Aaron M. Savage

Nascent vascular networks adapt to the increasing metabolic demands of growing tissues by expanding via angiogenesis. As vascular networks expand, blood vessels remodel, progressively refining vascular connectivity to generate a more haemodynamically efficient network. This is driven by interplay between endothelial cell (EC) signalling and blood flow. While much is known about angiogenesis, less is understood of how blood flow remodels vessels. Here, we employ the zebrafish sub-intestinal venous plexus (SIVP) to characterise blood flow-dependent remodelling. Using live imaging to track ECs, we show that blood flow controls SIVP remodelling by coordinating collective EC migration within the developing plexus. Blood flow opposes continuous ventral EC migration within the SIVP and is required for angiogenic sprout regression to support plexus growth. Sprout regression occurs by coordinated migration of ECs from non-perfused leading sprouts, which migrate against blood flow and incorporate into the sub-intestinal vein. Sprout regression remains compatible with low blood flow but requires flt4 function under these conditions. Collectively, these studies reveal how blood flow sculpts a developing vascular plexus by coordinating EC migration and balancing vascular remodelling via flt4.

The axolotl is a popular model organism in regenerative biology owing to its ability to regenerate amputated limbs and internal organs. The role of injury-derived signals in initiating the regenerative response is still not well understood, but the potential involvement of the stress response is of interest, as injury and stress are temporally linked. The dominant glucocorticoid response to stress varies among species, with corticosterone generally considered dominant in most amphibians, whereas cortisol predominates in others. Here we characterize the adrenal stress response in the axolotl and describe methods to measure axolotl stress hormones to facilitate their inclusion in future research involving axolotl development and regeneration. We describe an intricate and unexpected axolotl stress response that involves cortisol and corticosterone, each being dominant under different conditions. Corticosterone is preferably activated by the classical hypothalamus–pituitary–interrenal axis pathway, with both arginine vasotocin and adrenocorticotropic hormone promoting its synthesis and release. Under manual stress and direct stimuli with acetylcholine, cortisol is more prominent, suggesting an alternative mechanism involving sympathetic nerve signaling. In response to an amputation injury, both cortisol and corticosterone are increased, with corticosterone being dominant, suggesting an injury-specific response. Finally, when administering glucocorticoids directly and measuring classical physiological effects of glucocorticoid signaling, cortisol is more potent. We propose a hypothesis that axolotls rely on cortisol as their dominant glucocorticoid, functioning in part as an extension of the catecholamine system. By contrast, corticosterone is mainly regulated classically via the hypothalamus–pituitary–interrenal axis.

Background Diabetes is a condition characterized by a loss of pancreatic β−cell function which results in the dysregulation of insulin homeostasis. Using a partial pancreatectomy model in axolotl, we aimed to observe the pancreatic response to injury. Results Here we show a comprehensive histological assessment of pancreatic islets in axolotl. Following pancreatic injury, no apparent blastemal structure was observed. We found a significant, organ-wide increase in cellular proliferation post-resection in the pancreas compared to sham-operated controls. This proliferative response was most robust at the site of injury. We found that β−cells actively contributed to the increased rates of proliferation upon injury. β−cell proliferation manifested in increased β−cell mass in injured tissue at two weeks post injury. At four weeks post injury, we found organ-wide proliferation to be extinguished while proliferation at the injury site persisted, corresponding to pancreatic tissue recovery. Similarly, total β−cell mass was comparable to sham after four weeks. Conclusions Our findings suggest a non-blastema-mediated regeneration process takes place in the pancreas, by which pancreatic resection induces whole-organ β−cell proliferation without the formation of a blastemal structure. This process is analogous to other models of compensatory growth in axolotl, including liver regeneration.

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.

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. GRAPHICAL

SUMMARY: 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 _2A -adrenergic receptor, and local regeneration responses via β-adrenergic receptors. Both α _2A – 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.

In vitro production of tissue-specific stem cells [e.g. haematopoietic stem cells (HSCs)] is a key goal of regenerative medicine. However, recent efforts to produce fully functional tissue-specific stem cells have fallen short. One possible cause of shortcomings may be that model organisms used to characterize basic vertebrate embryology (Xenopus, zebrafish, chick) may employ molecular mechanisms for stem cell specification that are not conserved in humans, a prominent example being the specification of primordial germ cells (PGCs). Germ plasm irreversibly specifies PGCs in many models; however, it is not conserved in humans, which produce PGCs from tissue termed germline-competent mesoderm (GLCM). GLCM is not conserved in organisms containing germ plasm, or even in mice, but understanding its developmental potential could unlock successful production of other stem cell types. GLCM was first discovered in embryos from the axolotl and its conservation has since been demonstrated in pigs, which develop from a flat-disc embryo like humans. Together these findings suggest that GLCM is a conserved basal trait of vertebrate embryos. Moreover, the immortal nature of germ cells suggests that immortality is retained during GLCM specification; here we suggest that the demonstrated pluripotency of GLCM accounts for retention of immortality in somatic stem cell types as well.This article has an associated Future Leaders to Watch interview with the author of the paper.

Nascent vascular networks adapt to the increasing metabolic demands of growing tissues by expanding via angiogenesis. As vascular networks expand, blood vessels remodel, progressively refining vascular connectivity to generate a more haemodynamically efficient network. This process is driven by interplay between endothelial cell (EC) signalling and blood flow. While much is known about angiogenesis, considerably less is understood of the mechanisms underlying vessel remodelling by blood flow. Here we employ the zebrafish sub-intestinal venous plexus (SIVP) to characterise the mechanisms underlying blood flow-dependent remodelling. Using live imaging to track ECs we show that blood flow controls SIVP remodelling by coordinating collective migration of ECs within the developing plexus. Blood flow opposes continuous ventral EC migration within the SIVP and is required for regression of angiogenic sprouts to support plexus growth. Sprout regression occurs by coordinated polarisation and migration of ECs from non-perfused leading sprouts, which migrate in opposition to blood flow and incorporate into the SIV. Sprout regression is compatible with low blood flow and is dependent upon vegfr3/flt4 function under these conditions. Collectively, these studies reveal how blood flow sculpts a developing vascular plexus by coordinating EC migration and balancing vascular remodelling via vegfr3/flt4 .

Vertebrate heart development requires the complex morphogenesis of a linear tube to form the mature organ, a process essential for correct cardiac form and function, requiring coordination of embryonic laterality, cardiac growth, and regionalized cellular changes. While previous studies have demonstrated broad requirements for extracellular matrix (ECM) components in cardiac morphogenesis, we hypothesized that ECM regionalization may fine tune cardiac shape during heart development. Using live in vivo light sheet imaging of zebrafish embryos, we describe a left-sided expansion of the ECM between the myocardium and endocardium prior to the onset of heart looping and chamber ballooning. Analysis using an ECM sensor revealed the cardiac ECM is further regionalized along the atrioventricular axis. Spatial transcriptomic analysis of gene expression in the heart tube identified candidate genes that may drive ECM expansion. This approach identified regionalized expression of hapln1a, encoding an ECM cross-linking protein. Validation of transcriptomic data by in situ hybridization confirmed regionalized hapln1a expression in the heart, with highest levels of expression in the future atrium and on the left side of the tube, overlapping with the observed ECM expansion. Analysis of CRISPR-Cas9-generated hapln1a mutants revealed a reduction in atrial size and reduced chamber ballooning. Loss-of-function analysis demonstrated that ECM expansion is dependent upon Hapln1a, together supporting a role for Hapln1a in regionalized ECM modulation and cardiac morphogenesis. Analysis of hapln1a expression in zebrafish mutants with randomized or absent embryonic left–right asymmetry revealed that laterality cues position hapln1a-expressing cells asymmetrically in the left side of the heart tube. We identify a regionalized ECM expansion in the heart tube which promotes correct heart development, and propose a novel model whereby embryonic laterality cues orient the axis of ECM asymmetry in the heart, suggesting these two pathways interact to promote robust cardiac morphogenesis.