We examined how correlated firing controls axon remodeling, using in vivo time-lapse imaging and electrophysiological analysis of individual retinal ganglion cell (RGC) axons that were visually stimulated either synchronously or asynchronously relative to neighboring inputs in the Xenopus laevis optic tectum. RGCs stimulated out of synchrony rapidly lost the ability to drive tectal postsynaptic partners while their axons grew and added many new branches. In contrast, synchronously activated RGCs produced fewer new branches, but these were more stable. The effects of synchronous activation were prevented by the inhibition of neurotransmitter release and N-methyl-D-aspartate receptor (NMDAR) blockade, which is consistent with a role for synaptic NMDAR activation in the stabilization of axonal branches and suppression of further exploratory branch addition.

The calcium-regulated protein phosphatase Calcineurin (CaN) participates in synaptic plasticity and the regulation of transcription factors, including Nuclear Factor of Activated T cells (NFAT). To understand how CaN contributes to neuronal circuit development, whole-cell mEPSC recordings and multiphoton imaging were performed in the visual system of living Xenopus laevis tadpoles electroporated to express either a CaN phosphatase inhibitor or N-VIVIT, a nuclear localization sequence-tagged VIVIT peptide that blocks the binding of CaN to select substrates including NFAT. Both strategies increased mEPSC frequency and dendritic arbor complexity in tectal neurons over 3 days. Expression of either of two constitutively active Xenopus NFATs (CA-NFATs) restored normal synaptic properties in neurons expressing N-VIVIT. However, the morphological phenotype was only rescued by a CA-NFAT bearing an intact regulatory domain, implying that transcriptional control of morphological and electrophysiological properties of neurons is mediated by distinct NFAT interactions.

Axin is a negative regulator of the Wnt pathway essential for down-regulation of ␤-catenin. Axin has been considered so far as a cytoplasmic protein. Here we show that, although cytoplasmic at steady state, Axin shuttles in fact in and out of the nucleus; Axin accumulates in the nucleus of cells treated with leptomycin B, a specific inhibitor of the CRM1-mediated nuclear export pathway and is efficiently exported from Xenopus oocyte nuclei in a RanGTP-and CRM1-dependent manner. We have characterized the sequence requirement for export and identified two export domains, which do not contain classical nuclear export consensus sequences, and we show that Axin binds directly to the export factor CRM1 in the presence of RanGTP.

Mesoderm formation results from an inducing process that requires maternal and zygotic FGF/MAPK and TGFb activities, while maternal activation of the Wnt/ b-catenin pathway determines the anterior±dorsal axis. Here, we show a new role of Wnt/b-catenin signaling in mesoderm induction. We ®nd that maternal b-catenin signaling is not only active dorsally but also all around the equatorial region, coinciding with the prospective mesoderm. Maternal b-catenin function is required both for expression of dorsal genes and for activation of MAPK and the mesodermal markers Xbra and eomesodermin. b-catenin acts in a noncell-autonomous manner upstream of zygotic FGF and nodal signals. The Wnt/b-catenin activity in the equatorial region of the early embryo is the ®rst example of a maternally provided mesoderm inducer restricted to the prospective mesoderm. Keywords: embryonic development/FGF/MAPK/ Wnt pathway/Xnr IntroductionThe basic subdivision of the vertebrate embryo into the three germ layers, ectoderm, mesoderm and endoderm, is established very early during development. In Xenopus, the three layers form along the primary animal±vegetal symmetry axis of the embryo: the pigmented animal pole becomes ectoderm, the equatorial region becomes mesoderm, and the yolk-rich vegetal pole becomes endoderm. A second axis is created at fertilization, which de®nes the dorso-ventral polarity, and the superimposition of these two patterns can account for the basic body plan of the embryo. Both patterns appear to rely on inductive signals, which are generated by pre-localized maternal determinants. The vegetal hemisphere seems to be the major source of such determinants: in particular, the vegetal pole emits a signal which can redirect the fate of animal cells from ectoderm to mesoderm and endoderm (Nieuwkoop, 1969). Another vegetally localized determinant is translocated on one side of the egg after fertilization and later induces the various dorsalizing signals, which constitute the activity of Spemann's organizer (Harland and Gerhart, 1997).Mesoderm±endoderm induction has been extensively studied, and the molecular nature of the signal has been at least partly elucidated (Harland and Gerhart, 1997;Zhang et al., 1998;Kimelman and Grif®n, 2000). Experiments using dominant-negative receptor constructs have revealed an essential role for secreted growth factors of the FGF and TGFb/activin family: FGF signaling was found to be necessary for induction of part of the mesodermal genes, while TGFb/activin-like signaling is required for formation of all mesoderm and endoderm. Most of the recent studies have focused on the role of TFGb-like factors of the Nodal sub-family, so-called Xenopus Nodal-related proteins (Xnrs). Xnrs are expressed zygotically under the control of a maternal, vegetally localized transcription factor, VegT (Clements et al., 1999). Knock down experiments have shown that depletion of maternal VegT has profound effects on early patterning: endoderm is not induced, mesoderm formation is delayed and, when some mesoderm eventua...

Near-infrared (NIR) genetically encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.

In the developing Xenopus tadpole, conditioning with 20 min of visual stimulation leads to increased proBDNF protein levels in the tectum measured 4 hr later. Following conditioning, the ability to induce direction selectivity in tectal neurons, as well as both retinotectal long-term potentiation and depression, thought to underlie this phenomenon, was strongly facilitated. This facilitation was blocked by knockdown of BDNF expression in tectal neurons. Animals that had been exposed to visual conditioning and subsequently received normal visual input for 7-11 hr exhibited higher spatial frequency thresholds of tectal cell responses to counterphasing gratings than nonconditioned control animals. An improvement in visual acuity was confirmed by enhanced sensitivity to counterphasing gratings in a behavioral test. These results indicate that brief sensory stimulation, by initiating nuclear transcription and de novo protein synthesis of BDNF, can facilitate the refinement of response properties in the developing visual system.

Radial glia in the developing optic tectum express the key guidance molecules responsible for topographic targeting of retinal axons. However, the extent to which the radial glia are themselves influenced by retinal inputs and visual experience remains unknown. Using multiphoton live imaging of radial glia in the optic tectum of intact Xenopus laevis tadpoles in conjunction with manipulations of neural activity and sensory stimuli, radial glia were observed to exhibit spontaneous calcium transients that were modulated by visual stimulation. Structurally, radial glia extended and retracted many filopodial processes within the tectal neuropil over minutes. These processes interacted with retinotectal synapses and their motility was modulated by nitric oxide (NO) signaling downstream of neuronal NMDA receptor (NMDAR) activation and visual stimulation. These findings provide the first in vivo demonstration that radial glia actively respond both structurally and functionally to neural activity, via NMDAR-dependent NO release during the period of retinal axon ingrowth.

In Xenopus laevis, patterning of the trunk mesoderm into the dorsal notochord and lateral somites depends on differential regulation of Wnt–β-catenin signaling. To study the cellular requirements for the physical separation of these tissues, we manipulated β-catenin activity in individual cells that were scattered within the trunk mesoderm. We found that high activity led to efficient cell sorting from the notochord to the somites, whereas reduced activity led to sorting in the opposite direction. Analysis of individual cells overexpressing β-catenin revealed that these cells were unable to establish stable contacts with notochord cells but could freely cross the boundary to integrate within the somitic tissue. Interference with cadherin-mediated adhesion disrupted tissue architecture, but it did not affect sorting and boundary formation. Based on these results, we propose that the boundary itself is the result of cell-autonomous changes in contact behavior that do not rely on differences in absolute levels of adhesion.