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  1. Evaluation Summary:

    This paper claims to identify a long-sought ligand for the receptor protein tyrosine phosphatase Lar that mediates its functions in neuromuscular junction development, mushroom body development, and photoreceptor axon targeting. If correct, this would be of interest to many developmental neurobiologists, However, further evidence is needed to strongly support this claim.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    In this paper, Bali et al. use an assay in which alkaline phosphatase-tagged cell surface proteins are used to stain live embryos overexpressing other cell surface proteins to identify Sticks and stones (Sns) as a possible ligand for the Lar receptor protein tyrosine phosphatase. Direct binding between the two proteins was confirmed in vitro, although it requires extensive multimerization of Lar, and is conserved between PTPRF/D and Nephrin homologues. The authors use GAL4 insertions to compare the expression patterns of Lar and sns in the nervous system, and observe genetic interactions in transheterozygotes as well as similar knockdown phenotypes. They conclude that Lar and Sns both act in motor neurons to promote NMJ growth, but act in different neuronal populations to control mushroom body morphogenesis and R7 photoreceptor axon targeting.

    Although it would be of great interest to confirm a receptor-ligand interaction between Lar and Sns, this paper falls short of that goal. Transheterozygous phenotypes are suggestive but do not prove that the two proteins act as a receptor-ligand pair. The physical interaction is quite weak and the evidence that Sns acts in the cells most likely to provide a Lar ligand is not compelling.

    First, Sns has a close homologue, Hibris. The authors do not examine whether Lar also binds to Hbs, or specifically interacts only with Sns.

    Second, only a single allele of sns is used to generate tran-sheterozygotes. It is possible that this chromosome carries another interacting mutation or even an allele of Lar.

    Third, the authors show that loss of Sns has a similar phenotype to loss of Lar in NMJ growth and mushroom body development, but do not demonstrate this for R7 axon targeting.

    Fourth, it is possible that the sns phenotypes are due to interactions with its known binding partners, Roughest and Kirre, and the transheterozygous interaction with Lar simply reflects the involvement of both Sns and Lar in the same process. The authors should more directly test the ligand hypothesis.

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  3. Reviewer #2 (Public Review):

    This manuscript from Bali, Lee and Zinn addresses a long-standing mystery in the receptor protein tyrosine phosphatase (RPTPs) field regarding the conserved extracellular protein binding partners of the LAR subfamily of RPTPs. Although mammalian LAR-family proteins bind and interact with several ligand molecules, and LAR-family receptors associate with heparan sulfate across species, the question of what protein ligands might represent the conserved partners of this well-conserved set of RPTPs has lingered. The authors present an exciting discovery that the Sns/Nephrin family of transmembrane proteins bind to LAR-family RPTPs in situ and in vitro. A powerful in situ tissue-binding assay that can overcome low-affinity interactions was developed by this group for screening and analysis of new ligands. Compelling Drosophila binding data implicating Sns as a candidate LAR ligand led the authors to analysis of mammalian Sns (Nephrin) for in situ binding in the same assay. By creating MIMIC reporters of the endogenous transcription units, the authors show that Sns and LAR are both expressed in larval motor neurons, where they are both required to promote NMJ morphogenesis. Consistent with the binding and very similar NMJ phenotypes of Sns and LAR, transheterozygous mutants show a very clear 'synthetic' phenotype strongly suggesting the type of strong functional interdependence characteristic of genes in a common signaling pathway. In contrast to the cis requirement in motor neurons, the authors discover that other regions of the nervous system display largely complementary expression of the reporters, raising the possibility that Sns and LAR act in trans in the larval mushroom body, and the visual system. The authors go on to confirm the same type of strong genetic interaction between Sns and LAR in these other contexts, leading to a working model that the two proteins function as a ligand-receptor pair in these other circuits at different stages of larval and pupal development. The data are very high in quality and the manuscript is clearly written. The study represents an exciting advance in this field from a leading lab in RPTP and receptor-ligand function in the fly nervous system.

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