2, 3 . Rod/cone photoreceptors and ipRGCs are complementary in signalling light intensity for NIF functions 4-12 . The ipRGCs, in addition to being directly photosensitive, also receive synaptic input from rod/cone networks 13, 14 . To determine how the ipRGCs relay rod/cone light information for both image and non-image forming functions, we genetically ablated ipRGCs in mice. Here we show that animals lacking ipRGCs retain pattern vision, but have deficits in both PLR and circadian photoentrainment that are more extensive than those observed in melanopsin knockouts 8,10,11 . The defects in PLR and photoentrainment resemble those observed in animals that lack phototransduction in all three †To whom correspondence should be addressed.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and regulate a wide array of light-dependent physiological processes1–11. Genetic ablation of ipRGCs eliminates circadian photoentrainment and severely disrupts the pupillary light reflex (PLR)12,13. Here we show that ipRGCs consist of distinct subpopulations that differentially express the Brn3b transcription factor, and can be functionally distinguished. Brn3b-negative M1 ipRGCs innervate the suprachiasmatic nucleus (SCN) of the hypothalamus, whereas Brn3b-positive ipRGCs innervate all other known brain targets, including the olivary pretectal nucleus. Consistent with these innervation patterns, selective ablation of Brn3b-positive ipRGCs severely disrupts the PLR, but does not impair circadian photoentrainment. Thus, we find that molecularly distinct subpopulations of M1 ipRGCs, which are morphologically and electrophysiologically similar, innervate different brain regions to execute specific light-induced functions.

SUMMARY Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled4 (Fz4) receptor, Lrp5 co-receptor, or Norrin ligand cause retinal hypovascularization, but the role of Norrin/Fz4/Lrp signaling in vascular development has not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, while excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is up-regulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.

SUMMARY Transcriptional regulatory networks that control the morphologic and functional diversity of mammalian neurons are still largely undefined. Here we dissect the roles of the highly homologous POU-domain transcription factors Brn3a and Brn3b in retinal ganglion cell (RGC) development and function using conditional Brn3a and Brn3b alleles that permit the visualization of individual wild type or mutant cells. We show that Brn3a- and Brn3b-expressing RGCs exhibit overlapping but distinct dendritic stratifications and central projections. Deletion of Brn3a alters dendritic stratification and the ratio of monostratified: bistratified RGCs, with little or no change in central projections. In contrast, deletion of Brn3b leads to RGC transdifferentiation and loss, axon defects in the eye and brain, and defects in central projections that differentially compromise a variety of visually-driven behaviors. These findings reveal distinct roles for Brn3a and Brn3b in programming RGC diversity, and they illustrate the broad utility of germ-line methods for genetically manipulating and visualizing individual identified mammalian neurons.

An alkaline phosphatase (AP) reporter has been used to visualize detailed morphologies for all major classes of retinal neurons in the adult mouse. The analysis was performed on retinas in which AP expression was activated by Cre-mediated DNA recombination in a small fraction of cells. Recombination was controlled pharmacologically and, to a first approximation, appears to have occurred randomly. The morphologies of 794 inner retinal neurons have been analyzed by measuring arbor area, stratification level, and neurite branching patterns. When analyzed in this multidimensional parametric space, the cells can be clustered into subgroups by visual inspection and by using the Ward's and K-means algorithms. One application of this cell morphology data set and cluster analysis is as a standard for comparison with the retinas of genetically altered mice. This work illustrates the utility and feasibility of genetically directed marking methods for large-scale surveys of neuronal morphology.

Analysis of cellular morphology is the most general approach to neuronal classification. With the increased use of genetically engineered mice, there is a growing need for methods that can selectively visualize the morphologies of specified subsets of neurons. This capability is needed both to define cell morphologic phenotypes and to mark cells in a noninvasive manner for lineage studies. To this end, we describe a bipartite genetic system based on a Cre-estrogen receptor (ER) fusion protein that irreversibly activates a plasma membrane-bound alkaline phosphatase reporter gene by site-specific recombination. Because the efficiency and timing of gene rearrangement is controlled pharmacologically, a sparse subset of labeled cells can be generated from the set of CreER-expressing cells at any time during development. Histochemical visualization of alkaline phosphatase activity reveals neuronal morphology with strong and uniform labeling of all processes.

In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells, and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL)1–3: a laminar region that is conventionally divided into five major parallel sublaminae1,2. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here, we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs), and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo with respect to the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes.

The mammalian retina contains more than 50 distinct neuronal types, which are broadly classified into several major classes: photoreceptor, bipolar, horizontal, amacrine, and ganglion cells. Although some of the developmental mechanisms involved in the differentiation of retinal ganglion cells (RGCs) are beginning to be understood, there is little information regarding the genetic and molecular determinants of the distinct morphologies of the 15 – 20 mammalian RGC cell types. Previous work has shown that the transcription factor Brn3b/Pou4f2 plays a major role in the development and survival of many RGCs. The roles of the closely related family members, Brn3a/Pou4f1 and Brn3c/Pou4f3 in RGC development are less clear. Using a genetically-directed method for sparse cell labeling and sparse conditional gene ablation in mice, we describe here the sets of RGC types in which each of the three Brn3/Pou4f transcription factors are expressed and the consequences of ablating these factors on the development of RGC morphologies.