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The tuba1a gene encodes a neural-specific alpha-tubulin isoform whose expression is restricted to the developing and regenerating nervous system. 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, due to 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:CreER T2 and β-actin2:loxP-mCherrry-loxP-GFP double transgenic fish that allowed us to conditionally and permanently label tuba1a-expressing cells 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. Following retinal injury, recombination showed tuba1a expression in Müller glia that had reentered the cell cycle and lineage tracing indicated 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.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are inner retinal photoreceptors that mediate non-image-forming visual functions, e.g. pupillary constriction, regulation of pineal melatonin release, and circadian photoentrainment. Five types of ipRGCs were recently discovered in mouse, but whether they exist in other mammals remained unknown. We report that the rat also has five types of ipRGCs, whose morphologies match those of mouse ipRGCs; this is the first demonstration of all five cell types in a non-mouse species. Through immunostaining and λmax measurements, we showed that melanopsin is likely the photopigment of all rat ipRGCs. The various cell types exhibited diverse spontaneous spike rates, with the M1 type spiking the least and M4 spiking the most, just like we had observed for their mouse counterparts. Also similar to mouse, all ipRGCs in rat generated not only sluggish intrinsic photoresponses but also fast, synaptically driven ones. However, we noticed two significant differences between these species. First, whereas we learned previously that all mouse ipRGCs had equally sustained synaptic light responses, rat M1 cells’ synaptic photoresponses were far more transient than those of M2–M5. Since M1 cells provide all input to the circadian clock, this rat-versus-mouse discrepancy could explain the difference in photoentrainment threshold between mouse and other species. Second, rat ipRGCs’ melanopsin-based spiking photoresponses could be classified into three varieties, but only two were discerned for mouse ipRGCs. This correlation of spiking photoresponses with cell types will help researchers classify ipRGCs in multielectrode-array (MEA) spike recordings.

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.bistability | genetic network | synthetic biology | ultrasensitivity | hysteresis B istable genetic systems display a discontinuity of expression states, where two distinct stable steady states are obtained without the presence of stable intermediate steady states. The previous history of the system determines which stable steady state is occupied. One of the important problems in systems biology is to understand how genetic bistability is established and regulated. This is because bistable genetic switches play an important role in a variety of cellular processes, such as cellular oscillators, progression through the eukaryotic cell cycle, and the development of differentiated cell and tissue types in organisms ranging from the temperate bacteriophage to the human (1-7). Many previous studies have focused on whether a given circuit topology has the capacity to display bistability for some range of environmental conditions (e.g., refs. 8-10). Although the possibility of bistable behavior is important, it is also important that the range of environmental conditions at which it occurs be large enough to achieve practical control of biological processes. Here, we focus upon identification and manipulation of the parameters that control the range of environmental conditions at which bistablity is obtained for systems known to be capable of bistability. We use the methods of synthetic biology to create model experimental systems to address the functions of multiple positive feedback loops in bistability.Theoretical studies have argued that the minimal requirements for genetic bistability are twofold. First, there must be some type of positive feedback controlling gene expression. Examples of positive feedback are when an activa...