The fluorescent protein toolbox has revolutionized experimental biology. Despite this advance, no fluorescent proteins have been identified from vertebrates, nor has chromogenic ligand-inducible activation or clinical utility been demonstrated. Here, we report the cloning and characterization of UnaG, a fluorescent protein from Japanese eel. UnaG belongs to the fatty-acid-binding protein (FABP) family, and expression in eel is restricted to small-diameter muscle fibers. On heterologous expression in cell lines or mouse brain, UnaG produces oxygen-independent green fluorescence. Remarkably, UnaG fluorescence is triggered by an endogenous ligand, bilirubin, a membrane-permeable heme metabolite and clinical health biomarker. The holoUnaG structure at 1.2 Å revealed a biplanar coordination of bilirubin by reversible π-conjugation, and we used this high-affinity and high-specificity interaction to establish a fluorescence-based human bilirubin assay with promising clinical utility. UnaG will be the prototype for a versatile class of ligand-activated fluorescent proteins, with applications in research, medicine, and bioengineering.

Recent findings implicate embryonic signaling centers in patterning the mammalian cerebral cortex. We used mouse in utero electroporation and mutant analysis to test whether cortical signaling sources interact to regulate one another. We identified interactions between the cortical hem, rich in Wingless-Int (WNT) proteins and bone morphogenetic proteins (BMPs), and an anterior telencephalic source of fibroblast growth factors (FGFs). Expanding the FGF8 domain suppressed Wnt2b, Wnt3a and Wnt5a expression in the hem. Next to the hem, the hippocampus was shrunken, consistent with its dependence for growth on a hem-derived WNT signal. Maintenance of hem WNT signaling and hippocampal development thus require a constraint on the FGF8 source, which is likely to be supplied by BMP activity. When endogenous BMP signaling is inhibited by noggin, robust Fgf8 expression appears ectopically in the cortical primordium. Abnormal signaling centers were further investigated in mice lacking the transcription factor EMX2, in which FGF8 activity is increased, WNT expression reduced, and the hippocampus defective. Suggesting that these defects are causally related, sequestering FGF8 in Emx2 homozygous mutants substantially recovered WNT expression in the hem and partially rescued hippocampal development. Because noggin can induce Fgf8 expression, we examined noggin and BMP signaling in the Emx2 mutant. As the telencephalic vesicle closed, Nog expression was expanded and BMP activity reduced,potentially leading to FGF8 upregulation. Our findings point to a cross-regulation of BMP, FGF, and WNT signaling in the early telencephalon,integrated by EMX2, and required for normal cortical development.

Selective autophagy adaptor proteins, including p62/SQSTM1, play pivotal roles in the targeted degradation of ubiquitinated proteins or organelles through the autophagy-lysosome system. However, how autophagy adaptors promote the autophagosomal engulfment of selected substrates is poorly understood. Here, we show that p62 phosphorylation at S403 is required for the efficient autophagosomal engulfment of polyubiquitinated mitochondria during Parkin-dependent mitophagy. p62 is able to interact with Parkin-recruited mitochondria without S403 phosphorylation under mitophagy-inducing conditions, but those mitochondria are not enclosed by autophagosomes. Intriguingly, the S403 phosphorylation occurs only in the early period of mitochondrial depolarization. Optineurin and TANK-binding kinase 1 (TBK1) are transiently recruited to the polyubiquitinated mitochondria, and the activated TBK1 phosphorylates p62 at S403. TBK1 inhibitor, BX795, prevents the p62-mediated autophagosomal engulfment of Parkin-recruited mitochondria. Our results suggest that TBK1-mediated S403 phosphorylation regulates the efficient autophagosomal engulfment of ubiquitinated mitochondria as an immediate response to the mitochondrial depolarization.

Diffusion tensor imaging is gaining increasing importance for anatomical imaging of the developing mouse brain. However, the application of diffusion tensor imaging to mouse brain imaging at microscopic levels is hindered by the limitation on achievable spatial resolution. In this study, fast diffusion tensor microimaging of the mouse brain, based on a diffusionweighted gradient and spin echo technique with twin-navigator echo phase correction, is presented. Compared to echo planar and spin echo acquisition, the diffusion-weighted gradient and spin echo acquisition resulted in significant reduction in scan time and had minimal image distortion, thereby allowing acquisition at higher spatial resolution. In this study, three-dimensional diffusion tensor microimaging of the mouse brains at spatial resolutions of 50-60 mm revealed unprecedented anatomical details. Thin fiber bundles in the adult striatum and white matter tracts in the embryonic day 12 mouse brains were visualized for the first time. The study demonstrated that data acquired using the diffusion tensor microimaging technique allow three-dimensional mapping of gene expression data and can serve as a platform to study gene expression patterns in the context of neuroanatomy in the developing mouse brain. Magn Reson Med 64:249-261,

The functions of Wingless-Int (Wnt) signaling, studied intensely in embryonic brain development, have been comparatively little investigated in the postnatal brain. We report remarkably patterned gene expression of Wnt signaling components in postnatal mouse cerebral cortex, lasting into young adulthood. Wnt genes are expressed in gene-specific regional and lamina patterns in each of the major subdivisions of the cerebral cortex: the olfactory bulb (OB), the hippocampal formation, and the neocortex. Genes encoding Frizzled (Fz) Wnt receptors, or secreted Frizzled-related proteins (sFrps), are also expressed in regional and lamina patterns. These findings suggest that Wnt signaling is active and regulated in the postnatal cortex and that different cortical cell populations have varying requirements for a Wnt signal. The OB, in particular, shows gene expression of a large variety of Wnt signaling components, making it a prime target for future functional studies. The penultimate components of the canonical Wnt pathway are the Tcf/Lef1 transcription factors, which regulate transcription of Wnt signaling target genes. Surprisingly, we found little Tcf/Lef1 expression in the postnatal neocortex. These observations suggest that noncanonical Wnt pathways predominate, which will require functional testing. However, Lef1 is widely expressed in the dorsal thalamus, and Wnt ligands and receptors are expressed, respectively, in cortical areas and thalamic nuclei that are interconnected. Thus, canonical Wnt signaling could be utilized in a major cortical input by Fz- and Lef1-expressing thalamic cells that innervate the Wnt-expressing cortex.

Thalamic innervation of each neocortical area is vital to cortical function, but the developmental strategies that guide axons to specific areas remain unclear. We took a new approach to determine the contribution of intracortical cues. The cortical patterning molecule fibroblast growth factor 8 (FGF8) was misexpressed in the cortical primordium to rearrange the area map. Thalamic axons faithfully tracked changes in area position and innervated duplicated somatosensory barrel fields induced by an ectopic source of FGF8, indicating that thalamic axons indeed use intracortical positional information. Because cortical layers are generated in temporal order, FGF8 misexpression at different ages could be used to shift regional identity in the subplate and cortical plate either in or out of register. Thalamic axons showed strikingly different responses in the two different conditions, disclosing sources of positional guidance in both subplate and cortical plate. Unexpectedly, axon trajectories indicated that an individual neocortical layer could provide not only laminar but also area-specific guidance. Our findings demonstrate that thalamocortical axons are directed by sequential, positional cues within the cortex and implicate FGF8 as an indirect regulator of thalamocortical innervation.

Voltage-gated Na(+) channel β-subunits are multifunctional molecules that modulate Na(+) channel activity and regulate cell adhesion, migration and neurite outgrowth. β-subunits including β4 are known to be highly concentrated in the nodes of Ranvier and axon initial segments in myelinated axons. Here we show diffuse β4 localization in striatal projection fibres using transgenic mice that express fluorescent protein in those fibres. These axons are unmyelinated, forming large, inhibitory fibre bundles. Furthermore, we report β4 dimer expression in the mouse brain, with high levels of β4 dimers in the striatal projection fascicles, suggesting a specific role of β4 in those fibres. Scn4b-deficient mice show a resurgent Na(+) current reduction, decreased repetitive firing frequency in medium spiny neurons and increased failure rates of inhibitory postsynaptic currents evoked with repetitive stimulation, indicating an in vivo channel regulatory role of β4 in the striatum.

IntroductionFUS/TLS is an RNA-binding protein whose genetic mutations or pathological inclusions are associated with neurological diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration, and essential tremor (ET). It is unclear whether their pathogenesis is mediated by gain or loss of function of FUS/TLS.ResultsHere, we established outbred FUS/TLS knockout mice to clarify the effects of FUS/TLS dysfunction in vivo. We obtained homozygous knockout mice that grew into adulthood. Importantly, they did not manifest ALS- or ET-like phenotypes until nearly two years. Instead, they showed distinct histological and behavioral alterations including vacuolation in hippocampus, hyperactivity, and reduction in anxiety-like behavior. Knockout mice showed transcriptome alterations including upregulation of Taf15 and Hnrnpa1, while they have normal morphology of RNA-related granules such as Gems.ConclusionsCollectively, FUS/TLS depletion causes phenotypes possibly related to neuropsychiatric and neurodegenerative conditions, but distinct from ALS and ET, together with specific alterations in RNA metabolisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0202-6) contains supplementary material, which is available to authorized users.