14-3-3 binding motif phosphorylation disrupts Hdac4-organized condensates to stimulate cardiac reprogramming.

Cell fate conversion is associated with extensive post-translational modifications (PTMs) and architectural changes of sub-organelles, yet how these events are interconnected remains unknown. We report here the identification of a phosphorylation code in 14-3-3 binding motifs (PC14-3-3) that greatly stimulates induced cardiomyocyte (iCM) formation from fibroblasts. PC14-3-3 is identified in pivotal functional proteins for iCM reprogramming, including transcription factors and chromatin modifiers. Akt1 kinase and protein phosphatase 2A are the key writer and key eraser of the PC14-3-3 code, respectively. PC14-3-3 activation induces iCM formation with the presence of only Tbx5. In contrast, PC14-3-3 inhibition by mutagenesis or inhibitor-mediated code removal abolishes reprogramming. We discover that key PC14-3-3-embedded factors, such as histone deacetylase 4 (Hdac4), Mef2c, and Foxo1, form Hdac4-organized inhibitory nuclear condensates. PC14-3-3 activation disrupts Hdac4 condensates to promote cardiac gene expression. Our study suggests that sub-organelle dynamics regulated by a PTM code could be a general mechanism for stimulating cell reprogramming.

14-3-3 binding motif phosphorylation disrupts Hdac4 organized condensates to stimulate cardiac reprogramming.

Cell fate conversion is associated with extensive epigenetic and post translational modifications (PTMs) and architectural changes of sub-organelles and organelles, yet how these events are interconnected remains unknown. We report here the identification of a phosphorylation code in 14-3-3 binding motifs (PC14-3-3) that greatly stimulates induced cardiomyocyte (iCM) formation from fibroblasts. PC14-3-3 was identified in pivotal functional proteins for iCM reprogramming, including transcription factors and epigenetic factors. Akt1 kinase and PP2A phosphatase were a key writer and eraser of the PC14-3-3 code, respectively. PC14-3-3 activation induces iCM formation with the presence of only Tbx5. In contrast, PC14-3-3 inhibition by mutagenesis or inhibitor-mediated code removal abolished reprogramming. We discovered that key PC14-3-3 embedded factors, such as Hdac4, Mef2c, Nrip1, and Foxo1, formed Hdac4 organized inhibitory nuclear condensates. Notably, PC14-3-3 activation disrupted Hdac4 condensates to promote cardiac gene expression. Our study suggests that sub-organelle dynamics regulated by a post-translational modification code could be a general mechanism for stimulating cell reprogramming and organ regeneration. Highlights: A PC14-3-3 (phosphorylation code in 14-3-3 binding motifs) is identified in pivotal functional proteins, such as HDAC4 and Mef2c, that stimulates iCM formation.Akt1 kinase and PP2A phosphatase are a key writer and a key eraser of the PC14-3-3 code, respectively, and PC14-3-3 code activation can replace Mef2c and Gata4 in cardiac reprogramming.PC14-3-3 activation disrupts Hdac4 organized condensates which results in releasing multiple 14-3-3 motif embedded proteins from the condensates to stimulate cardiac reprogramming.Sub-organelle dynamics and function regulated by a post-translational modification code could be a general mechanism in stimulating cell reprogramming and organ regeneration.

Head removal enhances planarian electrotaxis.

Certain animal species utilize electric fields for communication, hunting and spatial orientation. Freshwater planarians move toward the cathode in a static electric field (cathodic electrotaxis). This planarian behavior was first described by Raymond Pearl more than a century ago. However, planarian electrotaxis has received little attention since, and the underlying mechanisms and evolutionary significance remain unknown. To close this knowledge gap, we developed an apparatus and scoring metrics for automated quantitative and mechanistic studies of planarian behavior upon exposure to a static electric field. Using this automated setup, we characterized electrotaxis in the planarian Dugesia japonica and found that this species responds to voltage instead of current, in contrast to results from previous studies using other planarian species. Surprisingly, we found differences in electrotaxis ability between small (shorter) and large (longer) planarians. To determine the cause of these differences, we took advantage of the regenerative abilities of planarians and compared electrotaxis in head, tail and trunk fragments of various lengths. We found that tail and trunk fragments electrotaxed, whereas head fragments did not, regardless of size. Based on these data, we hypothesized that signals from the head may interfere with electrotaxis when the head area/body area reached a critical threshold. In support of this hypothesis, we found that (1) smaller intact planarians that cannot electrotax have a relatively larger head-to-body-ratio than large planarians that can electrotax, and (2) the electrotaxis behavior of cut head fragments was negatively correlated with the head-to-body ratio of the fragments. Moreover, we could restore cathodic electrotaxis in head fragments via decapitation, directly demonstrating inhibition of electrotaxis by the head.

Planarian fragments behave as whole animals.

Behavioral responses of freshwater planarians have been studied for over a century.1 In recent decades, behavior has been used as a readout to study planarian development and regeneration,2-6 wound healing,7,8 molecular evolution,4,9,10 neurotoxicology,11-13 and learning and memory.14-17The planarian nervous system is among the simplest of the bilaterally symmetric animals,18 with an anterior brain attached to two ventral nerve cords interconnected by multiple commissures. We found that, in response to mechanical and near-UV stimulation, head stimulation produces turning, tail stimulation produces contraction, and trunk stimulation produces midbody elongation in the planarian Dugesia japonica. When cut into two or three pieces, the anterior end of each headless piece switched its behavior to turning instead of elongation; i.e., it responded as though it were the head. In addition, posterior ends of the head and midbody pieces sometimes produced contraction instead of elongation. Thus, each severed piece acts like an intact animal, with each midbody region having nearly complete behavioral capabilities. These observations show that each midbody region reads the global state of the organism and adapts its response to incoming signals from the remaining tissue. Selective lateral incisions showed that the changes in behavior are not due to nonselective pain responses and that the ventral nerve cords and cross-connectives are responsible for coordinating local behaviors. Our findings highlight a fast functional reorganization of the planarian nervous system that complements the slower repairs provided by regeneration. This reorganization provides needed behavioral responses for survival as regeneration proceeds.

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing.

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Pharmacological or genetic targeting of Transient Receptor Potential (TRP) channels can disrupt the planarian escape response.

In response to noxious stimuli, planarians cease their typical ciliary gliding and exhibit an oscillatory type of locomotion called scrunching. We have previously characterized the biomechanics of scrunching and shown that it is induced by specific stimuli, such as amputation, noxious heat, and extreme pH. Because these specific inducers are known to activate Transient Receptor Potential (TRP) channels in other systems, we hypothesized that TRP channels control scrunching. We found that chemicals known to activate TRPA1 (allyl isothiocyanate (AITC) and hydrogen peroxide) and TRPV (capsaicin and anandamide) in other systems induce scrunching in the planarian species Dugesia japonica and, except for anandamide, in Schmidtea mediterranea. To confirm that these responses were specific to either TRPA1 or TRPV, respectively, we tried to block scrunching using selective TRPA1 or TRPV antagonists and RNA interference (RNAi) mediated knockdown. Unexpectedly, co-treatment with a mammalian TRPA1 antagonist, HC-030031, enhanced AITC-induced scrunching by decreasing the latency time, suggesting an agonistic relationship in planarians. We further confirmed that TRPA1 in both planarian species is necessary for AITC-induced scrunching using RNAi. Conversely, while co-treatment of a mammalian TRPV antagonist, SB-366791, also enhanced capsaicin-induced reactions in D. japonica, combined knockdown of two previously identified D. japonica TRPV genes (DjTRPVa and DjTRPVb) did not inhibit capsaicin-induced scrunching. RNAi of DjTRPVa/DjTRPVb attenuated scrunching induced by the endocannabinoid and TRPV agonist, anandamide. Overall, our results show that although scrunching induction can involve different initial pathways for sensing stimuli, this behavior's signature dynamical features are independent of the inducer, implying that scrunching is a stereotypical planarian escape behavior in response to various noxious stimuli that converge on a single downstream pathway. Understanding which aspects of nociception are conserved or not across different organisms can provide insight into the underlying regulatory mechanisms to better understand pain sensation.

Mouth Function Determines the Shape Oscillation Pattern in Regenerating Hydra Tissue Spheres.

Hydra is a small freshwater polyp capable of regeneration from small tissue pieces and from aggregates of cells. During regeneration, a hollow bilayered sphere is formed that undergoes osmotically driven shape oscillations of inflation and rupture. These oscillations are necessary for successful regeneration. Eventually, the oscillating sphere breaks rotational symmetry along the future head-foot axis of the animal. Notably, the shape oscillations show an abrupt shift from large-amplitude, long-period oscillations to small-amplitude, short-period oscillations. It has been widely accepted that this shift in oscillation pattern is linked to symmetry breaking and axis formation, and current theoretical models of Hydra symmetry breaking use this assumption as a model constraint. However, a mechanistic explanation for the shift in oscillation pattern is lacking. Using in vivo manipulation and imaging, we quantified the shape oscillation dynamics and dissected the timing and triggers of the pattern shift. Our experiments demonstrate that the shift in the shape oscillation pattern in regenerating Hydra tissue pieces is caused by the formation of a functional mouth and not by shape symmetry breaking as previously assumed. Thus, model assumptions must be revised in light of these new experimental data, which can be used to constrain and validate improved theoretical models of pattern formation in Hydra.