Dissociation and reaggregation of Hydra vulgaris for studies of self-organization.

The remarkable regenerative abilities of the small cnidarian Hydra vulgaris include the capacity to reassemble itself after dissociation into individual cells. Here, we present a robust protocol for the dissociation and reaggregation of Hydra tissue that addresses many common challenges encountered during the preparation and execution of the dissociation, as well as the formation and care of regenerating cellular aggregates. Analysis of the process provides insight into the mechanisms of nervous system self-organization. For complete details on the use and execution of this protocol, please refer to Lovas and Yuste (2021).

Ensemble synchronization in the reassembly of Hydra's nervous system.

Although much is known about how the structure of the nervous system develops, it is still unclear how its functional modularity arises. A dream experiment would be to observe the entire development of a nervous system, correlating the emergence of functional units with their associated behaviors. This is possible in the cnidarian Hydra vulgaris, which, after its complete dissociation into individual cells, can reassemble itself back together into a normal animal. We used calcium imaging to monitor the complete neuronal activity of dissociated Hydra as they reaggregated over several days. Initially uncoordinated neuronal activity became synchronized into coactive neuronal ensembles. These local modules then synchronized with others, building larger functional ensembles that eventually extended throughout the entire reaggregate, generating neuronal rhythms similar to those of intact animals. Global synchronization was not due to neurite outgrowth but to strengthening of functional connections between ensembles. We conclude that Hydra's nervous system achieves its functional reassembly through the hierarchical modularity of neuronal ensembles. VIDEO ABSTRACT.

Defined factors to reactivate cell cycle activity in adult mouse cardiomyocytes.

Adult mammalian cardiomyocytes exit the cell cycle during the neonatal period, commensurate with the loss of regenerative capacity in adult mammalian hearts. We established conditions for long-term culture of adult mouse cardiomyocytes that are genetically labeled with fluorescence. This technique permits reliable analyses of proliferation of pre-existing cardiomyocytes without complications from cardiomyocyte marker expression loss due to dedifferentiation or significant contribution from cardiac progenitor cell expansion and differentiation in culture. Using this system, we took a candidate gene approach to screen for fetal-specific proliferative gene programs that can induce proliferation of adult mouse cardiomyocytes. Using pooled gene delivery and subtractive gene elimination, we identified a novel functional interaction between E2f Transcription Factor 2 (E2f2) and Brain Expressed X-Linked (Bex)/Transcription elongation factor A-like (Tceal) superfamily members Bex1 and Tceal8. Specifically, Bex1 and Tceal8 both preserved cell viability during E2f2-induced cell cycle re-entry. Although Tceal8 inhibited E2f2-induced S-phase re-entry, Bex1 facilitated DNA synthesis while inhibiting cell death. In sum, our study provides a valuable method for adult cardiomyocyte proliferation research and suggests that Bex family proteins may function in modulating cell proliferation and death decisions during cardiomyocyte development and maturation.

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

PINK1-mediated phosphorylation of Miro inhibits synaptic growth and protects dopaminergic neurons in Drosophila.

Mutations in the mitochondrial Ser/Thr kinase PINK1 cause Parkinson's disease. One of the substrates of PINK1 is the outer mitochondrial membrane protein Miro, which regulates mitochondrial transport. In this study, we uncovered novel physiological functions of PINK1-mediated phosphorylation of Miro, using Drosophila as a model. We replaced endogenous Drosophila Miro (DMiro) with transgenically expressed wildtype, or mutant DMiro predicted to resist PINK1-mediated phosphorylation. We found that the expression of phospho-resistant DMiro in a DMiro null mutant background phenocopied a subset of phenotypes of PINK1 null. Specifically, phospho-resistant DMiro increased mitochondrial movement and synaptic growth at larval neuromuscular junctions, and decreased the number of dopaminergic neurons in adult brains. Therefore, PINK1 may inhibit synaptic growth and protect dopaminergic neurons by phosphorylating DMiro. Furthermore, muscle degeneration, swollen mitochondria and locomotor defects found in PINK1 null flies were not observed in phospho-resistant DMiro flies. Thus, our study established an in vivo platform to define functional consequences of PINK1-mediated phosphorylation of its substrates.

The meaning of mitochondrial movement to a neuron's life.

Cells precisely regulate mitochondrial movement in order to balance energy needs and avoid cell death. Neurons are particularly susceptible to disturbance of mitochondrial motility and distribution due to their highly extended structures and specialized function. Regulation of mitochondrial motility plays a vital role in neuronal health and death. Here we review the current understanding of regulatory mechanisms that govern neuronal mitochondrial transport and probe their implication in health and disease. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.