Panza et al. Neural Development (2015) 10:23 DOI 10.1186/s13064-015-0050-x RESEARCH ARTICLE Open Access The LRR receptor Islr2 is required for retinal axon routing at the vertebrate optic chiasm Paolo Panza1* , Austen A. Sitko3, Hans-Martin Maischein1,4, Iris Koch5, Matthias Flötenmeyer5, Gavin J. Wright6, Kenji Mandai7, Carol A. Mason2,3 and Christian Söllner1 Abstract Background: In the visual system of most binocular vertebrates, the axons of retinal ganglion cells (RGCs) diverge at the diencephalic midline and extend to targets on both ipsi- and contralateral sides of the brain. While a molecular mechanism explaining ipsilateral guidance decisions has been characterized, less is known of how RGC axons cross the midline. Results: Here, we took advantage of the zebrafish, in which all RGC axons project contralaterally at the optic chiasm, to characterize Islr2 as an RGC receptor required for complete retinal axon midline crossing. We used a systematic extracellular protein-protein interaction screening assay to identify two Vasorin paralogs, Vasna and Vasnb, as specific Islr2 ligands. Antibodies against Vasna and Vasnb reveal cellular populations surrounding the retinal axon pathway, suggesting the involvement of these proteins in guidance decisions made by axons of the optic nerve. Specifically, Vasnb marks the membranes of a cellular barricade located anteriorly to the optic chiasm, a structure termed the “glial knot” in higher vertebrates. Loss of function mutations in either vasorin paralog, individually or combined, however, do not exhibit an overt retinal axon projection phenotype, suggesting that additional midline factors, acting either independently or redundantly, compensate for their loss. Analysis of Islr2 knockout mice supports a scenario in which Islr2 controls the coherence of RGC axons through the ventral midline and optic tract. Conclusions: Although stereotypic guidance of RGC axons at the vertebrate optic chiasm is controlled by multiple, redundant mechanisms, and despite the differences in ventral diencephalic tissue architecture, we identify a novel role for the LRR receptor Islr2 in ensuring proper axon navigation at the optic chiasm of both zebrafish and mouse. Keywords: Islr2, Vasorin, Slit-like2, Optic chiasm, Glial knot, Midline crossing, Axon coherence, Zebrafish, Mouse Background midbrain targets. In most binocular animals, retinal The stereotypical trajectory of developing axons is the axons approaching the optic chiasm – the crossing point outcome of chemotactic guidance. However, it also sub- at the midline – diverge into ipsi- and contralateral pro- stantially depends on axon extension by local substrate jections. While the latter is always predominant in size, adhesion and axon-axon fasciculation, and regulated ipsilaterally-projecting axons vary in number in different passage through signaling landmarks [38]. One of the organisms, ranging from zero in zebrafish to ~3 % in most clearly defined and best studied choice points rodents and ~45 % in primates [25]. encountered en route by commissural axons is the In vitro studies using retinal explants showed that the embryonic midline. In the vertebrate visual system, ret- cells of the optic chiasm suppress retinal axon growth, inal ganglion cell (RGCs) axons project into the optic irrespective of their laterality of projection [27, 51, 52, 55]. nerve, across the ventral diencephalic midline and into Accordingly, growth cones proceed in a saltatory fashion the optic tracts towards their dorsal thalamic and across the ventral diencephalon and slow down when approaching the midline [20, 22, 32]. In amphibians and * Correspondence: [email protected] mice, a specific population of midline radial glial cells 1Max-Planck-Institut für Entwicklungsbiologie, Abteilung Genetik, Spemannstraße 35, 72076 Tübingen, Germany expresses Ephrin-B2, which repels ventro-temporal retinal Full list of author information is available at the end of the article axons [33, 54]. As a consequence, growth cones © 2015 Panza et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Panza et al. Neural Development (2015) 10:23 Page 2 of 17 expressing the Ephrin-B2 receptor EphB1 abruptly change annotated LRR receptors listed by the zebrafish model morphology to turn and ultimately follow the ipsilateral organism database, ZFIN [5], starting from the collection trajectory. Conversely, evidence to date argues that retinal of mammalian orthologs for these proteins [13]. Within axons with a contralateral trajectory cross the midline by our dataset of 116 genes, we identified 23 receptors overcoming chemosuppression at the chiasm. In the mur- expressed in the zebrafish RGC layer that we hypothe- ine anterior hypothalamus, a cluster of CD44/SSEA1-ex- sized may play a role in retinal axon guidance (Table 1). pressing early neurons is required for retinal axon entry Because we are interested in cellular recognition into the chiasm [48], providing the first evidence that the events at the midline, we focused our attention on chiasmatic territory is not exclusively refractory to axon receptor-encoding genes that are expressed in RGCs growth. Indeed, the expression of Plexin-A1 in these neu- between 30 and 36 h, the time between the beginning of rons, in conjunction with NrCAM on radial glia, reverses RGC differentiation and chiasm establishment. Most the inhibitory effect of midline-derived Semaphorin-6D, genes (e.g. ntrk2b, lrrc4.1, flrt1a, lrtm2)wereexpressedat thereby promoting axon growth [27, 53]. Similarly, a later stages (48 hpf onwards), leaving a single candidate, VEGF isoform expressed at the mouse optic chiasm acts islr2, fulfilling the temporal expression pattern suitable for as an attractant to support crossing of Neuropilin1- further study. In general, islr2 is expressed in brain nuclei positive retinal fibers [15]. associated with differentiated neurons (Fig. 1a), similar to In contrast to rodents and primates, fish have an en- descriptions of mouse and chick Islr2 expression [18, 21]. tirely contralateral retinal projection, making it an ideal The dynamic pattern of islr2 transcription and its correl- system in which to dissect mechanisms mediating RGC ation with the birthdate of RGCs are suggestive of a post- axon crossing at the midline. In order to identify new differentiation function held by this protein. Because islr2 molecular and cellular players in this process, we consid- expression starts in the very first cohort of RGCs develop- ered that both axon growth and guidance crucially ing in the retina (Fig. 1a), we hypothesized that Islr2 might depend on cell-cell and cell-extracellular matrix (ECM) be involved in early guidance decisions that these cells interactions mediated by cell surface and secreted pro- take, such as midline crossing. teins [38]. In this respect, proteins belonging to the leucine-rich repeat (LRR) superfamily – including Slits Islr2 is necessary for retinal axon midline crossing in and Trks – meet important requirements to participate zebrafish in precise and dynamic processes in neurodevelopment To investigate the function of Islr2 in vivo, we selected a [12]. These molecules display highly specific and zebrafish mutant strain harboring a germline mutation dynamic expression patterns, particularly in the nervous in islr2, generated by TILLING. The islr2sa82 locus con- system. Cell surface LRR proteins can mediate low affin- tains a premature nonsense mutation that leads to a pro- ity interactions with their binding partners. Finally, the tein truncation before the end of the LRR domain number of cell surface LRR proteins is greatly expanded (Fig. 1b). As a consequence, this allele is expected to in vertebrates, correlating with the increased complexity behave as a functional null. of nervous and immune system organization. To score for phenotypes related to retinal axon guid- Here, by analyzing the spatiotemporal expression pat- ance, we anterogradely labeled the optic nerves by terns of cell surface LRR superfamily members in zebra- intraretinal lipophilic dye injections at 5 dpf, at which fish, we identified islr2 as a LRR receptor-encoding gene time this axon tract is stable, since most error correction expressed in RGCs. Zebrafish larvae mutant for islr2 events have terminated by 72 hpf [22] and robust visual aberrantly display ipsilateral retinal projections, demon- behaviors are displayed by wild-type larvae [14, 34]. strating a role for Islr2 in RGC axon midline crossing. In Strikingly, despite being mostly normal morphologically, Islr2 (alias Linx) mutant mice, many retinal axons mis- islr2 homozygous mutant larvae clearly display project at and distal to the chiasm, confirming the role ipsilaterally-projecting retinal axons (Fig. 1c, asterisks). of Islr2 in ensuring proper axon routing at this choice We also observed that a subset of islr2 mutants had point. We also identify two midline factors expressed in thinner optic nerves compared to wild-type siblings the zebrafish optic pathway, Vasna