Philip R. Williams

Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina

Cellular mechanisms underlying the precision by which neurons target their synaptic partners have largely been determined based on the study of projection neurons. By contrast, little is known about how interneurons establish their local connections in vivo. Here, we investigated how developing amacrine interneurons selectively innervate the appropriate region of the synaptic neuropil in the inner retina, the inner plexiform layer (IPL). Increases (ON) and decreases (OFF) in light intensity are processed by circuits that are structurally confined to separate ON and OFF synaptic sublaminae within the IPL. Using transgenic zebrafish in which the majority of amacrine cells express fluorescent protein, we determined that the earliest amacrine-derived neuritic plexus formed between two cell populations whose somata, at maturity, resided on opposite sides of this plexus. When we followed the behavior of individual amacrine cells over time, we discovered that they exhibited distinct patterns of structural dynamics at different stages of development. During cellular migration, amacrine cells exhibited an exuberant outgrowth of neurites that was undirected. Upon reaching the forming IPL, neurites extending towards the ganglion cell layer were relatively more stable. Importantly, when an arbor first formed, it preferentially ramified in either the inner or outer IPL corresponding to the future ON and OFF sublaminae, and maintained this stratification pattern. The specificity by which ON and OFF amacrine interneurons innervate their respective sublaminae in the IPL contrasts with that observed for projection neurons in the retina and elsewhere in the central nervous system.

In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells

Targeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity.

Patterns of Gene Expression Reveal a Temporally Orchestrated Wound Healing Response in the Injured Spinal Cord

Spinal cord injury (SCI) induces a progressive pathophysiology affecting cell survival and neurological integrity via complex and evolving molecular cascades whose interrelationships are not fully understood. The present experiments were designed to: (1) determine potential functional interactions within transcriptional expression profiles obtained after a clinically relevant SCI and (2) test the consistency of transcript expression after SCI in two genetically and immunologically diverse rat strains characterized by differences in T cell competence and associated inflammatory responses. By interrogating Affymetrix U34A rat genome GeneChip microarrays, we defined the transcriptional expression patterns in midcervical contusion lesion sites between 1 and 90 d postinjury of athymic nude (AN) and Sprague Dawley (SD) strains. Stringent statistical analyses detected significant changes in 3638 probe sets, with 80 genes differing between the AN and SD groups. Subsequent detailed functional categorization of these transcripts unveiled an overall tissue remodeling response that was common to both strains. The functionally organized gene profiles were temporally distinct and correlated with repair indices observed microscopically and by magnetic resonance microimaging. Our molecular and anatomical observations have identified a novel, longitudinal perspective of the post-SCI response, namely, that of a highly orchestrated tissue repair and remodeling repertoire with a prominent cutaneous wound healing signature that is conserved between two widely differing rat strains. These results have significant bearing on the continuing development of cellular and pharmacological therapeutics directed at tissue rescue and neuronal regeneration in the injured spinal cord.

Nonapical Symmetric Divisions Underlie Horizontal Cell Layer Formation in the Developing Retina In Vivo

Symmetric cell divisions have been proposed to rapidly increase neuronal number late in neurogenesis, but how critical this mode of division is to establishing a specific neuronal layer is unknown. Using in vivo time-lapse imaging methods, we discovered that in the laminated zebrafish retina, the horizontal cell (HC) layer forms quickly during embryonic development upon division of a precursor cell population. The precursor cells morphologically resemble immature, postmitotic HCs and express HC markers such as ptf1a and Prox1 prior to division. These precursors undergo nonapical symmetric division at the laminar location where mature HCs contact photoreceptors. Strikingly, the precursor cell type we observed generates exclusively HCs. We have thus identified a dedicated HC precursor, and our findings suggest a mechanism of neuronal layer formation whereby the location of mitosis could facilitate rapid contact between synaptic partners.

Cone photoreceptor types in zebrafish are generated by symmetric terminal divisions of dedicated precursors

Significance Color vision requires multiple types of cone photoreceptors, each with peak sensitivity to a specific wavelength. How different cone types are generated in vivo is not clear. We show that there are precursor cells individually dedicated to producing a single cone type. We tracked cone genesis in vivo in transgenic zebrafish in which red cones and their progenitors express fluorescent protein driven by the thyroid hormone receptor β2 promoter. We discovered that red cones are generated by symmetric terminal divisions of a red-cone precursor. Moreover, UV, blue, and green cones also have their own dedicated precursors. Thyroid hormone receptor β2 expression in cone precursors is required to produce pure red cones, whereas expression after cell division results in cones with mixed opsins. Proper functioning of sensory systems requires the generation of appropriate numbers and proportions of neuronal subtypes that encode distinct information. Perception of color relies on signals from multiple cone photoreceptor types. In cone-dominated retinas, each cone expresses a single opsin type with peak sensitivity to UV, long (L) (red), medium (M) (green), or short (S) (blue) wavelengths. The modes of cell division generating distinct cone types are unknown. We report here a mechanism whereby zebrafish cone photoreceptors of the same type are produced by symmetric division of dedicated precursors. Transgenic fish in which the thyroid hormone receptor β2 (trβ2) promoter drives fluorescent protein expression before L-cone precursors themselves are produced permitted tracking of their division in vivo. Every L cone in a local region resulted from the terminal division of an L-cone precursor, suggesting that such divisions contribute significantly to L-cone production. Analysis of the fate of isolated pairs of cones and time-lapse observations suggest that other cone types can also arise by symmetric terminal divisions. Such divisions of dedicated precursors may help to rapidly attain the final numbers and proportions of cone types (L > M, UV > S) in zebrafish larvae. Loss- and gain-of-function experiments show that L-opsin expression requires trβ2 activity before cone differentiation. Ectopic expression of trβ2 after cone differentiation produces cones with mixed opsins. Temporal differences in the onset of trβ2 expression could explain why some species have mixed, and others have pure, cone types.

An assay to image neuronal microtubule dynamics in mice

Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.

In Vivo Development of Outer Retinal Synapses in the Absence of Glial Contact

Astroglia secrete factors that promote synapse formation and maintenance. In culture, glial contact has also been shown to facilitate synaptogenesis. Here, we examined whether glial contact is important for establishing circuits in vivo by simultaneously monitoring differentiation of glial cells and local synaptogenesis over time. Photoreceptor circuits of the vertebrate retina are particularly suitable for this study because of the relatively simple, laminar organization of their connectivity with their target neurons, horizontal cells and bipolar cells. Also, individual photoreceptor terminals are ensheathed within the outer plexiform layer (OPL) by the processes of one type of glia, Müller glia cells (MGs). We conducted in vivo time-lapse multiphoton imaging of the rapidly developing and relatively transparent zebrafish retina to ascertain the time course of MG development relative to OPL synaptogenesis. The emergence of synaptic triads, indicative of functional photoreceptor circuits, and structural association with glial processes were also examined across ages by electron microscopy. We first show that MG processes form territories that tile within the inner and outer synaptic layers. We then demonstrate that cone photoreceptor synapses are assembled before the elaboration of MG processes in the OPL. Using a targeted cell ablation approach, we also determined whether the maintenance of photoreceptor synapses is perturbed when local MGs are absent. We found that removal of MGs had no appreciable effect on the stability of newly formed cone synapses. Thus, in contrast to other CNS circuits, contact from glia is not necessary for the formation or immediate stabilization of outer retinal synapses.

A recoverable state of axon injury persists for hours after spinal cord contusion in vivo

Therapeutic strategies for spinal cord injury (SCI) commonly focus on regenerating disconnected axons. An alternative approach would be to maintain continuity of damaged axons, especially after contusion. The viability of such neuropreservative strategies depends on the degree to which initially injured axons can recover. Here we use morphological and molecular in vivo imaging after contusion SCI in mice to show that injured axons persist in a metastable state for hours. Intra-axonal calcium dynamics influence fate, but the outcome is not invariably destructive in that many axons with calcium elevations recover homeostasis without intervention. Calcium enters axons primarily through mechanopores. Spontaneous pore resealing allows calcium levels to normalize and axons to survive long term. Axon loss can be halted by blocking calcium influx or calpain, even with delayed initiation. Our data identify an inherent self-preservation process in contused axons and a window of opportunity for rescuing connectivity after nontransecting SCI.

Wild-Type Cone Photoreceptors Persist Despite Neighboring Mutant Cone Degeneration

In many retinal diseases, the malfunction that results in photoreceptor loss occurs only in either rods or cones, but degeneration can progress from the affected cell type to its healthy neighbors. Specifically, in human and mouse models of Retinitis Pigmentosa the loss of rods results in the death of neighboring healthy cones. Significantly less is known about cone-initiated degenerations and their affect on neighboring cells. Sometimes rods remain normal after cone death, whereas other patients experience a loss of scotopic vision over time. The affect of cone death on neighboring cones is unknown. The zebrafish is a cone-rich animal model in which the potential for dying cones to kill neighboring healthy cones can be evaluated. We previously reported that the zebrafish cone phosphodiesterase mutant (pde6c w59) displays a rapid death of cones soon after their formation and a subsequent loss of rods in the central retina. In this study we examine morphological changes associated with cone death in vivo in pde6c w59 fish. We then use blastulae transplantations to create chimeric fish with a photoreceptor layer of mixed wild-type (WT) and pde6c w59 cones. We find that the death of inoperative cones does not cause neighboring WT cone loss. The survival of WT cones is independent of transplant size and location within the retina. Furthermore, transplanted WT cones persist at least several weeks after the initial death of dysfunctional mutant cones. Our results suggest a potential for the therapeutic transplantation of healthy cones into an environment of damaged cones.

Transmission from the dominant input shapes the stereotypic ratio of photoreceptor inputs onto horizontal cells

Many neurons receive synapses in stereotypic proportions from converging but functionally distinct afferents. However, developmental mechanisms regulating synaptic convergence are not well understood. Here we describe a heterotypic mechanism by which one afferent controls synaptogenesis of another afferent, but not vice-versa. Like other CNS circuits, zebrafish retinal H3 horizontal cells undergo an initial period of remodeling, establishing synapses with UV and blue cones while eliminating red and green cone contacts. As development progresses, the horizontal cells selectively synapse with UV cones to generate a 5:1 UV-to-blue cone synapse ratio. Blue cone synaptogenesis increases in mutants lacking UV cones, and when transmitter release or visual stimulation of UV cones is perturbed. Connectivity is unaltered when blue cone transmission is suppressed. Moreover, there is no homotypic regulation of cone synaptogenesis by neurotransmission. Thus, biased connectivity in this circuit is established by an unusual activity-dependent, unidirectional control of synaptogenesis exerted by the dominant input.