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.

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.