Prolonged, brain-wide expression of nuclear-localized GCaMP3 for functional circuit mapping
FRONTIERS IN NEURAL CIRCUITS
Mitotic Position and Morphology of Committed Precursor Cells in the Zebrafish Retina Adapt to Architectural Changes upon Tissue Maturation
2014; 7 (2): 386-397
Prolonged, brain-wide expression of nuclear-localized GCaMP3 for functional circuit mapping.
Frontiers in neural circuits
2014; 8: 138-?
The development of complex neuronal tissues like the vertebrate retina requires the tight orchestration of cell proliferation and differentiation. Although the complexity of transcription factors and signaling pathways involved in retinogenesis has been studied extensively, the influence of tissue maturation itself has not yet been systematically explored. Here, we present a quantitative analysis of mitotic events during zebrafish retinogenesis that reveals three types of committed neuronal precursors in addition to the previously known apical progenitors. The identified precursor types present at distinct developmental stages and exhibit different mitotic location (apical versus nonapical), cleavage plane orientation, and morphology. Interestingly, the emergence of nonapically dividing committed bipolar cell precursors can be linked to an increase in apical crowding caused by the developing photoreceptor cell layer. Furthermore, genetic interference with neuronal subset specification induces ectopic divisions of committed precursors, underlining the finding that progressing morphogenesis can effect precursor division position.
View details for DOI 10.1016/j.celrep.2014.03.014
View details for Web of Science ID 000335442300010
View details for PubMedID 24703843
Imaging zebrafish neural circuitry from whole brain to synapse
FRONTIERS IN NEURAL CIRCUITS
Coupling of NF-protocadherin signaling to axon guidance by cue-induced translation
2013; 16 (2): 166-173
Larval zebrafish offer the potential for large-scale optical imaging of neural activity throughout the central nervous system; however, several barriers challenge their utility. First, ~panneuronal probe expression has to date only been demonstrated at early larval stages up to 7 days post-fertilization (dpf), precluding imaging at later time points when circuits are more mature. Second, nuclear exclusion of genetically-encoded calcium indicators (GECIs) limits the resolution of functional fluorescence signals collected during imaging. Here, we report the creation of transgenic zebrafish strains exhibiting robust, nuclearly targeted expression of GCaMP3 across the brain up to at least 14 dpf utilizing a previously described optimized Gal4-UAS system. We confirmed both nuclear targeting and functionality of the modified probe in vitro and measured its kinetics in response to action potentials (APs). We then demonstrated in vivo functionality of nuclear-localized GCaMP3 in transgenic zebrafish strains by identifying eye position-sensitive fluorescence fluctuations in caudal hindbrain neurons during spontaneous eye movements. Our methodological approach will facilitate studies of larval zebrafish circuitry by both improving resolution of functional Ca(2+) signals and by allowing brain-wide expression of improved GECIs, or potentially any probe, further into development.
View details for DOI 10.3389/fncir.2014.00138
View details for PubMedID 25505384
Imaging zebrafish neural circuitry from whole brain to synapse.
Frontiers in neural circuits
2013; 7: 76-?
Cell adhesion molecules and diffusible cues both regulate axon pathfinding, yet how these two modes of signaling interact is poorly understood. The homophilic cell adhesion molecule NF-protocadherin (NFPC) is expressed in the mid-dorsal optic tract neuroepithelium and in the axons of developing retinal ganglion cells (RGC) in Xenopus laevis. Here we report that targeted disruption of NFPC function in RGC axons or the optic tract neuroepithelium results in unexpectedly localized pathfinding defects at the caudal turn in the mid-optic tract. Semaphorin 3A (Sema3A), which lies adjacent to this turn, stimulates rapid, protein synthesis-dependent increases in growth cone NFPC and its cofactor, TAF1, in vitro. In vivo, growth cones exhibit marked increases in NFPC translation reporter activity in this mid-optic tract region that are attenuated by blocking neuropilin-1 function. Our results suggest that translation-linked coupling between regionally localized diffusible cues and cell adhesion can help axons navigate discrete segments of the pathway.
View details for DOI 10.1038/nn.3290
View details for Web of Science ID 000314260200013
View details for PubMedID 23292679
Imaging axon pathfinding in zebrafish in vivo.
Cold Spring Harbor protocols
2012; 2012 (9): 992-997
Recent advances in imaging tools are inspiring zebrafish researchers to tackle ever more ambitious questions in the neurosciences. Behaviorally fundamental conserved neural networks can now be potentially studied using zebrafish from a brain-wide scale to molecular resolution. In this perspective, we offer a roadmap by which a zebrafish researcher can navigate the course from collecting neural activities across the brain associated with a behavior, to unraveling molecular identities and testing the functional relevance of active neurons. In doing so, important insights will be gained as to how neural networks generate behaviors and assimilate changes in synaptic connectivity.
View details for DOI 10.3389/fncir.2013.00076
View details for PubMedID 23630470
Imaging axon pathfinding in Xenopus in vivo.
Cold Spring Harbor protocols
2012; 2012 (9): 984-991
Axon pathfinding in the developing animal involves a highly dynamic process in which the axonal growth cone makes continuous decisions as it navigates toward its target. Changes occurring in the growth cone with respect to retracting from or extending into complex new territories can occur in minutes. Thus, the advent of strategies to visualize axon path-finding in vivo in a live intact animal is crucial for a better understanding of how the growth cone makes such rapid decisions in response to multiple cues. Combining these strategies with loss-of-function and/or gain-of-function techniques, one can gain some insight as to which molecules are crucial to particular growth cone behaviors at specific choice points during navigation. The major advantage of using zebrafish lies in the accessibility of major axon tracts for live microscopy, as their embryonic development occurs ex utero. Furthermore, the robust embryos remain healthy during immobilization and allow for good imaging for long periods. This protocol describes the method for stabilizing and preparing live zebrafish embryos for imaging labeled axonal tracts at high spatial and temporal resolution for up to 72 h. It has been used for retinotectal axon pathfinding, but can be adapted to visualize other axon tracts of interest.
View details for DOI 10.1101/pdb.prot070011
View details for PubMedID 22949713
Slit1b-Robo3 Signaling and N-Cadherin Regulate Apical Process Retraction in Developing Retinal Ganglion Cells
JOURNAL OF NEUROSCIENCE
2012; 32 (1): 223-228
Axon pathfinding in the developing animal involves a highly dynamic process in which the axonal growth cone makes continuous decisions as it navigates toward its target. Changes occurring in the growth cone with respect to retracting from or extending into complex new territories can occur in minutes. Thus, the advent of strategies to visualize axon path-finding in vivo in a live intact animal is crucial for a better understanding of how the growth cone makes such rapid decisions in response to multiple cues. Combining these strategies with loss-of-function and/or gain-of-function techniques allows one to gain some insight as to which molecules are crucial to particular growth cone behaviors at specific choice points during navigation. The main advantage of using Xenopus lies in the accessibility of major axon tracts for live microscopy, as their embryonic development occurs ex utero. Furthermore, the robust embryos remain healthy during immobilization and allow for good imaging for long periods. This protocol describes the methods for stabilizing and preparing live Xenopus embryos for imaging labeled axonal tracts at high spatial and temporal resolution for up to 72 h. This approach can been used to investigate how the knockdown of certain gene functions can affect the speed of navigation through the well-studied Xenopus retinotectal pathway. It can be adapted to visualize other axon tracts of interest.
View details for DOI 10.1101/pdb.prot070003
View details for PubMedID 22949712
Apical migration of nuclei during G2 is a prerequisite for all nuclear motion in zebrafish neuroepithelia
2011; 138 (22): 5003-5013
When neurons exit the cell cycle after their terminal mitosis, they detach from the apical surface of the neuroepithelium. Despite the fact that this detachment is crucial for further neurogenesis and neuronal migration, the underlying mechanisms are still not understood. Here, taking advantage of the genetics and imaging possibilities of the zebrafish retina as a model system, we show by knockdown experiments that the guidance molecule Slit1b and its receptor Robo3 are required for apical retraction of retinal ganglion cells (RGCs). In contrast, N-cadherin seems to be responsible for maintenance of apical attachment, as expression of dominant-negative N-cadherin causes RGCs to lose apical attachments prematurely and rescues retraction in slit1b morphants. These results suggest that Slit-Robo signaling downregulates N-cadherin activity to allow apical retraction in newly generated RGCs.
View details for DOI 10.1523/JNEUROSCI.2596-11.2012
View details for Web of Science ID 000299119700021
View details for PubMedID 22219284
NF-Protocadherin and TAF1 regulate retinal axon initiation and elongation in vivo
JOURNAL OF NEUROSCIENCE
2008; 28 (1): 100-105
Nuclei in the proliferative pseudostratified epithelia of vastly different organisms exhibit a characteristic dynamics - the so-called interkinetic nuclear migration (IKNM). Although these movements are thought to be intimately tied to the cell cycle, little is known about the relationship between IKNM and distinct phases of the cell cycle and the role that this association plays in ensuring balanced proliferation and subsequent differentiation. Here, we perform a quantitative analysis of modes of nuclear migration during the cell cycle using a marker that enables the first unequivocal differentiation of all four phases in proliferating neuroepithelial cells in vivo. In zebrafish neuroepithelia, nuclei spend the majority of the cell cycle in S phase, less time in G1, with G2 and M being noticeably shorter still in comparison. Correlating cell cycle phases with nuclear movements shows that IKNM comprises rapid apical nuclear migration during G2 phase and stochastic nuclear motion during G1 and S phases. The rapid apical migration coincides with the onset of G2, during which we find basal actomyosin accumulation. Inhibiting the transition from G2 to M phase induces a complete stalling of nuclei, indicating that IKNM and cell cycle continuation cannot be uncoupled and that progression from G2 to M is a prerequisite for rapid apical migration. Taken together, these results suggest that IKNM involves an actomyosin-driven contraction of cytoplasm basal to the nucleus during G2, and that the stochastic nuclear movements observed in other phases arise passively due to apical migration in neighboring cells.
View details for DOI 10.1242/dev.071522
View details for Web of Science ID 000296576700018
View details for PubMedID 22028032
NF-protocadherin (NFPC)-mediated cell-cell adhesion plays a critical role in vertebrate neural tube formation. NFPC is also expressed during the period of axon tract formation, but little is known about its function in axonogenesis. Here we have tested the role of NFPC and its cytosolic cofactor template-activating factor 1 (TAF1) in the emergence of the Xenopus retinotectal projection. NFPC is expressed in the developing retina and optic pathway and is abundant in growing retinal axons. Inhibition of NFPC function in developing retinal ganglion cells (RGCs) severely reduces axon initiation and elongation and suppresses dendrite genesis. Furthermore, an identical phenotype occurs when TAF1 function is blocked. These data provide evidence that NFPC regulates axon initiation and elongation and indicate a conserved role for TAF1, a transcriptional regulator, as a downstream cytosolic effector of NFPC in RGCs.
View details for DOI 10.1523/JNEUROSCI.4490-07.2008
View details for Web of Science ID 000252242900012
View details for PubMedID 18171927