A Novel Perspective on Neuronal Control of Anatomical Patterning, Remodeling, and Maintenance.

While the nervous system may be best known as the sensory communication center of an organism, recent research has revealed a myriad of multifaceted roles for both the CNS and PNS from early development to adult regeneration and remodeling. These systems work to orchestrate tissue pattern formation during embryonic development and continue shaping pattering through transitional periods such as metamorphosis and growth. During periods of injury or wounding, the nervous system has also been shown to influence remodeling and wound healing. The neuronal mechanisms responsible for these events are largely conserved across species, suggesting this evidence may be important in understanding and resolving many human defects and diseases. By unraveling these diverse roles, this paper highlights the necessity of broadening our perspective on the nervous system beyond its conventional functions. A comprehensive understanding of the complex interactions and contributions of the nervous system throughout development and adulthood has the potential to revolutionize therapeutic strategies and open new avenues for regenerative medicine and tissue engineering. This review highlights an important role for the nervous system during the patterning and maintenance of complex tissues and provides a potential avenue for advancing biomedical applications.

Adaptive correction of craniofacial defects in pre-metamorphic Xenopus laevis tadpoles involves thyroid hormone-independent tissue remodeling.

Although it is well established that some organisms can regenerate lost structures, the ability to remodel existing malformed structures has been less well studied. Therefore, in this study we examined the ability of pre-metamorphic Xenopus laevis tadpoles to self-correct malformed craniofacial tissues. We found that tadpoles can adaptively improve and normalize abnormal craniofacial morphology caused by numerous developmental perturbations. We then investigated the tissue-level and molecular mechanisms that mediate the self-correction of craniofacial defects in pre-metamorphic X. laevis tadpoles. Our studies revealed that this adaptive response involves morphological changes and the remodeling of cartilage tissue, prior to metamorphosis. RT-qPCR and RNA-seq analysis of gene expression suggests a thyroid hormone-independent endocrine signaling pathway as the potential mechanism responsible for triggering the adaptive and corrective remodeling response in these larvae that involves mmp1 and mmp13 upregulation. Thus, investigating how malformed craniofacial tissues are naturally corrected in X. laevis tadpoles has provided valuable insights into the maintenance and manipulation of craniofacial morphology in a vertebrate system. These insights may help in the development of novel therapies for developmental craniofacial anomalies in humans.

Mechanisms of physiological tissue remodeling in animals: Manipulating tissue, organ, and organism morphology.

Tissue remodeling is broadly defined as the reorganization or restoration of existing tissues. Tissue remodeling processes are responsible for directing the development and maintenance of tissues, organs, and overall morphology of an organism. Therefore, studying the regulatory and mechanistic aspects of tissue remodeling allows one to decipher how tissue structure and function is manipulated in animals. As such, research focused on investigating natural tissue reorganization in animal model organisms has great potential for advancing medical therapies, in conjunction with tissue engineering and regenerative medicine. Here we discuss the molecular and cellular mechanisms responsible for tissue remodeling events that occur across several animal phyla. Notably, this review emphasizes the molecular and cellular mechanisms involved in embryonic and postnatal physiological tissue remodeling events, ranging from metamorphosis to bone remodeling during functional adaptation.

Recovery of the Xenopus laevis heart from ROS-induced stress utilizes conserved pathways of cardiac regeneration.

Urodele amphibians and some fish are capable of regenerating up to a quarter of their heart tissue after cardiac injury. While many anuran amphibians like Xenopus laevis are not capable of such feats, they are able to repair lesser levels of cardiac damage, such as that caused by oxidative stress, to a far greater degree than mammals. Using an optogenetic stress induction model that utilizes the protein KillerRed, we have investigated the extent to which mechanisms of cardiac regeneration are conserved during the restoration of normal heart morphology post oxidative stress in X. laevis tadpoles. We focused particularly on the processes of cardiomyocyte proliferation and dedifferentiation, as well as the pathways that facilitate the regulation of these processes. The cardiac response to KillerRed-induced injury in X. laevis tadpole hearts consists of a phase dominated by indicators of cardiac stress, followed by a repair-like phase with characteristics similar to mechanisms of cardiac regeneration in urodeles and fish. In the latter phase, we found markers associated with partial dedifferentiation and cardiomyocyte proliferation in the injured tadpole heart, which, unlike in regenerating hearts, are not dependent on Notch or retinoic acid signaling. Ultimately, the X. laevis cardiac response to KillerRed-induced oxidative stress shares characteristics with both mammalian and urodele/fish repair mechanisms, but is nonetheless a unique form of recovery, occupying an intermediate place on the spectrum of cardiac regenerative ability. An understanding of how Xenopus repairs cardiac damage can help bridge the gap between mammals and urodeles and contribute to new methods of treating heart disease.

Bioelectric signaling in regeneration: Mechanisms of ionic controls of growth and form.

The ability to control pattern formation is critical for the both the embryonic development of complex structures as well as for the regeneration/repair of damaged or missing tissues and organs. In addition to chemical gradients and gene regulatory networks, endogenous ion flows are key regulators of cell behavior. Not only do bioelectric cues provide information needed for the initial development of structures, they also enable the robust restoration of normal pattern after injury. In order to expand our basic understanding of morphogenetic processes responsible for the repair of complex anatomy, we need to identify the roles of endogenous voltage gradients, ion flows, and electric fields. In complement to the current focus on molecular genetics, decoding the information transduced by bioelectric cues enhances our knowledge of the dynamic control of growth and pattern formation. Recent advances in science and technology place us in an exciting time to elucidate the interplay between molecular-genetic inputs and important biophysical cues that direct the creation of tissues and organs. Moving forward, these new insights enable additional approaches to direct cell behavior and may result in profound advances in augmentation of regenerative capacity.

HCN4 ion channel function is required for early events that regulate anatomical left-right patterning in a nodal and lefty asymmetric gene expression-independent manner.

Laterality is a basic characteristic of all life forms, from single cell organisms to complex plants and animals. For many metazoans, consistent left-right asymmetric patterning is essential for the correct anatomy of internal organs, such as the heart, gut, and brain; disruption of left-right asymmetry patterning leads to an important class of birth defects in human patients. Laterality functions across multiple scales, where early embryonic, subcellular and chiral cytoskeletal events are coupled with asymmetric amplification mechanisms and gene regulatory networks leading to asymmetric physical forces that ultimately result in distinct left and right anatomical organ patterning. Recent studies have suggested the existence of multiple parallel pathways regulating organ asymmetry. Here, we show that an isoform of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of ion channels (hyperpolarization-activated cyclic nucleotide-gated channel 4, HCN4) is important for correct left-right patterning. HCN4 channels are present very early in Xenopus embryos. Blocking HCN channels (Ih currents) with pharmacological inhibitors leads to errors in organ situs. This effect is only seen when HCN4 channels are blocked early (pre-stage 10) and not by a later block (post-stage 10). Injections of HCN4-DN (dominant-negative) mRNA induce left-right defects only when injected in both blastomeres no later than the 2-cell stage. Analysis of key asymmetric genes' expression showed that the sidedness of Nodal, Lefty, and Pitx2 expression is largely unchanged by HCN4 blockade, despite the randomization of subsequent organ situs, although the area of Pitx2 expression was significantly reduced. Together these data identify a novel, developmental role for HCN4 channels and reveal a new Nodal-Lefty-Pitx2 asymmetric gene expression-independent mechanism upstream of organ positioning during embryonic left-right patterning.

Coordinating heart morphogenesis: A novel role for hyperpolarization-activated cyclic nucleotide-gated (HCN) channels during cardiogenesis in Xenopus laevis.

Hyperpolarization-activated cyclic-nucleotide gated channel (HCN) proteins are important regulators of both neuronal and cardiac excitability. Among the 4 HCN isoforms, HCN4 is known as a pacemaker channel, because it helps control the periodicity of contractions in vertebrate hearts. Although the physiological role of HCN4 channel has been studied in adult mammalian hearts, an earlier role during embryogenesis has not been clearly established. Here, we probe the embryonic roles of HCN4 channels, providing the first characterization of the expression profile of any of the HCN isoforms during Xenopus laevis development and investigate the consequences of altering HCN4 function on embryonic pattern formation. We demonstrate that both overexpression of HCN4 and injection of dominant-negative HCN4 mRNA during early embryogenesis results in improper expression of key patterning genes and severely malformed hearts. Our results suggest that HCN4 serves to coordinate morphogenetic control factors that provide positional information during heart morphogenesis in Xenopus.

Use of Xenopus Frogs to Study Renal Development/Repair.

The Xenopus genus includes several members of aquatic frogs native to Africa but is perhaps best known for the species Xenopus laevis and Xenopus tropicalis. These species were popularized as model organisms from as early as the 1800s and have been instrumental in expanding several biological fields including cell biology, environmental toxicology, regenerative biology, and developmental biology. In fact, much of what we know about the formation and maturation of the vertebrate renal system has been acquired by examining the intricate genetic and morphological patterns that epitomize nephrogenesis in Xenopus. From these numerous reports, we have learned that the process of kidney development is as unique among organs as it is conserved among vertebrates. While development of most organs involves increases in size at a single location, development of the kidney occurs through a series of three increasingly complex nephric structures that are temporally distinct from one another and which occupy discrete spatial locales within the body. These three renal systems all serve to provide homeostatic, osmoregulatory, and excretory functions in animals. Importantly, the kidneys in amphibians, such as Xenopus, are less complex and more easily accessed than those in mammals, and thus tadpoles and frogs provide useful models for understanding our own kidney development. Several descriptive and mechanistic studies conducted with the Xenopus model system have allowed us to elucidate the cellular and molecular mediators of renal patterning and have also laid the foundation for our current understanding of kidney repair mechanisms in vertebrates. While some species-specific responses to renal injury have been observed, we still recognize the advantage of the Xenopus system due to its distinctive similarity to mammalian wound healing, reparative, and regenerative responses. In addition, the first evidence of renal regeneration in an amphibian system was recently demonstrated in Xenopus laevis. As genetic and molecular tools continue to advance, our appreciation for and utilization of this amphibian model organism can only intensify and will certainly provide ample opportunities to further our understanding of renal development and repair.

Beyond the Mammalian Heart: Fish and Amphibians as a Model for Cardiac Repair and Regeneration.

The epidemic of heart disease, the leading cause of death worldwide, is made worse by the fact that the adult mammalian heart is especially poor at repair. Damage to the mammal heart-such as that caused by myocardial infarction-leads to scarring, resulting in cardiac dysfunction and heart failure. In contrast, the hearts of fish and urodele amphibians are capable of complete regeneration of cardiac tissue from multiple types of damage, with full restoration of functionality. In the last decades, research has revealed a wealth of information on how these animals are able to perform this remarkable feat, and non-mammalian models of heart repair have become a burgeoning new source of data on the morphological, cellular, and molecular processes necessary to heal cardiac damage. In this review we present the major findings from recent research on the underlying mechanisms of fish and amphibian heart regeneration. We also discuss the tools and techniques that have been developed to answer these important questions.

Optogenetic Control of Apoptosis in Targeted Tissues of Xenopus laevis Embryos.

KillerRed (KR) is a recently discovered fluorescent protein that, when activated with green light, releases reactive oxygen species (ROS) into the cytoplasm, triggering apoptosis in a KR-expressing cell. This property allows for the use of KR as a means of killing cells in an organism with great temporal and spatial specificity, while minimizing the nonspecific effects that can result from mechanical or chemical exposure damage techniques. Such optogenetic control of cell death, and the resulting ability to induce the targeted death of specific tissues, is invaluable for regeneration/repair studies-particularly in Xenopus laevis, where apoptosis plays a key role in regeneration and repair. We here describe a method by which membrane-bound KR, introduced to Xenopus embryos by mRNA microinjection, can be activated with green light to induce apoptosis in specific organs and tissues, with a focus on the developing eye and pronephric kidney.

Regeneration of functional pronephric proximal tubules after partial nephrectomy in Xenopus laevis.

BACKGROUND: While the renal system is critical for maintaining homeostatic equilibrium within the body, it is also susceptible to various kinds of damage. Tubule dysfunction in particular contributes to acute renal injury and chronic kidney disease in millions of patients worldwide. Because current treatments are highly invasive and often unavailable, gaining a better understanding of the regenerative capacity of renal structures is vital. Although the effects of various types of acute damage have been previously studied, the ability of the excretory system to repair itself after dramatic tissue loss due to mechanical damage is less well characterized. RESULTS: A novel unilateral nephrectomy technique was developed to excise pronephric proximal tubules from Xenopus laevis tadpoles to study tubule repair after injury. Immunohistochemical detection of protein expression and renal uptake assays demonstrated that X. laevis larvae have the capacity to regenerate functional proximal tubules following resection. CONCLUSIONS: We have validated the renal identity of the restored tubules and demonstrated their ability to functional normally providing the first evidence of regeneration of renal tissue in an amphibian system. Importantly, this tubule restoration occurs by means of a process involving an early apoptotic event and the biphasic expression of the matrix metalloproteinase, Xmmp-9.

Low concentrations of atrazine, glyphosate, 2,4-dichlorophenoxyacetic acid, and triadimefon exposures have diverse effects on Xenopus laevis organ morphogenesis.

Many chemicals are released into the environment, and chemical contamination has been suggested as a contributing factor to amphibian declines. To add to a growing body of knowledge about the impact of individual chemicals on non-target organisms, we examined the specificity of deformities induced by exposure to four pesticides (atrazine, 2,4-dichloropheoxyacetic acid (2,4-D), triadimefon, and glyphosate) in the model amphibian species, Xenopus laevis. We focused on the period of organ morphogenesis, as it is frequently found to be particularly sensitive to chemical exposure yet also commonly overlooked. We found similar levels of intestine malformations and edemas, as well as disruption of skeletal muscle, in atrazine and triadimefon exposed tadpoles. The effects of 2,4-D were only apparent at the highest concentrations we examined; glyphosate did not induce dramatic malformations at the concentrations tested. While researchers have shown that it is important to understand how chemical mixtures affect non-target organisms, our results suggest that it is first crucial to determine how these chemicals act independently in order to be able to identify consequences of individual pesticide exposure.

Acute atrazine exposure disrupts matrix metalloproteinases and retinoid signaling during organ morphogenesis in Xenopus laevis.

Exposure to the herbicide atrazine disrupts many developmental processes in non-target animals. Atrazine exposure during organ morphogenesis in amphibians results in dramatic malformations; the mechanism by which this happens has not been described. We have taken a candidate gene approach to explore two possible mechanisms by which acute atrazine exposure causes extensive malformations in several tissues in Xenopus laevis tadpoles. Using a static renewal system, we exposed tadpoles to atrazine for 6-48 h during organ morphogenesis (Nieuwkoop and Faber stage 42). We observed degradation of cranial cartilage and differentiated muscle in the head, gut and somites of exposed tadpoles. Additionally, transcript levels of matrix metalloproteinases (MMPs), specifically both MMP9TH and MMP18, increased in atrazine-exposed tadpoles in a dose-response test, and MMP18 increased as early as 6 h after exposure began. Gelatinase MMP activity was also altered by atrazine exposure, indicating that atrazine disrupts gene function at the level of transcription and protein activity. Furthermore, transcript levels of the enzyme Xcyp26, an enzyme in the retinoic acid signaling pathway, significantly decreased in the intestines of tadpoles exposed to 10 or 35 mg l(-1) atrazine for 48 h. Our results suggest two mechanisms by which atrazine can disrupt tissue morphogenesis: through misregulation of MMPs that are critical in extracellular matrix remodeling throughout development and the disruption of retinoic acid signaling. This study begins to describe conserved vertebrate developmental processes that are disrupted by atrazine exposure.

Bioelectric controls of cell proliferation: ion channels, membrane voltage and the cell cycle.

All cells possess long-term, steady-state voltage gradients across the plasma membrane. These transmembrane potentials arise from the combined activity of numerous ion channels, pumps and gap junction complexes. Increasing data from molecular physiology now reveal that the role of changes in membrane voltage controls, and is in turn controlled by, progression through the cell cycle. We review recent functional data on the regulation of mitosis by bioelectric signals, and the function of membrane voltage and specific potassium, sodium and chloride ion channels in the proliferation of embryonic, somatic and neoplastic cells. Its unique properties place this powerful, well-conserved, but still poorly-understood signaling system at the center of the coordinated cellular interactions required for complex pattern formation. Moreover, disregulation of ion channel expression and function is increasingly observed to be not only a useful marker but likely a functional element in oncogenesis. New advances in genomics and the development of in vivo biophysical techniques suggest exciting opportunities for molecular medicine, bioengineering and regenerative approaches to human health.

Coordinating the timing of cardiac precursor development during gastrulation: a new role for Notch signaling.

Notch signaling has been shown to mediate a wide array of cell fate decisions during development. While previous work has demonstrated that Notch signaling plays an important role in regulating cardiac differentiation and morphogenesis, an earlier role during cardiac field formation has not yet been fully characterized. Previously, our lab demonstrated that perturbations in Notch signaling beginning at the onset of gastrulation affect the subdivision of germ layers. However due to the potential additive effects of misregulating Notch signaling over multiple stages of development, it was not possible to distinguish a specific role for this pathway during heart field specification. Here, we developed an innovative approach that takes advantage of temporally inducible constructs to isolate our manipulations to specific windows of development. In particular, we focused our studies on some of the earliest stages of cardiogenesis when heart field specification occurs. Our findings demonstrate a novel role for Notch signaling during the prepatterning of the cardiac mesoderm. Specifically, once relieved of aberrantly activated Notch signaling following gastrulation, cardiac precursors retain the ability to express markers of the cardiac field. Conversely, downregulating Notch signaling in cells fated to become heart tissue results in the induction of cardiac field genes in gastrula embryos. Finally, we provide evidence suggesting that this new role for Notch signaling is mediated at least in part via the Notch effector protein, Esr9 and the transcription factor GATA4. Taken together, these findings provide strong evidence for a novel role for Notch signaling in regulating the timing of heart field specification during early cardiogenesis.

Perturbation of organogenesis by the herbicide atrazine in the amphibian Xenopus laevis.

BACKGROUND: Exposure to anthropogenic chemicals during development can disrupt the morphogenesis of organ systems. Use of the herbicide atrazine has been debated in recent years because of its implicated, but poorly characterized, effects on vertebrates. Previous studies primarily examined the effects of atrazine exposure during metamorphosis or early developmental stages of amphibians. OBJECTIVES: We sought to identify and characterize the susceptibility during the often-overlooked developmental stage of organ morphogenesis. METHODS: We used a static renewal experimental treatment to investigate the effects of 10, 25, and 35 mg/L atrazine from early organ morphogenesis through the onset of tadpole feeding in the aquatic amphibian model system, Xenopus laevis. We quantified malformations of the body axis, heart, and intestine, as well as apoptosis in the midbrain and pronephric kidney. RESULTS: We found a significant dose-dependent increase in the percentage of atrazine-exposed tadpoles with malformations of multiple tissues including the main body axis, circulatory system, kidney, and digestive system. Incidence of apoptotic cells also increased in the both midbrain and kidney of atrazine-exposed tadpoles. CONCLUSIONS: Our results demonstrate that acute atrazine exposure (10-35 mg/L for < or = 48 hr) during early organ morphogenesis disrupts proper organ development in an amphibian model system. The concurrent atrazine-induced apoptosis in the pronephric kidney and midbrain begins to elucidate a mechanism by which atrazine may disrupt developmental processes in nontarget organisms.

Patterning the embryonic kidney: BMP signaling mediates the differentiation of the pronephric tubules and duct in Xenopus laevis.

The Bone morphogenetic proteins (BMPs) mediate a wide range of diverse cellular behaviors throughout development. Previous studies implicated an important role for BMP signaling during the differentiation of the definitive mammalian kidney, the metanephros. In order to examine whether BMP signaling also plays an important role during the patterning of earlier renal systems, we examined the development of the earliest nephric system, the pronephros. Using the amphibian model system Xenopus laevis, in combination with reagents designed to inhibit BMP signaling during specific stages of nephric development, we revealed an evolutionarily conserved role for this signaling pathway during renal morphogenesis. Our results demonstrate that conditional BMP inhibition after specification of the pronephric anlagen is completed, but prior to the onset of morphogenesis and differentiation of renal tissues, results in the severe malformation of both the pronephric duct and tubules. Importantly, the effects of BMP signaling on the developing nephron during this developmental window are specific, only affecting the developing duct and tubules, but not the glomus. These data, combined with previous studies examining metanephric development in mice, provide further support that BMP functions to mediate morphogenesis of the specified renal field during vertebrate embryogenesis. Specifically, BMP signaling is required for the differentiation of two types of nephric structures, the pronephric tubules and duct.

Subdividing the embryo: a role for Notch signaling during germ layer patterning in Xenopus laevis.

The development of all vertebrate embryos requires the establishment of a three-dimensional coordinate system in order to pattern embryonic structures and create the complex shape of the adult organism. During the process of gastrulation, the three primary germ layers are created under the guidance of numerous signaling pathways, allowing cells to communicate during development. Cell-cell communication, mediated by receptors of the Notch family, has been shown to be involved in mediating diverse cellular behaviors during development and has been implicated in the regulation of cell fate decisions in both vertebrate and invertebrate organisms. In order to investigate a role for Notch signaling during boundary formation between the mesoderm and endoderm during gastrulation, we manipulated Notch signaling in gastrula stage embryos and examined gene expression in resultant tissues and organs. Our findings demonstrate a much broader role for Notch signaling during germ layer determination than previously reported in a vertebrate organism. Activation of the Notch pathway, specifically in gastrula stage embryos, results in a dramatic decrease in the expression of genes necessary to create many different types of mesodermal tissues while causing a dramatic expansion of endodermal tissue markers. Conversely, temporally controlled suppression of this pathway results in a loss of endodermal cell types and an expansion of molecular markers of mesoderm. Thus, our data are consistent with and significantly extend the implications of prior observations suggesting roles for Notch signaling during germ layer formation and establish an evolutionarily conserved role for Notch signaling in mediating mesoderm-endoderm boundaries during early vertebrate development.

The mitochondrial-apoptotic pathway is triggered in Xenopus mesoderm cells deprived of PDGF receptor signaling during gastrulation.

Platelet-derived growth factor receptor (PDGFR) signaling is required for normal gastrulation in Xenopus laevis. Embryos deprived of PDGFR signaling develop with a range of gastrulation-specific defects including spina bifida, shortened anteroposterior axis, and reduced anterior structures. These defects arise because the involuting mesoderm fails to move appropriately. In this study, we determine that inhibition of PDGFR signaling causes prospective head mesoderm cells to appear in the blastocoel cavity at the onset of gastrulation, stage 10. These aberrant cells undergo apoptosis via the caspase 3 pathway at an embryonic checkpoint called the early gastrula transition (EGT). They are TUNEL-positive and have increased levels of caspase 3 activity compared to control embryos. Apoptotic death of these mesoderm cells can be prevented by co-injection of mRNA encoding Bcl-2 or by injection of either a general caspase inhibitor or a caspase 3-specific inhibitor. Prevention of cell death, however, is not sufficient to rescue gastrulation defects in these embryos. Based on these data, we propose that PDGFR signaling is necessary for survival of prospective head mesoderm cells, and also plays an essential role in the control of their cell movement during gastrulation.