Acute multidrug delivery via a wearable bioreactor facilitates long-term limb regeneration and functional recovery in adult Xenopus laevis.

Limb regeneration is a frontier in biomedical science. Identifying triggers of innate morphogenetic responses in vivo to induce the growth of healthy patterned tissue would address the needs of millions of patients, from diabetics to victims of trauma. Organisms such as Xenopus laevis-whose limited regenerative capacities in adulthood mirror those of humans-are important models with which to test interventions that can restore form and function. Here, we demonstrate long-term (18 months) regrowth, marked tissue repatterning, and functional restoration of an amputated X. laevis hindlimb following a 24-hour exposure to a multidrug, pro-regenerative treatment delivered by a wearable bioreactor. Regenerated tissues composed of skin, bone, vasculature, and nerves significantly exceeded the complexity and sensorimotor capacities of untreated and control animals' hypomorphic spikes. RNA sequencing of early tissue buds revealed activation of developmental pathways such as Wnt/ß-catenin, TGF-ß, hedgehog, and Notch. These data demonstrate the successful "kickstarting" of endogenous regenerative pathways in a vertebrate model.

Nervous system and tissue polarity dynamically adapt to new morphologies in planaria.

The coordination of tissue-level polarity with organism-level polarity is crucial in development, disease, and regeneration. Here, we characterize a new example of large-scale control of dynamic remodeling of body polarity. Exploiting the flexibility of the body plan in regenerating planarians, we used mirror duplication of the primary axis to show how established tissue-level polarity adapts to new organism-level polarity. Characterization of epithelial planar cell polarity revealed a remarkable reorientation of tissue polarity in double-headed planarians. This reorientation of cilia occurs even following irradiation-induced loss of all stem cells, suggesting independence of the polarity change from the formation of new cells. The presence of the two heads plays an important role in regulating the rate of change in overall polarity. We further present data that suggest that the nervous system itself adapts its polarity to match the new organismal anatomy as revealed by changes in nerve transport driving distinct regenerative outcomes. Thus, in planaria tissue-level polarity can dynamically reorient to match the organism-level anatomical configuration.

Regulation of axial and head patterning during planarian regeneration by a commensal bacterium.

Some animals, such as planaria, can regenerate complex anatomical structures in a process regulated by genetic and biophysical factors, but additional external inputs into regeneration remain to be uncovered. Microbial communities inhabiting metazoan organisms are important for metabolic, immune, and disease processes, but their instructive influence over host structures remains largely unexplored. Here, we show that Aquitalea sp. FJL05, an endogenous commensal bacterium of Dugesia japonica planarians, and one of the small molecules it produces, indole, can influence axial and head patterning during regeneration, leading to regeneration of permanently two-headed animals. Testing the impact of indole on planaria tissues via RNA sequencing, we find that indole alters the regenerative outcomes in planarians through changes in expression to patterning genes, including a downregulation of Wnt pathway genes. These data provide a unique example of the product of a commensal bacterium modulating transcription of patterning genes to affect the host's anatomical structure during regeneration.

Regenerative Adaptation to Electrochemical Perturbation in Planaria: A Molecular Analysis of Physiological Plasticity.

Anatomical homeostasis results from dynamic interactions between gene expression, physiology, and the external environment. Owing to its complexity, this cellular and organism-level phenotypic plasticity is still poorly understood. We establish planarian regeneration as a model for acquired tolerance to environments that alter endogenous physiology. Exposure to barium chloride (BaCl2) results in a rapid degeneration of anterior tissue in Dugesia japonica. Remarkably, continued exposure to fresh solution of BaCl2 results in regeneration of heads that are insensitive to BaCl2. RNA-seq revealed transcriptional changes in BaCl2-adapted heads that suggests a model of adaptation to excitotoxicity. Loss-of-function experiments confirmed several predictions: blockage of chloride and calcium channels allowed heads to survive initial BaCl2 exposure, inducing adaptation without prior exposure, whereas blockade of TRPM channels reversed adaptation. Such highly adaptive plasticity may represent an attractive target for biomedical strategies in a wide range of applications beyond its immediate relevance to excitotoxicity preconditioning.

The effects of stress, cortisol administration and cortisol inhibition on black sea bass (Centropristis striata) sex differentiation.

Sex differentiation in many lower vertebrates (e.g. reptiles, amphibians, and fishes) can be influenced by environmental factors experienced during sensitive developmental periods. Environmental stressors, acting through cortisol, masculinize some teleost fishes during development by limiting gonadal cytochrome P450 aromatase (cyp19a1a), the enzyme that irreversibly converts testosterone to 17ß-estradiol. In this study, we examined the influence of cortisol, cortisol inhibitors and a repeated, acute stressor (net-chasing) on sex differentiation in black sea bass (BSB; Centropristis striata), a protogynous hermaphroditic teleost. Wild-caught, sexually-undifferentiated, BSB juveniles (~90 mm) were collected from Rhode Island waters, raised in recirculating systems and fed diets supplemented with cortisol, a cortisol receptor antagonist (mifepristone), a cortisol synthesis inhibitor (metyrapone), or net-chased twice a week for two min until gonads were differentiated (77-89 days). Long term cortisol administration partially masculinized all female fish, but repeated net-chasing did not alter sex differentiation relative to the control group. Blocking cortisol receptor binding delayed sex differentiation in some individuals, but overall led to increased masculinization compared to control fish. The proportion of treatment fish that developed as males suggests a functionally, diandric protogynous reproductive strategy in this species. We also identified a glucocorticoid response element in the gonadal aromatase (cyp19a1a) promoter, indicating a possible relationship between cortisol and cyp19a1a gene expression.

Do Verbal and Tactile Cueing Selectively Alter Gluteus Maximus and Hamstring Recruitment During a Supine Bridging Exercise in Active Females? A Randomized Controlled Trial.

CONTEXT: Hip extension with hamstring-dominant rather than gluteus maximus-dominant recruitment may increase anterior femoracetabular forces and contribute to conditions that cause hip pain. Cueing methods during hip extension exercises may facilitate greater gluteus maximus recruitment. OBJECTIVE: We examined whether specific verbal and tactile cues facilitate gluteus maximus recruitment while inhibiting hamstring recruitment during a bridging exercise. DESIGN: Randomized controlled trial. SETTING: Biomechanics laboratory. PARTICIPANTS: 30 young adult women (age 24 [3] y; BMI 22.2 [2.4] kg/m2). INTERVENTION: Participants were tested over 2 sessions, 1 week apart, while performing 5 repetitions of a bridging exercise. At their second visit, participants in the experimental group received verbal and tactile cues intended to facilitate gluteus maximus recruitment and inhibit hamstring recruitment. Control group participants received no additional cues beyond original instructions. MAIN OUTCOME MEASURES: Gluteus maximus and hamstring recruitment were measured with surface electromyography, normalized to maximal voluntary isometric contractions (MVICs). RESULTS: Gluteus maximus recruitment was unchanged in the control group and increased from 16.8 to 33.0% MVIC in the cueing group (F = 33.369, P < .001). Hamstring recruitment was unchanged in the control group but also increased from 16.5 to 29.8% MVIC in the cueing group (F = 6.400, P = .02). The effect size of the change in gluteus maximus recruitment in the cueing group (Cohen's d = 1.5, 95% CI = 0.9 to 2.2) was not significantly greater than the effect size in hamstring recruitment (Cohen's d = 0.8, 95% CI = 0.1 to 1.5). CONCLUSIONS: Verbal and tactile cues hypothesized to facilitate gluteus maximus recruitment yielded comparable increases in both gluteus maximus and hamstring recruitment. If one intends to promote hip extension by facilitating gluteus maximus recruitment while inhibiting hamstring recruitment during bridging exercises, the cueing methods employed in this study may not produce desired effects.