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Experiments by three independent groups on zebrafish have clarified the role of two signaling factors, Nodal and Gdf3, during the early stages of development.Experiments by three independent groups on zebrafish have clarified the role of two signaling factors, Nodal and Gdf3, during the early stages of development.

Fibrodysplasia ossificans progressiva (FOP) is a rare human genetic disorder characterized by altered skeletal development and extraskeletal ossification. All cases of FOP are caused by activating mutations in the type I BMP/TGFβ cell surface receptor ACVR1, which over-activates signaling through phospho-Smad1/5 (pSmad1/5). To investigate the mechanism by which FOP-ACVR1 enhances pSmad1/5 activation, we used zebrafish embryonic dorsoventral (DV) patterning as an assay for BMP signaling. We determined that the FOP mutants ACVR1-R206H and -G328R do not require their ligand binding domain to over-activate BMP signaling in DV patterning. However, intact ACVR1-R206H has the ability to respond to both Bmp7 and Activin A ligands. Additionally, BMPR1, a type I BMP receptor normally required for BMP-mediated patterning of the embryo, is dispensable for both ligand-independent signaling pathway activation and ligand-responsive signaling hyperactivation by ACVR1-R206H. These results demonstrate that FOP-ACVR1 is not constrained by the same receptor/ligand partner requirements as WT-ACVR1.

Throughout the animal kingdom regenerative ability varies greatly from species to species, and even tissue to tissue within the same organism. The sheer diversity of structures and mechanisms renders a thorough comparison of molecular processes truly daunting. Are “blastemas” found in organisms as distantly related as planarians and axolotls derived from the same ancestral process, or did they arise convergently and independently? Is a mouse digit tip blastema orthologous to a salamander limb blastema? In other fields, the thorough characterization of a reference model has greatly facilitated these comparisons. For example, the amphibian Spemann-Mangold organizer has served as an amazingly useful comparative template within the field of developmental biology, allowing researchers to draw analogies between distantly related species, and developmental processes which are superficially quite different. The salamander limb blastema may serve as the best starting point for a comparative analysis of regeneration, as it has been characterized by over 200 years of research and is supported by a growing arsenal of molecular tools. The anatomical and evolutionary closeness of the salamander and human limb also add value from a translational and therapeutic standpoint. Tracing the evolutionary origins of the salamander blastema, and its relatedness to other regenerative processes throughout the animal kingdom, will both enhance our basic biological understanding of regeneration and inform our selection of regenerative model systems.

Heterodimeric TGF-β ligands outperform homodimers in a variety of developmental, cell culture, and therapeutic contexts; however, the mechanisms underlying this increased potency remain uncharacterized. Here, we use dorsal–ventral axial patterning of the zebrafish embryo to interrogate the BMP2/7 heterodimer signaling mechanism. We demonstrate that differential interactions with BMP antagonists do not account for the reduced signaling ability of homodimers. Instead, we find that while overexpressed BMP2 homodimers can signal, they require two nonredundant type I receptors, one from the Acvr1 subfamily and one from the Bmpr1 subfamily. This implies that all BMP signaling within the zebrafish gastrula, even BMP2 homodimer signaling, requires Acvr1. This is particularly surprising as BMP2 homodimers do not bind Acvr1 in vitro. Furthermore, we find that the roles of the two type I receptors are subfunctionalized within the heterodimer signaling complex, with the kinase activity of Acvr1 being essential, while that of Bmpr1 is not. These results suggest that the potency of the Bmp2/7 heterodimer arises from the ability to recruit both Acvr1 and Bmpr1 into the same signaling complex.

Heterodimeric TGF-β ligands outperform homodimers in a variety of developmental, cell culture, and therapeutic contexts; however, the mechanisms underlying this difference in signaling remain uncharacterized. Here we use dorsal-ventral axial patterning of the zebrafish embryo to interrogate the BMP2/7 heterodimer signaling mechanism. We find that the roles of the two type I receptors are subfunctionalized within the heterodimer signaling complex, with the kinase activity of Acvr1 being essential, while that of Bmpr1 is not. These results suggest that the Bmp2/7 heterodimer signals exclusively, due to its ability to recruit both Acvr1 and Bmpr1 into the same signaling complex. We also find that even though Bmpr1 kinase activity is not required for DV patterning, the Bmpr1 intracellular domain is required for this process, potentially for an uncharacterized non-kinase signaling function. We further find that the Acvr1 and Bmpr1 intracellular domains are not interchangeable, suggesting that heterodimer signaling requires specific motifs within both the Acvr1 and Bmpr1 intracellular domains. To explore the possibility that BMP2/7 heterodimers also integrate two distinct Type II receptors into the same signaling complex, we generated mutations in four of the Type II receptors and investigated the functions of five of the zebrafish BMP type II receptors, including acvr2aa, acvr2ab, acvr2ba, acvr2bb, and bmpr2b. We found that zygotic acvr2 quadruple mutants are moderately dorsalized, demonstrating that Acvr2 is involved in DV patterning. Conversely, we did not observe Bmpr2b expression in the zebrafish embryo. We did, however, find that homozygous bmpr2b mutant females are infertile, with defects in oogenesis. Cumulatively these results suggest that the BMP2/7 heterodimer assembles a complex containing Acvr1, Bmpr1, and Acvr2 during zebrafish patterning, and that Bmpr1 and Acvr1 each contribute distinct intracellular functions to this complex

Previous studies have reported that amputation invokes body-wide responses in regenerative organisms, but most have not examined the implications of these changes beyond the region of tissue regrowth. Specifically, long-range epidermal responses to amputation are largely uncharacterized, with research on amputation-induced epidermal responses in regenerative organisms traditionally being restricted to the wound site. Here, we investigate the effect of amputation on long-range epidermal permeability in two evolutionarily distant, regenerative organisms: axolotls and planarians. We find that amputation triggers a long-range increase in epidermal permeability in axolotls, accompanied by a long-range epidermal downregulation in MAPK signaling. Additionally, we provide functional evidence that pharmacologically inhibiting MAPK signaling in regenerating planarians increases long-range epidermal permeability. These findings advance our knowledge of body-wide changes due to amputation in regenerative organisms and warrant further study on whether epidermal permeability dysregulation in the context of amputation may lead to pathology in both regenerative and non-regenerative organisms.

BMP signaling drives dorsoventral (DV) axial patterning in vertebrates and invertebrates, with BMP dimers recruiting tetrameric receptor complexes to phosphorylate SMADs that activate ventral target gene expression. In zebrafish DV patterning, BMP2/7 heterodimers exclusively signal, assembling a receptor complex of two distinct type I receptors, Bmpr1 and Acvr1l, that canonically bind Bmp2 and Bmp7 ligands, respectively. We tested if the two distinct classes of BMP type II receptors, Bmpr2 and Acvr2, also act in the signaling complex. We determined that Acvr2 receptors solely transduce BMP signaling in DV patterning. We mutated all four and genes in the zebrafish and found that maternal-zygotic depletion of just Acvr2b receptors abrogates all BMP signaling, indicating that Acvr2b is the primary type II receptor transducing BMP signaling in the gastrula. We further demonstrated that hyperactive signaling through the ACVR1-R206H Fibrodysplasia Ossificans Progressiva human disease-causing mutant receptor is restricted when maternal and zygotic contributions of either Acvr2ba or Acvr2bb are absent. This reveals an increased sensitivity of ACVR1-R206H signaling to Acvr2b dosage, compared to wild-type ACVR1. These findings support a model in which Acvr2b receptors mediate the endogenous BMP signaling in the gastrula and that hyperactivity of ACVR1-R206H is limited in a dose-dependent manner by the relative concentration of Acvr2b.

Many species regenerate lost body parts following amputation. Most limb regeneration research has focused on the immediate injury site. Meanwhile, body-wide injury responses remain largely unexplored but may be critical for regeneration. Here, we discovered a role for the sympathetic nervous system in stimulating a body-wide stem cell activation response to amputation that drives enhanced limb regeneration in axolotls. This response is mediated by adrenergic signaling, which coordinates distant cellular activation responses via the α -adrenergic receptor, and local regeneration responses via β-adrenergic receptors. Both α - and β-adrenergic signaling act upstream of mTOR signaling. Notably, systemically-activated axolotls regenerate limbs faster than naïve animals, suggesting a potential selective advantage in environments where injury from cannibalism or predation is common. This work challenges the predominant view that cellular responses underlying regeneration are confined to the injury site and argues instead for body-wide cellular priming as a foundational step that enables localized tissue regrowth.

Salamanders are capable of regenerating whole limbs throughout life, a feat that is unmatched within tetrapods. Limb regeneration is dependent upon the formation of a blastema, which contains undifferentiated cells capable of giving rise to most cells of the regenerated limb. Innervation is required for regeneration, along with many signaling pathways, including FGF, BMP and Wnt, but the role of VEGF signaling during salamander limb regeneration is not well understood. Here we show that VEGF signaling is essential for limb regeneration and that blastema cells and limb fibroblasts display impaired proliferation in the absence of VEGF signaling. By performing analogous experiments in planaria, which lack vasculature, we show a potential evolutionarily conserved role for VEGF in the expansion of blastema tissues that is separable from angiogenesis. Moreover, loss of VEGF signaling reduces induction of EMT-like processes, suggesting VEGF signaling functions upstream of the expression of EMT transcription factors, including Snai2. These findings highlight potential roles for VEGF signaling during regeneration which may extend beyond typical findings related to angiogenesis.