Cell motility is essential for processes such as immune responses, wound healing, and fertilization. Caenorhabditis elegans is an excellent model for studying sperm motility, due to the worm's transparency and high sperm production. C. elegans amoeboid sperm move by extending and retracting pseudopods, which are powered internally by the dynamic polymerization and depolymerization of the nematode Major Sperm Protein (MSP). Previous biochemical studies in the parasitic nematode Ascaris suum identified MSP polymerization-activating kinase (MPAK) as a key regulator of MSP assembly within the pseudopod. Here we investigate two C. elegans homologs, Tau Tubulin Kinase 8.1 (ttbk-8.1) and Tau Tubulin Kinase 8.2 (ttbk-8.2), that arose from a C. elegans-specific gene duplication. We generated both single and double knockout mutants; single knockout animals have fertility comparable to wildtype. Self-fertilizing double knockout (ttbk-8.1/2) hermaphrodites are severely sub-fertile, and males are completely infertile. ttbk-8.1/2 males successfully transfer sperm to females; however, their sperm cannot crawl to the site of fertilization. During sperm activation, ttbk-8.1/2 sperm undergo the two key events of sperm activation (membranous organelle fusion and pseudopod extension), however the process is abnormally slow. Furthermore, the resulting pseudopods are non-motile and abnormally smooth. Localization of key proteins to the pseudopods remains unaffected. TTBK-8.1/2 colocalizes with MSP to the fibrous bodies in mature spermatocytes, distributes throughout the cytoplasm in spermatids, and is confined to pseudopods of spermatozoa. These findings suggest an essential role for TTBK-8.1/2 in timely sperm activation and pseudopod motility.

Some major discoveries come as the culmination of a long progression of incremental findings and evolving ideas. The discovery of maternal mRNA was different, its instancy noteworthy as a matter of epistemology. But it had a second impact, one that was far greater than the fast kinetics of the discovery per se. This was because it was the moment when embryology became molecular biology. This key event then soon aligned with genetics, deployed particularly in Drosophila and C. elegans, there proving this approach to be the emperor of all disciplines for the study of development. But the 1960's discovery of maternal mRNA, in the sea urchin, was the milestone in catalyzing this later heuristic vector.

One-carbon (1C) metabolism is a central metabolic network that integrates nutrient availability with biosynthetic and epigenetic processes essential for embryonic and placental development. By transferring carbon units derived from amino acids and folate metabolism, this pathway generates nucleotides, methyl donors, and other intermediates required for cell proliferation and differentiation. The pathway is compartmentalized between mitochondria and cytosol, with mitochondrial reactions frequently supplying formate and other 1C units to sustain cytosolic biosynthesis in rapidly dividing cells. Pluripotent stem cells illustrate the strong dependence of cell identity on 1C metabolism. Mouse embryonic stem cells rely on threonine catabolism to produce glycine, acetyl-CoA, and S-adenosylmethionine (SAM), which supports chromatin methylation and self-renewal. In contrast, human ESCs lack a functional threonine catabolic pathway and instead depend on high methionine metabolism and glycine cleavage to maintain SAM levels and pluripotency. Genetic and metabolomic studies in diverse animal models have revealed stage-specific roles for 1C metabolism in processes including zygotic genome activation, DNA replication, epigenetic reprogramming, cell-cycle progression, organogenesis, cell migration, and longevity. Importantly, disruption of individual enzymes often produces defined developmental defects rather than general metabolic failure. Beyond early development, 1C metabolism also contributes to transgenerational epigenetic inheritance through its effects on germline DNA and histone methylation which are sensitive to perturbations in the folate and methionine pathways. Together, these findings show that 1C-metabolism links cellular metabolism with epigenetic regulation and developmental processes.

Receptor tyrosine kinase (RTK) signaling is a central mechanism by which local tissue interactions are translated into coordinated morphogenesis during eye development. The vertebrate eye provides a uniquely tractable system for studying this logic because it is assembled from neural epithelium, surface ectoderm, neural crest-derived mesenchyme, vascular endothelium, and specialized epithelial and endothelial derivatives across a prolonged developmental timeline. In this review, we synthesize how major RTK families, including FGF-FGFR, EGF-ErbB, PDGF-PDGFR, VEGF-VEGFR, Eph-ephrin, ANGPT-TEK/Tie2, IGF-INS receptors, and collagen-sensing DDRs, control optic cup formation, optic fissure closure, lens differentiation and architecture, corneal and anterior segment morphogenesis, retinal vascularization, Schlemm's canal development, and lacrimal gland branching. Rather than acting through pathway-specific outputs, these receptors repeatedly converge on shared MAPK/ERK, PI3K-AKT, PLCγ-PKC, SRC/FAK, and Rho-family cytoskeletal modules. Developmental specificity emerges from ligand geography, receptor dosage, co-receptor and extracellular matrix context, signal duration, receptor trafficking, mechanical state, and cross-talk with other signaling pathways. We also highlight how emerging single-cell, spatial, and multiomic atlases are shifting the field from static expression maps toward ligand-receptor source mapping and quantitative pathway-state inference. Finally, we connect developmental RTK mechanisms to congenital and pediatric eye disease. Together, these studies position the eye as a powerful model for understanding how reiterative RTK signaling builds complex organ form and informs therapeutic modulation, regenerative medicine, and disease mechanism.

Traditionally redox regulation has been interpreted through the lens of intracellular signaling, metabolic adaptation, or oxidative damage. In the vascular system, this view has been particularly influential: endothelial reactive oxygen species, nitric oxide, lipid peroxidation, and antioxidant pathways are commonly discussed as cell-autonomous regulators of vascular tone, permeability, inflammation, angiogenesis, dysfunction or aging. Here, we propose a broader perspective. We argue that the vasculature does not merely experience redox stress; it generates, edits, buffers, packages, and exports redox-active information beyond the traditionally recognized signaling molecules. This exported information-the secreted redoxome-may include oxidatively modified proteins, thiol/disulfide states, extracellular vesicle cargo, and additional circulating redox buffers. We first summarize what is known about vascular oxidative post-translational modifications and circulating redox systems. We then develop the opinion that secreted redox proteins may function as an angiocrine communication layer that instructs tissue homeostasis and disease transition. This view shifts the field from asking how vessels suffer oxidative stress to asking how vessels communicate regulated redox instructions.

Vision depends on the function of retinal circuits, supported by the retinal pigment epithelium (RPE) that lines the back of the eye. This single-cell layer thick epithelium provides growth factors and nutrients for the underlying photoreceptors, phagocytoses shed photoreceptor outer segment debris, and contributes to the blood-retinal barrier. Whether the cells of the RPE represent a single homogeneous population or represent distinct functional entities or states is poorly understood. Here, we use single cell RNA sequencing (scRNAseq) data from our mature 5-day post-fertilization zebrafish retina dataset and a publicly available adult zebrafish eye dataset, alongside in situ hybridization, to explore the transcriptomic diversity of RPE cells, its emergence over development, and its persistence into the adult. We identify 6 transcriptomically distinct RPE populations in the 5 day larval eye, representing either RPE subtypes or states, that exhibit different localization within the eye. Specifically, we find potential proliferative, iris-associated, dorso-ventral differentiating, and mature RPE subpopulations. These populations appear to emerge via separate developmental trajectories and may be distinct from the transcriptomic RPE subpopulations of the adult eye.

Mammalian limb development is a complex system involving several signaling centers and coordinated cell behaviors to sculpt a functioning limb capable of the diverse locomotory strategies that mammals exhibit. To investigate the changes in development that facilitate the generation of the wide array of limb phenotypes across mammals, we take a correlation network approach to investigate the developing limbs of mice, bats, and opossums, which represent typical limb development, a novel limb phenotype, and a shift in developmental timing, respectively. Using transcriptomic data of early limb development across these taxa, we build module correlation networks and identify a difference in network connectivity and the distribution of limb development genes across bat limb development. We identify a unique signature of increased modularity in the bat forelimb that is not detected in mouse or opossum. This modularity is not associated with increased specialization of limb development modules, but rather is marked by target limb development genes being spread evenly across several modules. The opossum, with its standard phenotype but altered developmental timing, does not show a difference in modularity relative to mouse. This work points toward the benefit of a network-minded approach to transcriptomic networks, which reveals developmental modularity and potential gene targets for exploration of developmental system evolution.

The cochlea resolves sound frequencies through molecular, structural, and functional specializations established along its tonotopic axis. Although transient morphogen gradients establish positional information during embryogenesis, the mechanisms that maintain and refine this information as cochlear function matures remain poorly understood. Here, we identify the chromatin modifier HMGA2 as a candidate molecular link between opposing hedgehog (HH) and retinoic acid (RA) signals and long-term auditory function. In vivo manipulation of RA and HH signaling demonstrated that RA suppresses, whereas HH activation promotes, Hmga2 expression, establishing its apex-to-base decreasing gradient. Conditional deletion of Hmga2 did not affect cochlear length, hair cell numbers, mechanotransduction, or Prestin localization but caused a delayed and persistent low-frequency hearing deficit. Longitudinal auditory measurements revealed two distinct functional phenotypes that may contribute to this deficit: non-tonotopic DPOAE abnormalities consistent with altered cochlear micromechanics and a late, low-frequency reduction in ABR P1 amplitude consistent with impaired inner hair cell-afferent signaling. Together, these findings identify HMGA2 as a molecular link between embryonic morphogen signaling and long-term low-frequency auditory function and suggest that persistent HMGA2-dependent regulation contributes to the maintenance of low-frequency auditory function.

Within the neuronal classes of the retina, amacrine cells (ACs) exhibit the greatest neuronal diversity in morphology and function. We show that the selective expression of the transcription factor Gbx2 is required for dendritic stratification of an individual AC subtype in the mouse retina. We identify Robo1 and Robo2 as downstream effectors that when deleted, phenocopy the dendritic misprojections seen in Gbx2 mutants. Slit1 and Slit2, the ligands of Robo receptors, are localized to the upper layers of the inner plexiform layer where we observe the dendritic misprojections in both Gbx2 and Robo1/2 mutants. We show that Robo receptors also are required for the proper dendritic stratification of additional AC subtypes, such as Vglut3+ ACs. These results show both that Gbx2 regulates the terminal differentiation of a single AC subtype and identify Slit-Robo signaling as a developmental mechanism for dendrite stratification in the retina.

Long noncoding RNAs (lncRNAs) display pervasive expression and function in the developing nervous system. Temporal profiling of gene expression in the retina has demonstrated differential expression of lncRNAs throughout development; however, determinations of lncRNA function during retinal development remain limited. In this study, we identify numerous lncRNAs with dynamic temporal expression and characterize the function of the lncRNA Gm11454, which we have named Peanut. Overexpression of Peanut in mouse retinas increases rod photoreceptor fate and neurogenesis of retinal progenitor cells (RPCs), inhibits Notch signaling, and inhibits expression of neighboring gene Tox2. A novel Peanut knockout mouse model demonstrates that Peanut is involved in the establishment of proper visual function and photoreceptor gene expression. Finally, we determined that Peanut modulates cell cycle progression and neurogenesis. Our results characterize the consequence of altered expression of a novel lncRNA as a putative regulator of RPC neurogenesis and differentiation and support the significance of lncRNAs in the developing retina.

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, as well as the consequences of secondarily losing metamorphosis.

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.

Mitochondria are dynamic organelles that can fragment or fuse to support different bioenergetic demands and cellular processes, although the role of mitochondrial fission and fusion during embryonic development is not well understood. Fluorescence lifetime imaging (FLIM) of the mitochondrial cofactor NADH can be used to visualize mitochondrial networks and infer aspects of cellular bioenergetics in a label-free manner. We used NADH FLIM to test whether germband cells undergo changes in cellular metabolism during Drosophila convergent extension (CE)--a process in which hundreds of epithelial cells undergo coordinated intercalation to elongate the embryo. Contrary to our expectations, we did not observe significant changes in NADH lifetime or mitochondrial topology during CE, suggesting that germband cells do not need to alter their baseline metabolism to fuel intercalation. Inhibiting mitochondrial fission led to hyper-fused basal networks and robustly increased NADH lifetime, whereas inhibiting fusion led to hyperfragmented apical networks and decreased NADH lifetime. Inhibiting either mitochondrial fission or fusion increased cell intercalation errors during CE, suggesting that a precise network topology is required for proper tissue elongation. These defects could be rescued by knocking down ROS scavengers, suggesting that one of the roles of mitochondria during CE is to create a ROS-rich environment to support cell motility. This study demonstrates the utility of NADH FLIM for visualizing mitochondria and characterizing bioenergetics during development in live embryos, and this technique should be broadly applicable to many other systems.

Drosophila melanogaster offers a genetically tractable model for dissecting the molecular logic of blood cell development. With a simple repertoire of blood cell types, Drosophila hematopoiesis relies on a conserved set of developmental pathways, among which Notch signaling emerges as a central regulator. In this review, we examine how Notch governs successive steps of blood cell development, including the initial specification of hematopoietic progenitors from the cardiogenic mesoderm, niche-dependent progenitor maintenance in the larval lymph gland, and fate decisions that include crystal cell specification, maturation, and transdifferentiation from plasmatocytes. We further address how Notch output is shaped by intracellular trafficking through the endolysosomal pathway, and how metabolic and environmental inputs are integrated to fine-tune lineage specification. Finally, we discuss how attenuation of Notch signaling in progenitors is required to permit lamellocyte differentiation in response to immune challenge. Throughout, we draw comparisons with vertebrate hematopoiesis, identifying conserved regulatory logic in progenitor emergence, niche-mediated stem cell maintenance, and binary fate decisions, while noting species-specific differences that reflect the distinct complexity of each system. Together, these analyses position Notch as a multifunctional developmental regulator whose activity underlies both homeostatic and adaptive blood cell production, and whose dysregulation is linked to hematological malignancies in humans.