The hemispheric, bi-layered optic cup forms from an oval optic vesicle during early vertebrate eye development through major morphological transformations. The overall basal surface, facing the developing lens, is increasing, while, at the same time, the space basally occupied by individual cells is decreasing. This cannot be explained by the classical view of eye development. Using zebrafish (Danio rerio) as a model, we show that the lens-averted epithelium functions as a reservoir that contributes to the growing neuroretina through epithelial flow around the distal rims of the optic cup. We propose that this flow couples morphogenesis and retinal determination. Our 4D data indicate that future stem cells flow from their origin in the lens-averted domain of the optic vesicle to their destination in the ciliary marginal zone. BMP-mediated inhibition of the flow results in ectopic neuroretina in the RPE domain. Ultimately the ventral fissure fails to close resulting in coloboma.DOI: http://dx.doi.org/10.7554/eLife.05216.001

Although there is increasing evidence that blood-derived macrophages support tumor progression, it is still unclear whether specialized resident macrophages, such as brain microglia, also play a prominent role in metastasis formation. Here, we show that microglia enhance invasion and colonization of brain tissue by breast cancer cells, serving both as active transporters and guiding rails. This is antagonized by inactivation of microglia as well as by the Wnt inhibitor Dickkopf-2. Proinvasive microglia demonstrate altered morphology, but neither upregulation of M2-like cytokines nor differential gene expression. Bacterial lipopolysacharide shifts tumor-educated microglia into a classical M1 phenotype, reduces their proinvasive function, and unmasks inflammatory and Wnt signaling as the most strongly regulated pathways. Histological findings in human brain metastases underline the significance of these results. In conclusion, microglia are critical for the successful colonization of the brain by epithelial cancer cells, suggesting inhibition of proinvasive microglia as a promising antimetastatic strategy. V V C

1Authors share senior authorship.Abbreviations used: 3MA, 3-methyladenine; ALLN, N-acetyl-leucylleucyl-norleucinal; EGFP, enhanced green fluorescent protein; PD, Parkinson's disease; WT, wildtype; DC, C-terminally truncated a-synuclein consisting of amino acids 1-108. AbstractAggregates of a-synuclein are the pathological hallmark of sporadic Parkinson's disease (PD), and mutations in the a-synuclein gene underlie familial forms of the disease. To characterize the formation of a-synuclein aggregates in living cells, we developed a new strategy to visualize a-synuclein by fluorescence microscopy: a-synuclein was tagged with a six amino acid PDZ binding motif and co-expressed with the corresponding PDZ domain fused to enhanced green fluorescent protein (EGFP). In contrast to the traditional approach of a-synuclein-EGFP fusion proteins, this technique provided several-fold higher sensitivity; this allowed us to compare a-synuclein variants and perform time-lapse imaging. A C-terminally truncated a-synuclein variant showed the highest prevalence of aggregates and toxicity, consistent with stabilization of the a-synuclein monomer by its C-terminus. Timelapse imaging illustrated how cells form and accumulate aggregates of a-synuclein. A substantial number of cells also reduced their aggregate load, primarily through formation of an aggresome, which could itself be cleared from the cell. The molecular chaperone Hsp70 not only prevented the formation of aggregates, but also increased their reduction and clearance, underlining the therapeutic potential of similar strategies. In contrast to earlier assumptions build-up, reduction and clearance of a-synuclein aggregation thus appear a highly dynamic process. Keywords: aggregation, aggresomes, molecular chaperones, time-lapse imaging, a-synuclein. J. Neurochem. (2008) 106, 529-540. JOURNAL OF NEUROCHEMISTRY | 2008 | 106 | 529-540 doi: 10.1111/j.1471-4159.2008 Next to WT and the two disease-related mutations A30P and A53T, we also used a C-terminally deleted variant, which had shown increased aggregation in vitro (Bertoncini et al. 2005b). Furthermore, we wanted to characterize the in vivo effects of the molecular chaperone Hsp70, which has been shown to reduce the formation of a-synuclein fibrils observed in vitro (Dedmon et al. 2005) and the prevalence of aggregates in vivo (McLean et al. 2002;Klucken et al. 2004). Experimental procedures CloningFor the fusion protein of PDZ domain and EGFP (PDZ-EGFP), the PDZ1 domain of S-SCAM (accession NP_446073, a gift from the lab of Nils Brose, MPI-em, Göttingen) was cloned into pEGFP-N1, resulting in PDZ-EGFP (Fw: 5¢-ATTAGATCTATGGGAACATTC-CTCAGCACCAC-3¢; Rev: 5¢-ATTACCGGTTCCTCCGGATATGT-GCCAT CTAGTTGG-3¢). The corresponding PDZ binding motif (amio acids HSTTRV, from neuroligin-1, accession NP_446320) was added to the C-termini of a-synuclein variants by PCR (Fw: 5¢-CTCAAGCTTATGGATGTATTCATG-3¢; Rev: 5¢-ATGG-ATATCTCACACTCTGGTGGTGCTGTGGGCTTCAGGTTCG-3¢; DCRev: 5¢-GTTGATATCTCACACTCTGGTGGTGCTGTGTGG-GGCTCCTTCTTC-3¢) and PDZ-EGFP was subclo...