It has long been thought that neurons are the only cells to utilize ion flux to temporally regulate secretion for cell-to-cell signaling, but we discovered that ectoderm and mesenchymal cells are excitable, undergo voltage-dependent calcium waves, and harness calcium influx to control secretion of essential morphogens, bone morphogenetic proteins (BMPs). Furthermore, altering ion flux during morphogenesis results in craniofacial and limb abnormalities in organisms as diverse as insects and mammals, suggesting that the role for electrical signaling is conserved. In neurons, calcium activates the SNARE complex to drive fusion of secretory vesicles to temporally regulate secretion. Our discoveries inspired the hypothesis that calcium induces the SNARE complex to control BMP secretion. Here, we use pharmacological inhibition of the SNARE complex (Botulinum toxin, BoNT-C) and genetic tools in Drosophila to test this hypothesis in the wing primordia (wing disc). To test whether the SNARE complex is important in epithelial cells for BMP secretion, we apply BoNT-C to the wing disc and measure BMP secretion and calcium activity. We found that BoNT-C inhibits BMP secretion and increases endogenous calcium activity in the epithelial cells of the wing disc, as it does in neurons. Furthermore, expression of BoNT-C in the BMP-producing cells significantly reduces measures of downstream signaling. These data open the possibility that the SNARE complex may control the secretion of other developmental morphogens. Furthermore, our data suggest that pharmacological inhibition of the SNARE complex may not be specific to neuronal targets.
Metamorphosis can provide an opportunity for animals with complex life cycles to transition between differing environments and still maintain optimal functionality across ontogeny. The structure and function of the amphibian visual system generally remodels to overcome differences in media (water, air) and fluctuations of available light. This in turn causes a shift in the selective pressures acting on the visual system at each life stage. Few comparative studies have evaluated how metamorphosis drives the evolution and expression of visual system genes across closely related species that occupy disparate habitats. Salamanders offer an excellent model to study visual system adaptations because of their diverse life cycles (biphasic and paedomorphic) and drastic habitat transitions (surface and cave-adapted). We sequenced whole-eye transcriptomes of larvae and adults across 13 lineages of Brook Salamanders to test whether there is a relationship between the expression of opsin and phototransduction genes, and lineages that differ in life cycle mode. Our study revealed that numerous differences in gene expression were lineage-specific. However, there was significant upregulation of rhodopsin and several rod-specific phototransduction genes in biphasic adults, whereas paedomorphic salamanders retained juvenilized expression patterns similar to larvae. The paedomorphic troglomorph species, Eurycea latitans exhibited aberrant expression patterns of rhodopsin and some rod-related genes. This study has implications for how metamorphosis allows salamanders to adaptively decouple their visual system in order to optimize to disparate larval and adult environments.
Semaphorins (Semas) are secreted and membrane-bound molecules classically characterized as axonal guidance cues in nervous system development. However, it is now widely appreciated that Semas have essential functions in the development of many systems including the cardiovascular, endothelial, and immune systems. Secreted Semas enable functionality distant from the site of release, while membrane-bound Semas mediate localized contact-dependent signaling. Interestingly, functional soluble ectodomains have been discovered in each of the four vertebrate membrane-bound classes which can drive change in the nature and reach of Sema signaling. This review highlights the discovery of these soluble Sema ectodomains, the mechanisms of their release, and their known roles in development and pathological states.
Biomolecular condensates formed by liquid-liquid phase separation (LLPS) have emerged as a major mode of intracellular organization, and their roles in craniofacial development are only beginning to be defined. In this review, we synthesize evidence that phase separation and condensate biology influences craniofacial morphogenesis at multiple levels, from transcriptional control in neural crest-derived lineages to signaling integration, mechanosensing, and the formation of mineralized tissues. Nuclear condensates formed by transcription factors and chromatin regulators are implicated in clefting and craniofacial syndromes are highlighted as modulators of local gene expression programs and cell fate decisions. Condensate-mediated regulation of mechanosensitive pathways and nucleolar function in osteogenic and chondrogenic cells is discussed in relation to craniofacial bone, cartilage, and sutural growth. In parallel, we examine extracellular phase-separation-like and LLPS-adjacent phenomena in enamel and dentin biomineralization, including self-assembly of enamel matrix proteins and polymer-induced liquid precursors that concentrate ions and guide apatite organization. Shared principles, conceptual distinctions, and outstanding questions are outlined, with emphasis on experimental strategies that link condensate or phase-behavior properties to craniofacial development, malformation and tissue repair.
Wakayama et al. describe an extraordinary 20-year serial cloning study in mice, concluding that serial cloning in mammals is ultimately limited by the accumulation of genetic anomalies. However, their whole-genome sequencing (WGS) analysis characterized selected cloned animals but did not include matched genomic profiling of the corresponding cumulus cell (CC)-donor mice, the source CC populations, or developmental stages. Because each reconstructed embryo originated from a single CC nucleus and re-cloned animals were used to advance the lineage, pre-existing somatic variation could have entered the lineage and subsequently been propagated. Consequently, variants detected in later generations cannot be assigned definitively to pre-existing donor-cell mosaicism, donor-cell handling, somatic cell nuclear transfer manipulation, or early embryogenesis. Thus, the observed decline cannot be attributed exclusively to genetic lesions arising during repeated cloning, but the unresolved genomic status of the lineage-founding donor cells remains a plausible but unproven contributor. The study therefore demonstrates the transmission and propagation of genetic lesions through serial cloning more directly than it establishes that all initiating lesions arose because of repeated cloning. Paired genomic profiling of donor-cell populations, embryos, and offspring would help resolve the origins of accumulated genetic lesions and determine whether donor-cell screening could extend serial cloning.
Initiated by NICHD and crafted with clinicians and laboratory scientists, the Structural Birth Defects (SBD) Initiative has supported research into the clinical, genetic, biochemical, mechanistic, developmental, and environmental basis of human disorders with structural anomalies for 25 years. The SBD Initiative has supported and continues to fund research teams studying a broad spectrum of single gene (Mendelian) disorders along with defining loci and susceptibility genes in oligogenic phenotypes, including genetic and environmental risk modifiers. The Initiative required and currently convenes biennial meetings of SBD investigators to share data, exchange ideas and initiate collaborations. In alternate years, online trainee symposia provide a platform for medical fellows, postdoctoral fellows and graduate students to present their data, with the goal of attracting and retaining this future generation of investigators in SBD research. In addition to determining their etiology, the SBD Initiative has supported remarkable progress in developing a fundamental mechanistic understanding of this diverse group of phenotypes. Together, scientific progress has led to translational benefits that include 1) widespread diagnostic testing for families with these disorders, both within the United States and across the world, and 2) pharmacological treatments for affected individuals. This progress has fulfilled the promise of the vision of the architects of the program, which is reviewed in this article, and continues to drive the field forward.
Thyroid hormones (THs) coordinate proliferation, apoptosis, and tissue remodeling during vertebrate development, yet how TH signaling constrains embryonic cell death in avian embryos remains unclear. Here, we used amiodarone, a pharmacological disruptor of TH signaling with reported effects on TH receptors and deiodinase-mediated hormone metabolism, to perturb TH signaling at the onset of incubation in chick embryos and integrate systems-level and functional readouts. In silico docking predicted favorable binding of amiodarone to all three deiodinase isoforms (DIO1-3), while whole-embryo assays showed a progressive reduction in total embryonic deiodinase capacity from day 2 to day 4. Treated embryos developed lateral plate mesoderm-associated craniofacial, limb, and ventral body-wall defects and exhibited widespread apoptosis, as evidenced by Nile blue sulfate staining, DNA laddering, TUNEL, Annexin V/propidium iodide flow cytometry, and cleaved CASPASE-3 immunolocalization. qRT-PCR and immunoblotting demonstrated downregulation of BCL2 and PCNA with upregulation of BAX, P53, BAD, caspases, and cleaved CASPASE-3, indicating activation of mitochondrial apoptotic signaling accompanied by increased P53 expression. High-resolution proteomics further supported induction of apoptotic and mitochondrial stress-associated pathways alongside suppression of vesicle trafficking and mitochondrial translation proteins. Cell-cycle profiling showed accumulation of cells in sub-G0/G1 and G0/G1 phases, indicating G1 checkpoint arrest and reduced proliferative capacity. Promoter analysis predicted putative thyroid hormone response elements in selected apoptotic regulators, suggesting potential TH-responsive regulatory sites requiring functional validation. Together, these data support an important role for early TH signaling in maintaining the proliferation-apoptosis balance during chick embryonic morphogenesis.
Precise neuromuscular connectivity depends on coordinated interactions between motor axons and their muscle targets during development. In the Drosophila embryo, dynamic muscle protrusions termed myopodia arise during early phases of muscle innervation and have been proposed to contribute to synaptic partner matching. However, whether similar structures occur across different muscle fibers and how they are developmentally regulated remain unclear. Here, we developed a genetic imaging toolkit based on GAL4 drivers that label defined subsets of embryonic muscle fibers or enable stochastic single-muscle visualization across the musculature. These reagents allow minimally invasive, high-resolution imaging of muscle membrane dynamics in both live and fixed embryos. Using this approach, we found that myopodial clustering is not restricted to a single muscle but is observed across multiple muscle fibers at presumptive synaptic contact sites. Importantly, clustered myopodia emerged during a defined developmental window coincident with motor axon arrival at target muscles, indicating that clustering is temporally associated with neuron-muscle interactions. While clustering was observed in multiple muscles, its frequency and morphology exhibited quantitative variation among muscle fibers. Together, these findings establish myopodial clustering as a recurrent and temporally regulated cellular behavior in the presumptive postsynaptic sites of ventral muscles during early neuromuscular contact development. This study provides a versatile toolkit and a framework for systematically analyzing muscle membrane dynamics during synaptic target recognition.
As a possible target for pro- or anti-angiogenic therapy, the process of angiogenesis has become more widely recognized in disease and is essential in normal growth and development. The current study examined the immunological activities of macrophages during angiogenesis in quail embryos. CD68 was used to identify perivascular macrophages, which also retained expression of some stem cell markers, including CD34 and CD117, and also CD21. The macrophages also expressed vascular endothelial growth factor (VEGF), a key promoter of angiogenesis, and matrix metalloproteinase-9 (MMP-9), which degrades matrix components to facilitate the formation of new blood vessels. The growth factor bone morphogenetic protein (BMP) and platelet-derived growth factor receptor-alpha (PDGF-α), which are often associated with mesenchymal cell recruitment and vascularization, were also expressed in the perivascular macrophages. More recently, studies investigating macrophages have become more prominent due to their proposed effects on endothelial cells. Macrophages are also increasingly considered potential targets for pro- or anti-angiogenic therapies, and this research provides insights into protein expression and roles in angiogenesis.
Archaeopteryx, the iconic 150-million-year-old basal bird, lies at the heart of the debate on the origin of avian flight. While its asymmetric wing feathers indicate some flight capability, its limited shoulder mobility and reduced feather asymmetry suggest it lacked the capacity for the single, powerful leap used by modern birds for rapid take-off. Observations of extant birds reveal that many species, including corvids, employ multiple bipedal leaps with minimal wing assistance to gradually accelerate during take-off, particularly in low-stress contexts. Given Archaeopteryx's robust hindlimbs, we hypothesized that it could have used a similar multi-leap strategy to become airborne. We therefore tested the hypothesis that Archaeopteryx could generate sufficient take-off velocity via multiple hops or leaps. To test this, we developed a biomechanical model based on experimental take-off data from living birds, adapted to Archaeopteryx's anatomy. By analysing joint moments at the hip, knee, and ankle, alongside muscle capacity, we estimated its take-off velocity. Our results demonstrate that Archaeopteryx could have achieved its minimum sustainable flight speed in as few as two to three leaps, without requiring the energetically demanding single leap of modern birds. This study provides the first quantitative support for a ground-up, multi-leap take-off mechanism in early birds, distinct from the single-leap strategy usually observed today. We propose that the modern avian take-off may have evolved from this primitive multi-leap behaviour, offering new insights into the biomechanical transition from terrestrial locomotion to powered flight.
Translating the bioelectric code remains one of the core challenges to widespread biomedical translation of bioelectric interventions, as well as a better evolutionary understanding of how developmental ionic signaling evolved to become the basis of neural intelligence. To advance our understanding, it is crucial to develop model systems that allow simultaneous, quantitative study of diverse bioelectric parameters in living states, which must also be linked to cell- and tissue-level outcomes. Here, we apply quantitative Fluorescent Lifetime Imaging (FLIM) optical estimation of membrane potential (Vmemoe) to map the bioelectric dynamics of spreading Xenopus laevis neural crest explants (NCEs) over > 17 h. We identify a slow hours-scale bioelectric component and distinct faster minutes-scale and seconds-scale components. These dynamics often span multiple cells, consistent with roles in the collective behavior of NCEs. We then use information theory to show that minutes-scale NCE Vmemoe dynamics are largely distinct from calcium dynamics. Finally, we provide a survey of diverse bioelectric events revealing a deep complexity in collective bioelectric dynamics, likely involving tunneling nanotubes in their transmission, which suggests numerous avenues for further investigation. These combined data demonstrate FLIM as a powerful tool for observing subtle bioelectric signals at the large temporal and spatial scale needed for developmental biology studies.
Vascularization of the central nervous system and the postnatal retina proceeds through angiogenic growth that relies in part on the basement membrane (BM) to support endothelial cell-cell interactions and maintain barrier integrity during vessel formation. In addition to the core structural components of the BM, matricellular proteins are present as minor constituents, however, their contributions to endothelial cohesion and barrier stability during angiogenesis remain poorly defined. Here, we identify the matricellular protein Fibulin-1 (Fbln1), a regulator of cell-matrix interactions, as an essential factor during mid-gestation that prevents the formation of dilated, tortuous capillaries that give rise to glomeruloid vascular lesions and intracerebral hemorrhage. Fbln1-deficient brains exhibit vascular malformations characterized by reduced Type IV collagen deposition and diminished CD31 localization at endothelial junctions. Although overall pericyte coverage across the vasculature is maintained, the glomeruloid lesions lack pericyte investment.In the developing retina, loss of Fbln1 similarly results in hemorrhage and tortuous capillaries with glomeruloid vascular abnormalities that disrupt the formation of both the superficial and deep vascular plexuses. Together, these findings demonstrate that Fbln1 is a critical determinant of brain and retinal vascular development and is required to preserve endothelial barrier integrity during angiogenesis.
Regeneration abilities vary among species, but they also vary within and across individuals of a single species. Many annelids (segmented worms) have variable regenerative ability along their anterior-posterior (AP) body axis. However, satisfactory explanations for the diverse regenerative outcomes at different amputation positions remain elusive. The annelid Capitella teleta demonstrates robust posterior regenerative ability but lacks anterior regenerative ability, although anterior regenerative ability has not been rigorously assessed at multiple amputation sites. In this study, we characterize regenerative ability along the AP axis of C. teleta by comparing the post-amputation response of tail fragments cut between segments 1 and 2, 4 and 5, 10 and 11, and 15 and 16. Cell proliferation, gene expression, and protein expression assays demonstrate that the anterior-most amputation site progresses to later stages of regeneration than more posterior amputation sites, but regeneration does not reach completion. The distribution of neoblast-like cells after amputation suggests that these cells do not substantially contribute to formation of the anterior-facing blastema. Additionally, regeneration outcome is not improved following activation of Wnt/β-catenin signaling in tail fragments amputated between segments 1 and 2. Conversely, inhibition of Wnt/β-catenin signaling at the same amputation position leads to increased regenerative ability with distinct phenotypes consistent with anterior regeneration. Although not all differentiated anterior structures form, this result hints at partial anteriorization. Taken together, our data show the value of comparing regenerative abilities of tissues within an individual organism and demonstrate potential of increasing regenerative ability in regeneration-deficient tissues.
The respiratory system of Drosophila melanogaster consists of a network of interconnected gas-filled epithelial tubes. Stellate-shaped terminal cells at the termini of each tracheal branch serve as the primary source of oxygen and grow continuously throughout larval stages in response to hypoxic signals. Each cytoplasmic branch of a terminal cell hollows out to form subcellular seamless tubes that lack epithelial junctions. It is known that over the course of larval development, tracheal terminal cells grow by extending new branches and lengthening existing branches. By the end of larval development, each branch of these highly ramified cells contains a subcellular lumen; the relationship between branch and tube extension over this period of immense growth has yet to be explored. Here we demonstrate that branch extension and intracellular lumen formation are spatially and temporally coupled throughout larval stages, however, nascent tubes formed during a larval instar are fluid-filled and do not inflate with air until the following molt. This has interesting implications for the delivery of oxygen to target tissues during the first 48 h of larval growth.
The vertebrate neural retina, the image forming component of the eye, offers a useful, clinically-relevant system for understanding nervous system development and regeneration. Within vertebrates, the teleost fish retina specifically has been the subject of intense study for its regenerative response to neuronal damage - a response that does not take place in mammals. While the field has recently focused upon molecular and cellular processes that initiate this response and generate new neurons, it is also important to evaluate the ultimate "success" of this process. True neural regeneration requires that the cells and tissues that replace those lost to damage, also restore their original structure and function. From this outcomes-based perspective, does retinal regeneration recapitulate the results of development? This paper reviews ∼50 years of effort and knowledge regarding the regeneration of retinal neuronal types and patterns, wiring of regenerated neurons within the retina and beyond, and physiological and behavioral studies of function of regenerated retina. Emphasis is placed upon goldfish and zebrafish model systems, as these have been popular, productive models for revealing both the process and the outcome of retinal regeneration. The outcome, as accumulated data suggest, is considered remarkable in the sense that several features of structure and function are restored. Regenerated retinas are structurally similar in comparison with undamaged retinas that developed and grew normally, with notable local patterning errors. A body of evidence suggests that these structural errors are not of great consequence for the re-establishment of retinal function that is centrally integrated to support simple, visually-mediated behaviors. However, functional tests have been limited in scope and scale, lacking rigorous applications of more sophisticated approaches to evaluate visual system circuitry. Such approaches are becoming possible in the zebrafish model. Fish models are upheld as outstanding resources for providing current and future knowledge of regeneration that can be translated to treat human retinal disorders.
The oculocutaneous albinism 2 protein is required for melanin synthesis in vertebrates and its loss of function leads to one of the most common forms of skin and eye albinism, Oculocutaneous albinism (OCA). Using a zebrafish disease model for OCA2, we explore developmental explanations for pleiotropic traits observed in humans with OCA2. At multiple stages, we find that oca2 mutant zebrafish exhibit abnormal number and location of iridophores, an accessory pigment cell type that arises from neural crest cells in fish and amphibians. In addition, oca2 mutants display defects during eye development including delayed choroid fissure closure and significant changes in retinal cell differentiation and organization. Our transcriptomic analysis of oca2 mutants revealed alterations in gene networks involved in differentiation of neural crest cells, retinal neurons, and the retinal pigment epithelium (RPE) including Notch and Wnt/β-catenin pathway genes. Together, our data provide information about developmental consequences of oca2 mutation and support the possibility that the Oca2 protein may function in neural crest differentiation and eye development.
Epigenetic regulation via DNA demethylation and histone modification is essential for tissue development. We investigated the role of the Drosophila ten-eleven translocation protein (dTet) in eye development and its interaction with the oncohistone H3.3K27M. dTet overexpression in eye imaginal discs caused strong downregulation of retinal determination genes (toy, ey, so, eya, dac) and developmental arrest, leading to partial or complete eye loss. Enhanced dTet knockdown using sensitized conditions also resulted in eye developmental defects, supporting a dosage-dependent role for dTet during retinal morphogenesis. DNA-binding analysis using published ChIP-seq data revealed that dTet binds the promoters of these genes as well as wingless (wg), a negative regulator of eye specification, explaining its dual role in repressing retinal programs and activating Wnt/Wg signaling. Co-expression with H3.3K27M suppressed the mutation-induced overgrowth phenotype, whereas dTet loss enhanced it, indicating context-dependent modulation of tissue growth. Many dTet targets have conserved human orthologs implicated in congenital eye disorders, highlighting evolutionary conservation and potential translational relevance. These findings establish dTet as an important regulator of eye development.
