Aaron Reifler

Building biological memory by linking positive feedback loops

A common topology found in many bistable genetic systems is two interacting positive feedback loops. Here we explore how this relatively simple topology can allow bistability over a large range of cellular conditions. On the basis of theoretical arguments, we predict that nonlinear interactions between two positive feedback loops can produce an ultrasensitive response that increases the range of cellular conditions at which bistability is observed. This prediction was experimentally tested by constructing a synthetic genetic circuit in Escherichia coli containing two well-characterized positive feedback loops, linked in a coherent fashion. The concerted action of both positive feedback loops resulted in bistable behavior over a broad range of inducer concentrations; when either of the feedback loops was removed, the range of inducer concentrations at which the system exhibited bistability was decreased by an order of magnitude. Furthermore, bistability of the system could be tuned by altering growth conditions that regulate the contribution of one of the feedback loops. Our theoretical and experimental work shows how linked positive feedback loops may produce the robust bistable responses required in cellular networks that regulate development, the cell cycle, and many other cellular responses.

Conditional gene expression and lineage tracing of tuba1a expressing cells during zebrafish development and retina regeneration

The tuba1a gene encodes a neural‐specific α‐tubulin isoform whose expression is restricted to the developing and regenerating nervous system. By using zebrafish as a model system for studying CNS regeneration, we recently showed that retinal injury induces tuba1a gene expression in Müller glia that reentered the cell cycle. However, because of the transient nature of tuba1a gene expression during development and regeneration, it was not possible to trace the lineage of the tuba1a‐expressing cells with a reporter directly under the control of the tuba1a promoter. To overcome this limitation, we generated tuba1a:CreERT2 and β‐actin2:loxP‐mCherrry‐loxP‐GFP double transgenic fish that allowed us to label tuba1a‐expressing cells conditionally and permanently via ligand‐induced recombination. During development, recombination revealed transient tuba1a expression in not only neural progenitors but also cells that contribute to skeletal muscle, heart, and intestine. In the adult, recombination revealed tuba1a expression in brain, olfactory neurons, and sensory cells of the lateral line, but not in the retina. After retinal injury, recombination showed tuba1a expression in Müller glia that had reentered the cell cycle, and lineage tracing indicated that these cells are responsible for regenerating retinal neurons and glia. These results suggest that tuba1a‐expressing progenitors contribute to multiple cell lineages during development and that tuba1a‐expressing Müller glia are retinal progenitors in the adult. J. Comp. Neurol. 518:4196–4212, 2010.

PIK3C2B inhibition improves function and prolongs survival in myotubular myopathy animal models

Myotubular myopathy (MTM) is a devastating pediatric neuromuscular disorder of phosphoinositide (PIP) metabolism resulting from mutations of the PIP phosphatase MTM1 for which there are no treatments. We have previously shown phosphatidylinositol-3-phosphate (PI3P) accumulation in animal models of MTM. Here, we tested the hypothesis that lowering PI3P levels may prevent or reverse the MTM disease process. To test this, we targeted class II and III PI3 kinases (PI3Ks) in an MTM1-deficient mouse model. Muscle-specific ablation of Pik3c2b, but not Pik3c3, resulted in complete prevention of the MTM phenotype, and postsymptomatic targeting promoted a striking rescue of disease. We confirmed this genetic interaction in zebrafish, and additionally showed that certain PI3K inhibitors prevented development of the zebrafish mtm phenotype. Finally, the PI3K inhibitor wortmannin improved motor function and prolonged lifespan of the Mtm1-deficient mice. In all, we have identified Pik3c2b as a genetic modifier of Mtm1 mutation and demonstrated that PIK3C2B inhibition is a potential treatment strategy for MTM. In addition, we set the groundwork for similar reciprocal inhibition approaches for treating other PIP metabolic disorders and highlight the importance of modifier gene pathways as therapeutic targets.

All Spiking, Sustained ON Displaced Amacrine Cells Receive Gap-Junction Input from Melanopsin Ganglion Cells

Retinal neurons exhibit sustained versus transient light responses, which are thought to encode low- and high-frequency stimuli, respectively. This dichotomy has been recognized since the earliest intracellular recordings from the 1960s, but the underlying mechanisms are not yet fully understood. We report that in the ganglion cell layer of rat retinas, all spiking amacrine interneurons with sustained ON photoresponses receive gap-junction input from intrinsically photosensitive retinal ganglion cells (ipRGCs), recently discovered photoreceptors that specialize in prolonged irradiance detection. This input presumably allows ipRGCs to regulate the secretion of neuromodulators from these interneurons. We have identified three morphological varieties of such ipRGC-driven displaced amacrine cells: (1) monostratified cells with dendrites terminating exclusively in sublamina S5 of the inner plexiform layer, (2) bistratified cells with dendrites in both S1 and S5, and (3) polyaxonal cells with dendrites and axons stratifying in S5. Most of these amacrine cells are wide field, although some are medium field. The three classes respond to light differently, suggesting that they probably perform diverse functions. These results demonstrate that ipRGCs are a major source of tonic visual information within the retina and exert widespread intraretinal influence. They also add to recent evidence that ganglion cells signal not only to the brain.

Conditional Knockout of Pik3c3 Causes a Murine Muscular Dystrophy

Abnormalities in phosphoinositide metabolism are an emerging theme in human neurodegenerative disease. Myotubular myopathy is a prototypical disorder of phosphoinositide dysregulation that is characterized by profound muscle pathology and weakness and that is caused by mutations in MTM1, which encodes a phosphatase that targets 3-position phosphoinositides, including phosphatidylinositol 3-phosphate. Although the association between MTM1 and muscle disease has become increasingly clarified, the normal role(s) of phosphatidylinositol 3-phosphate metabolism in muscle development and homeostasis remain poorly understood. To begin to address the function of phosphatidylinositol 3-phosphate in skeletal muscle, we focused on the primary kinase responsible for its production, and created a muscle-specific conditional knockout of the class III phosphatidylinositol 3-kinase, Pik3c3. Muscle-specific deletion of Pik3c3 did not disturb embryogenesis or early postnatal development, but resulted in progressive disease characterized by reduced activity and death by 2 months of age. Histopathological analysis demonstrated changes consistent with a murine muscular dystrophy. Examination for cellular mechanism(s) responsible for the dystrophic phenotype revealed significant alterations in the autophagolysosomal pathway with mislocation of known dystrophy proteins to the lysosomal compartment. In all, we present the first analysis of Pik3c3 in skeletal muscle, and report a novel association between deletion of Pik3c3 and muscular dystrophy.

Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures

Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na(+) and K(+) currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.

Characterizing and modeling the intrinsic light response of rat ganglion-cell photoreceptors

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate both image-forming vision and non-image-forming visual responses such as pupillary constriction and circadian photoentrainment. Five types of ipRGCs, named M1-M5, have been discovered in rodents. To further investigate their photoresponse properties, we made multielectrode array spike recordings from rat ipRGCs, classified them into M1, M2/M4, and M3/M5 clusters, and measured their intrinsic, melanopsin-based responses to single and flickering light pulses. Results showed that ipRGC spiking can track flickers up to ∼0.2 Hz in frequency and that flicker intervals between 5 and 14 s evoke the most spikes. We also learned that melanopsins integration time is intensity and cluster dependent. Using these data, we constructed a mathematical model for each clusters intrinsic photoresponse. We found that the data for the M1 cluster are best fit by a model that assumes a large photoresponse, causing the cell to enter depolarization block. Our models also led us to hypothesize that the M2/M4 and M3/M5 clusters experience comparable photoexcitation but that the M3/M5 cascade decays significantly faster than the M2/M4 cascade, resulting in different response waveforms between these clusters. These mathematical models will help predict how each ipRGC cluster might respond to stimuli of any waveform and could inform the invention of lighting technologies that promote health through melanopsin stimulation.

Application of Cre-loxP recombination for lineage tracing of adult zebrafish retinal stem cells.

The Cre-loxP recombination system is widely used as a genetic tool to achieve conditional gene expression and for lineage tracing. Though extensively used in mice, this technology has only recently been applied to zebrafish. Here we describe Cre-loxP methodology for conditional expression of transgenes in zebrafish and their use in lineage tracing Müller glia as they undergo cellular reprogramming and proliferation to repair damaged retinal circuitry following mechanical injury. This methodology can be used for conditional gene expression and lineage tracing at any stage of development and in any cell type.

Murine Fig4 is dispensable for muscle development but required for muscle function

BackgroundPhosphatidylinositol phosphates (PIPs) are low-abundance phospholipids that participate in a range of cellular processes, including cell migration and membrane traffic. PIP levels and subcellular distribution are regulated by a series of lipid kinases and phosphatases. In skeletal muscle, PIPs and their enzymatic regulators serve critically important functions exemplified by mutations of the PIP phosphatase MTM1 in myotubular myopathy (MTM), a severe muscle disease characterized by impaired muscle structure and abnormal excitation–contraction coupling. FIG4 functions as a PIP phosphatase that participates in both the synthesis and breakdown of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Mutation of FIG4 results in a severe neurodegenerative disorder in mice and a progressive peripheral polyneuropathy in humans. The effect of FIG4 mutation on skeletal muscle has yet to be examined.MethodsHerein we characterize the impact of FIG4 on skeletal muscle development and function using the spontaneously occurring mouse mutant pale tremor (plt), a mouse line with a loss of function mutation in Fig4.ResultsIn plt mice, we characterized abnormalities in skeletal muscle, including reduced muscle size and specific force generation. We also uncovered ultrastructural abnormalities and increased programmed cell death. Conversely, we detected no structural or functional abnormalities to suggest impairment of excitation–contraction coupling, a process previously shown to be influenced by PI(3,5)P2 levels. Conditional rescue of Fig4 mutation in neurons prevented overt muscle weakness and the development of obvious muscle abnormalities, suggesting that the changes observed in the plt mice were primarily related to denervation of skeletal muscle. On the basis of the ability of reduced FIG4 levels to rescue aspects of Mtmr2-dependent neuropathy, we evaluated the effect of Fig4 haploinsufficiency on the myopathy of Mtm1-knockout mice. Male mice with a compound Fig4+/−/Mtm1–/Y genotype displayed no improvements in muscle histology, muscle size or overall survival, indicating that FIG4 reduction does not ameliorate the Mtm1-knockout phenotype.ConclusionsOverall, these data indicate that loss of Fig4 impairs skeletal muscle function but does not significantly affect its structural development.

P.4.1 PIP kinases, muscle development, and the pathogenesis of myotubular myopathy

Myotubular myopathy (MTM) is a severe congenital myopathy with no currently identified treatment. MTM is caused by mutations in MTM1, a phosphatase that dephosphorylates 3-position phosphoinositides. Through the use of vertebrate model systems, the consequences (s) of MTM1 mutation in skeletal muscle in vivo are beginning to be unraveled. In particular, work from several laboratories (including our own) has demonstrated that loss of MTM1 (1) increases the levels of PI3P in skeletal muscle and (2) results in the disruption of the structure and function of the EC coupling apparatus. Based on these data, one hypothesis to explain MTM is that pathologic elevation of PI3P results in aberrant EC coupling, which in turn causes muscle weakness and severe neurologic disability. A correlate hypothesis is that lowering PI3P levels in MTM will enable normal muscle structure and will thus ameliorate the disease. These hypotheses lead to 2 critical questions: What is the consequence of reducing PI3P levels on normal skeletal muscle development? What is the impact of reducing PI3P levels on MTM pathogenesis? The goal of this study is to address these questions by studying models with reduced PI3P. We have created knockout mice that lack either Pik3c2b or Pik3c3, the kinases responsible for PI3P production in skeletal muscle. Using these animals, we have identified unique and non-overlapping functions for these kinases, including the observation that loss of Pik3c3 results in a muscular dystrophy phenotype. Furthermore, we have tested the concept of PI3P reduction as a therapeutic strategy for MTM by crossbreeding individual kinase knockouts with Mtm1 knockouts. Preliminary data shows that reduced expression of one of the kinases significantly improves the Mtm1 phenotype. In all, our data reveals for the first time the role of 3-PI kinases in skeletal muscle, and identifies a potential therapeutic target for the treatment of MTM.