SUMMARY Having the ability to coordinate the behavior of stem cells to induce regeneration of specific large-scale structures would have far reaching consequences in the treatment of degenerative diseases, acute injury, and aging. Thus, identifying and learning to manipulate the sequential steps that determine the fate of new tissue within the overall morphogenetic program of the organism is fundamental. We identified novel early signals, mediated by the central nervous system and 3 innexin proteins, which determine the fate and axial polarity of regenerated tissue in planarians. Modulation of gap junction-dependent and neural signals specifically induces ectopic anterior regeneration blastemas in posterior and lateral wounds. These ectopic anterior blastemas differentiate new brains that establish permanent primary axes re-established during subsequent rounds of unperturbed regeneration. These data reveal powerful novel controls of pattern formation and suggest a constructive model linking nervous inputs and polarity determination in early stages of regeneration.

SUMMARY Biophysical signaling is required for both embryonic polarity and regenerative outgrowth. Exploiting endogenous ion transport for regenerative therapies will require direct regulation of membrane voltage. Here, we develop a pharmacological method to target ion transporters, uncovering a novel role for membrane voltage as a key regulator of anterior polarity in regenerating planaria. Utilizing the highly specific inhibitor, SCH-28080, our data reveal that H+,K+-ATPase-mediated membrane depolarization is essential for anterior gene expression and brain induction. H+,K+-ATPase-independent manipulation of membrane potential with ivermectin confirms that depolarization drives head formation, even at posterior-facing wounds. Using this chemical genetics approach, we demonstrate that membrane voltage controls head-vs.-tail identity during planarian regeneration. Our data suggest well-characterized drugs (already approved for human use) might be exploited to control adult stem cell-driven pattern formation during the regeneration of complex structures.

SUMMARYA main goal of regenerative medicine is to replace lost or damaged tissues and organs with functional parts of the correct size and shape. But the proliferation of new cells is not sufficient; we will also need to understand how the scale and ultimate form of newly produced tissues are determined. Using the planarian model system, we report that membrane voltage-dependent bioelectric signaling determines both head size and organ scaling during regeneration. RNA interference of the H + ,K + -ATPase ion pump results in membrane hyperpolarization, which has no effect on the amount of new tissue (blastema) that is regenerated yet produces regenerates with tiny 'shrunken' heads and proportionally oversized pharynges. Our data show that this disproportionality results from a lack of the apoptosis required to adjust head and organ size and placement, highlighting apoptotic remodeling as the link between bioelectric signaling and the establishment of organ size during regeneration.

We show that regenerating planarians' normal anterior-posterior pattern can be permanently rewritten by a brief perturbation of endogenous bioelectrical networks. Temporary modulation of regenerative bioelectric dynamics in amputated trunk fragments of planaria stochastically results in a constant ratio of regenerates with two heads to regenerates with normal morphology. Remarkably, this is shown to be due not to partial penetrance of treatment, but a profound yet hidden alteration to the animals' patterning circuitry. Subsequent amputations of the morphologically normal regenerates in water result in the same ratio of double-headed to normal morphology, revealing a cryptic phenotype that is not apparent unless the animals are cut. These animals do not differ from wild-type worms in histology, expression of key polarity genes, or neoblast distribution. Instead, the altered regenerative bodyplan is stored in seemingly normal planaria via global patterns of cellular resting potential. This gradient is functionally instructive, and represents a multistable, epigenetic anatomical switch: experimental reversals of bioelectric state reset subsequent regenerative morphology back to wild-type. Hence, bioelectric properties can stably override genome-default target morphology, and provide a tractable control point for investigating cryptic phenotypes and the stochasticity of large-scale epigenetic controls.

The molecular mechanisms governing the cell behaviors underlying morphogenesis remain a major focus of research in both developmental biology and cancer biology. TGF-␤ ligands control cell fate specification via Smad-mediated signaling. However, their ability to guide cellular morphogenesis in a variety of biological contexts is poorly understood. We report on the discovery of a novel TGF-␤ signaling-mediated cellular morphogenesis occurring during vertebrate gastrulation. Activin/nodal members of the TGF-␤ superfamily induce the expression of two genes regulating cell adhesion during gastrulation: Fibronectin Leucine-rich Repeat Transmembrane 3 (FLRT3), a type I transmembrane protein containing extracellular leucine-rich repeats, and the small GTPase Rnd1. FLRT3 and Rnd1 interact physically and modulate cell adhesion during embryogenesis by controlling cell surface levels of cadherin through a dynamin-dependent endocytosis pathway. Our model suggests that cell adhesion can be dynamically regulated by sequestering cadherin through internalization, and subsequent redeploying internalized cadherin to the cell surface as needed. As numerous studies have linked aberrant expression of small GTPases, adhesion molecules such as cadherins, and TGF-␤ signaling to oncogenesis and metastasis, it is tempting to speculate that this FLRT3/Rnd1/cadherin pathway might also control cell behavior and morphogenesis in adult tissue homeostasis.[Keywords: GTPase; FLRT3; Rnd1; gastrulation; activin; Xenopus] Supplemental material is available at http://www.genesdev.org. During gastrulation, the three germ layers (ectoderm, mesoderm, and endoderm) establish new contacts, permitting new inductive interactions to specify the development of organ primordia. Some of the major movements that constitute gastrulation are convergence and extension, involution, and epiboly. The dynamic nature of cell-cell contacts in tissues undergoing these movements has been visualized using time-lapse video microscopy. Cells involved in convergence and extension movements are shown to continuously break and remake local adhesive contacts, via polarized membranous processes (e.g., lamellipodia), as they "slide" past one another during mediolateral intercalation (Davidson et al. 2002;Shook et al. 2004).Distinct adhesive properties conferred by adhesion molecules constitute a key feature of cells that undergo gastrulation movements. Type I cadherins are required for proper morphogenesis in sea urchin (Miller and McClay 1997), zebrafish (Montero et al. 2005;Shimizu et al. 2005), and mouse embryos (Riethmacher et al. 1995). Inactivation of C-cadherin, the primary mediator of adhesion in the Xenopus blastula, leads to both involution and convergent extension defects during gastrulation (Heasman et al. 1994). By changing the functional activity of C-cadherin at the cell surface, the morphogenetic elongation of "animal cap" ectodermal explants (mimicking the convergence and extension movements of the embryonic mesoderm) is altered (Brieher and Gumbiner 1994).The Wn...