Developmental Dynamics

The morphogenesis of the optic cup provides a robust system for studying how two apposed epithelial monolayers with distinct properties fold and stretch in a coordinated manner to form the primordial eye. While much research has been conducted on the temporal dynamics of retinal neuroepithelium invagination, the spatial organization and stretching of the retinal pigment epithelium has received less attention. The fish species Astyanax mexicanus offers a unique model to examine the mechanisms of optic tissue morphogenesis through a comparative lens, as it exhibits natural variation in eye development between its river-dwelling and cave-adapted morphs. Using quantitative 3D imaging of optic cups from both morphs, we found that RPE morphogenesis involves transient, graded, and anisotropic cell stretching that patterns the epithelium during optic cup shaping. Analyses of RPE nuclear spacing and cell morphology showed that tissue stretching gradually increases along the proximo-distal axis, suggesting maximal tension in the elongated distal RPE cells aligned along the optic cup meridians. Furthermore, nuclear volumes and apical surface areas of RPE cells scaled spatially along the same axis, independently of endoreplication. In the cavefish natural mutant, RPE expansion was delayed by over six hours and proximal stretching exhibited altered isotropy, indicative of disrupted temporal coordination and suggesting modified mechanical constraints. These results demonstrate that RPE morphogenesis is a highly heterogeneous process from a spatiotemporal perspective, offering new insights into the study of the biomechanical principles of eye development in vertebrates. Summary statementThis study reveals the emergence of cell morphology gradients within the retinal pigment epithelium during morphogenesis of the eye in two distinct populations of the same species of fish.

Periodic patterns are a frequent motif in biology that occurs across diverse tissues and species. In mammals, pigmentation patterns such as zebra stripes or tiger stripes are well-known examples of periodic patterns; more recently, the myenteric plexus (MP) of the enteric nervous system (ENS), which controls gastrointestinal motility, has been found to exhibit a striped organization in humans and laboratory mice. In domestic cats and other felids, the Transmembrane aminopeptidase Q (Taqpep) gene plays a key role in color pattern establishment during skin development, but its patterning role has not been examined in other tissues. Here, we show that, in laboratory mice, Taqpep is required for normal patterning of developing hair follicles and the MP. Using both sequencing and histologic techniques, we found Taqpep is expressed in mesenchymal cells in embryonic skin and intestine directly adjacent to where periodic patterning occurs. We generated Taqpep mutant mice, which exhibit disrupted epidermal patterning akin to the changes in periodic coat patterning observed in Taqpep mutant cats. The intestine of Taqpep mutants has irregularly periodicity of enteric neuronal stripes, and enteric neurons in Taqpep mutants exhibit disrupted Wnt signaling. This work provides new insight into the mechanism of enteric neuronal patterning and identify Taqpep as a common and conserved mediator of periodic patterning across mammalian tissues and organisms. Author summaryPeriodic patterning is a frequent motif in biology. Examples include pigmentation patterning such as tiger stripes and, as recently identified in both mouse and human, the striped organization of enteric neurons in the myenteric plexus of the intestine. In domestic and wild cats, the Transmembrane aminopeptidase Q (Taqpep) gene is essential for the establishment of periodic patterning. Whether this gene plays a conserved role in periodic patterning across other tissues and species has yet to be explored. We found that Taqpep is expressed in mesenchymal cells in embryonic mouse skin and intestine at key locations and developmental stages to instruct periodic patterning. We next generated Taqpep mutant mice that exhibit disrupted periodic patterns in both developing skin follicles and in enteric neuron organization. Thus, Taqpep is essential in establishing periodic patterning in diverse mammals and tissues.

Background: During vertebrate embryogenesis, the isthmic region spans the midbrain hindbrain-boundary of the neural tube and includes an organizer (IsO) that is essential for proper formation of adjacent brain regions, yet the molecular and cellular composition of the isthmic region remains unresolved. Results: We employed combined single-nucleus ATAC-seq and RNA-seq (scMultiome) in 13 and 16 hours-post-fertilization zebrafish embryos to molecularly resolve cell populations in the isthmic region and validated our findings in vivo by RNA fluorescence in situ hybridization. We identified two distinct isthmic cell populations (isthmic midbrain [IsMB] and isthmic hindbrain [IsHB]) that share expression of canonical isthmic genes, but that differ in their expression of midbrain vs hindbrain genes. We also uncovered a previously unrecognized heterogeneity within the IsHB, reflecting a canonical fgf8-expressing population anteriorly (IsO/r0a), and a novel fgf8-negative population posteriorly (r0p). We find that inhibition of Fgf signaling disrupts formation of the isthmic region, leading to loss of isthmic cell populations except a residual population characterized by a mixed neural identity. Conclusions: Using transcriptional and epigenetic characterization, we expand on prior anatomical and genetic analyses of the isthmic region to refine our understanding of its cellular organization and demonstrate that it consists of several subdomains.

Zebrafish combines the power of genetics and unparalleled in vivo imaging for investigating the dynamics of vertebrate hematopoietic development. Across species, the transcription factor Runx1 is essential for definitive hematopoiesis. We generated a zebrafish runx1-2A-creERT2 CRISPR knock-in for tamoxifen-regulated Cre recombinase Runx1 lineage tracing and characterized its activity using the ubi:Switch recombinase-dependent fluorescence reporter, microscopic live imaging and flow cytometry. Tamoxifen treatment beginning at gastrula stage labeled all expected Runx1 lineages in the early embryo, including neuroectodermal olfactory placode and Rohan-Beard neurons, primitive hematopoietic blood cells, and nascent hematopoietic stem and progenitor cells (HSPCs) in the dorsal aorta. Runx1 HSPCs colonized the larval caudal hematopoietic tissue and thymus from three to five days of development. Timed tamoxifen induction of Cre activity allowed separation of Runx1 primitive hematopoiesis from definitive HSPC emergence and larval stem cell niche colonization. Flow cytometry of kidney marrow and peripheral blood from adults treated with tamoxifen at gastrula stage revealed Runx1 embryonic hematopoietic cells contributed to adult hematopoietic precursors, myeloid, lymphoid, and peripheral blood lineages. Labeling of all blood lineages was also effective by tamoxifen treatment of 5-month-old adults. The zebrafish runx1-2A-creERT2 line provides a powerful tool for precise spatial and temporal analysis of Runx1 progenitor mechanisms in developmental and adult hematopoiesis. Key PointsO_LIzebrafish endogenous runx1-2A-creERT2 provides inducible Cre recombinase genetic analysis in all runx1 neuromesodermal and blood lineages C_LIO_LIzebrafish runx1-2A-creERT2 line enables in vivo spatial and temporal analysis of embryonic and adult hematopoiesis C_LI

The hallmark of sessile adult ascidians is a vase-like shape with a single oral and atrial siphon. Ciona, however, develops two atrial siphons after metamorphosis, which subsequently fuse into one. The mechanisms underlying this fusion are unknown. This study suggests that Hox2 controls this process. Hox2-knockout animals using Transcription-Activator-Like Effector Nuclease (TALEN) retain two atrial siphons throughout their lives. During normal fusion, epidermal cells between the siphons flatten along the anterior-posterior axis. This cellular flattening does not occur in Hox2-knockout animals, suggesting that the shape change in the epidermal cells produces tension, allowing the atrial siphon openings to converge at the midline for fusion. Hox2-knockout animals lack cupular organs, which are suspected hydrodynamic sensors in the internal epithelium of the fused atrial siphon and on the sperm duct. Among several knockout attempts, atrial siphon fusion was reproduced by only one TALEN pair, suggesting that this phenotype is driven by a mutation having a broader effect than those abolishing protein function. Many ascidians, unlike Ciona, develop a single atrial siphon shortly after metamorphosis. Our findings suggest that a phylogenetically conserved gene, Hox2, establishes this group-specific atrial siphon formation mechanism in Ciona.

Non-syndromic orofacial clefts (nsOFCs), such as cleft lip, cleft lip with or without cleft palate (CL/P), and cleft palate only, are among the most common craniofacial malformations in humans. We identified a range of rare and de novo structural variants (SVs) associated with nsOFCs in a cohort of children of African and Asian ancestry from the Gabriella Miller Kids First Data Resource Center. Most of these novel candidate SVs are located in non-coding regions. More specifically, we characterized SVs associated with nsOFCs across the DOT1L (disruptor of telomeric silencing 1-like) genomic locus. DOT1L is the sole histone methyltransferase that catalyzes the mono-, di-, and trimethylation of histone H3 at lysine 79 (H3K79). In addition, our data revealed associations between specific SVs and genes encoding DOT1L complex subunits as well as downstream target genes. Single-nuclear RNA sequencing analysis demonstrated that mouse Dot1L and its associated targets are highly expressed in neural crest-derived mesenchyme enriched for key osteogenic genes essential for palate formation. Depletion of a Dot1L allele resulted in cleft palate and micrognathia in mice. Furthermore, we identified several families carrying SVs in the DOT1L locus and downstream DOT1L targets genes. These findings support an oligogenic model in which the concurrent presence of several SVs enhances susceptibility to the nsOFC phenotype. We propose that DOT1L and its targets act within the same genetic pathways to influence CL/P such that the combined effects of variants affecting these genes may be more substantial than their individual effects. Collectively, these studies establish a pivotal role for DOT1L-mediated H3K79 methylation in craniofacial development and palate formation and identify this pathway as a key contributor to the etiology of CL/P.

UNC-6/Netrin is a conserved regulator of dorsal-ventral axon and cell migrations. UNC-6 is composed of a Laminin N-terminal domain (LN), three epidermal growth factor repeats (EGF), and a Netrin C terminal domain (NC). Here, we identified missense mutations in distinct UNC-6 domains and assessed their roles in dorsal VD/DD motor axon guidance and ventral AVM axon guidance. A missense mutation in a conserved residue of the LN domain (G289D) resulted in dorsal and ventral axon guidance defects similar to unc-6 null. A distinct missense mutation in the LN domain (S120F) was hypomorphic and strongly perturbed ventral AVM axon guidance with minimal effects on dorsal VD/DD axon guidance, showing that S120F is predominantly required for ventral guidance. Missense mutations altering conserved cysteine residues involved in di-sulfide bonding in the EGF domains were analyzed. EGF1(C321G) caused both ventral and dorsal axon guidance defects albeit weaker than unc-6 null, indicating that EGF1 is required for both. EGF2(C347Y) strongly affected dorsal VD/DD axon guidance similar to unc-6 null, with weaker perturbation of ventral AVM axon guidance. Previous results revealed that EGF3(C410Y) specifically disrupted dorsal axon guidance, a result that we confirmed. Our studies using missense mutations in the endogenous unc-6 locus complement previous structure-function studies using transgenic expression, and identify domains specifically required for ventral AVM guidance (S120Y in the LN domain) and dorsal VD/DD axon guidance (C410Y in EGF3). The crystal structure of UNC-6 indicates conserved N-linked glycosylation at N114 and N128. Mutation of these sites in UNC-6 had no effect on dorsal ventral axon guidance, showing that they do not play a major role. However, the N114 and N128 mutations interacted genetically with unc-40 and unc-5 mutations, indicating that these glycosylation sites indeed have a role in UNC-6 signaling. Our results will inform studies on how these distinct UNC-6 domains interact with guidance receptors (e.g. UNC-40/DCC and UNC-5) and other extracellular molecules to mediate dorsal-ventral axon guidance.

Col8a2Q455K/Q455K (Q455K) mice exhibit features of early-onset Fuchs endothelial corneal dystrophy (FECD), including decreased endothelial cell density (ECD) and guttae formation. Within the context of these clinical features, this study longitudinally evaluates ferroptosis in Q455K and wild-type (WT) mice using in vivo imaging, PCR and immunohistochemistry. Fifty-six Q455K and 56 WT mice were evaluated from 3 to 24 months of age with in vivo confocal microscopy; ECD and guttae were measured. Ferroptosis marker expression was determined with PCR and immunohistochemistry (IHC). Data were analyzed using two-way ANOVA with Tukeys post hoc test, Chi-square test and a paired t-test. The ECD significantly decreased in both groups from 3 to 24 months of age, but more markedly in Q455K (2285-/+317 to 1012-/+58 cells/mmSquare) versus WT mice (2714-/+139 to 2057-/+149 cells/mmSquare, P<0.0001). Guttae were observed exclusively in Q455K mice beginning at 3 months of age and increased over time (P=0.0003). The Q455K mice demonstrate guttae at the vertices of corneal endothelial cells rather than their centers (74.3% vs. 25.7%P<0.001). Expression of ferroptosis-related genes (Tfrc, Slc40a1, Ftl1, Gpx4) were significantly increased in the Q455K versus WT mice (P<0.05). Furthermore, corresponding protein expression (transferrin receptor 1, ferroportin, ferritin and glutathione peroxidase 4) was significantly elevated adjacent to guttae in Q455K versus WT mice (P<0.05). These findings implicate guttae in the initiation of ferroptosis as it relates to the pathophysiology of FECD and provide an optimal window for testing novel FECD therapies using this murine model, particularly those that target ferroptosis.

Orofacial clefts (OFCs), including cleft lip (CL), cleft palate (CP), and cleft lip with cleft palate (CLP), are among the most common craniofacial malformations in humans, with a birth prevalence of approximately 1 in 1,000 live births globally. Non-syndromic forms of OFC are predominantly genetic, with significant variability in prevalence across populations. Understanding the genetic underpinnings of OFCs remains a key public health priority, given the substantial medical and societal burden of these conditions. Recent genome-wide association studies (GWAS) have implicated numerous genetic loci, but challenges remain due to genetic heterogeneity and complex gene-environment interactions. This study aimed to identify sex-specific genetic risk factors for cleft lip with or without cleft palate (CL/P) through a meta-analysis of whole genome sequencing (WGS) data from 1,922 case-parent trios across eight diverse cohorts. Our approach revealed four SNPs in three distinct regions that showed genome-wide significant sex-specific effects. However, despite each of these SNPs passing standard quality control filters, follow-up analyses showed that these signals most likely were technical artifacts caused by sequencing errors, in particular mis-mapped reads due to sequence similarities with the sex chromosomes. These findings highlight the necessity for careful scrutiny when studying differences between the sexes in genetic association studies.

In a wide variety of animals, developmental crowding results in adults with smaller bodies. The crowding effect on body size in Drosophila melanogaster is canonically attributed to heightened competition for nutrition. However, whether other consequences of crowding also contribute to its effect on size remains an open question. We tested the relative contributions of nutritional competition, oxygen availability, and larval-generated metabolites to the crowding effect on size. We found that while nutrition explains most of the variation in body size due to crowding, oxygen also contributes in a sex- and nutrition-dependent manner. We found no evidence that larval-generated chemicals affect body size. These data confirm a widely suspected but untested role of nutrition in producing the crowding effect on size in D. melanogaster, while revealing an unexpected role of oxygen, and raise the possibility that behavior may be a mediator of density-dependent plasticity. Research HighlightsWe found that both nutrition and oxygen mediate the crowding effect on size in Drosophila melanogaster.

Histone proteins, which reside in the nuclei of eukaryotic cells, are involved in diverse cellular processes. The core histone H4 serves as a structural component of the nucleosome. Patients carrying mutations in H4Clustered histone (H4C) genes exhibit a broad spectrum of developmental abnormalities, including short stature, microcephaly, intellectual disability, growth retardation, and digital anomalies. However, the impact of H4 mutations on mammalian embryogenesis remains largely unclear. Here, we demonstrate that histone H4C genes play crucial roles in skeletal development and cortical neurogenesis. We found that mRNAs of the histone H4C gene family are specifically expressed in proliferating progenitor cells in the developing mouse neocortex and in human induced pluripotent stem cell-derived cortical organoids. CRISPR-mediated disruption of H4C3 in mice caused severe defects in skeletal formation and neocortical neurogenesis. Furthermore, overexpression of a mutant form of H4C3 resulted in altered expression of genes associated with cellular migration and motility. Together, these findings suggest that histone H4 plays a critical role in regulating the balance between proliferation and differentiation during mammalian embryonic development, thereby explaining the broad spectrum of patient phenotypes.

In contrast to many other developing systems, in which axon pathfinding and synaptogenesis are separated in time, the pioneering axon of the individually identifiable caudal primary motor neuron in embryonic zebrafish forms en passant synapses during its stereotypical ventral growth. How simultaneous synaptic differentiation and axon pathfinding are coordinated is not fully understood. Here we ask what the role of the tac1 gene, coding for the synaptic tachykinin neuropeptides, is in this unique axon differentiation process. The gene is expressed during axon outgrowth and its disruption results in increased branch length of CaP axons and subtle morphological defects of the pre-synapse. These abnormalities are accompanied by a robust [~]1.5-fold increase in motor neuron activity and in spontaneous early contractions in tac1-deficient embryos. Furthermore, pharmacological inhibition of the tachykinin receptor (Tacr1) leads to altered CaP axonal morphology, mimicking the axonal phenotype observed in tac1-deficient zebrafish. These findings suggest that tachykinin neuropeptides modulate formation and activity of en passant synapses and prevent aberrant axon branching during growth of zebrafish motor axons. HIGHLIGHTS- tac1 refines CaP primary motor axon development in zebrafish - Loss of tac1 disrupts presynaptic maturation at the horizontal myoseptum - tac1 mutants show elevated motor neuron activity and spontaneous contractions

Study questionHow does cryptorchidism affect germ cell development and UTF-1-mediated pluripotency potential at the time of orchiopexy? Summary answerCryptorchidism was associated with the following germ cell abnormalities: germ cell clustering with many cords/tubules lacking germ cells and reduced UTF-1 expression, suggesting limited germ cell differentiation into spermatogonia What is known alreadyCryptorchidism, affecting 1.6-9% of male newborns, is known to increase the risk of infertility and testicular cancer due to abnormal germ cell development. Germ cells and pluripotent stem cell gene, UTF-1, play critical roles in spermatogonia differentiation, self-renewal, and spermatogenesis. No prior study has evaluated the testicular development by immunohistochemically mapping of these cell populations, Study design, size, durationA cross-sectional study of 31 postnatal cryptorchid testis and 5 age-matched scrotal testicular biopsies obtained from UCSFs pathology department performed between 1993-2023. Participants/materials, setting, methodsSpecimens were grouped by age at surgery (6-18 months, 19 months-7 years, 8-12 years, and [&ge;]13 years) and testis location (palpable vs. non-palpable). Scrotal prepubertal testis biopsies were sourced through the Pedi-LIFE program, a fertility preservation research biobank, with at least one control per age group. Immunohistochemistry was performed to stain specimens for germ cell (DDX4, OCT4, TSPY), pluripotent cell marker (UTF-1), as well as other key testis cell markers (A-actin, AR, P450, Sox-9), with staining graded based on control expression levels. The number of germ cells per seminiferous tubule was quantified and compared across anatomical locations using appropriate statistical analyses. Main results and the role of chanceThis study included 36 specimens, comprising 31 cryptorchid testes (86%) and 5 scrotal control testes (16%). The cryptorchid group exhibited testicular dysgenesis and reduced germ cell expression, correlated with increased age and testis location. Qualitative assessment revealed reduced germ cell expression across all ages in cryptorchid testes. The number of germ cells per tubule was markedly reduced in cryptorchid compared with scrotal testes after 19 months of age for DDX4, TSPY, and UTF-1. Germ cell clusters were identified in 15 out of 31 cryptorchid specimens (48%) stained for DDX4 and TSPY. UTF-1 expression was lower in cryptorchid testes across all age groups. No significant differences were noted in other testicular cell markers. Large scale dataNA Limitations, reasons for cautionFirst, the power and generalizability of the study are limited by the availability of specimens within each age group, particularly for scrotal testes, as biopsies of these tissues are not routinely performed. Second, a cross-sectional study design limits a longitudinal comparison to evaluate changes in marker expression, delayed maturation, or irreversible germ cell loss. Third, immunohistochemistry data is semi-quantitative, and protein detection is affected by antibody sensitivity and tissue preservation and influenced by antibody sensitivity. Lastly, scrotal testis used as controls were obtained from cryopreserved tissue from patients with other unrelated pathology, which may influence histological profiles. Wider implications of the findingsCollectively, our findings support a model in which cryptorchidism involves both germ cell depletion and disrupted SSC lineage formation, with UTF-1 downregulation and germ cell clustering as early signatures of testicular dysgenesis. These features may help identify high-risk patients for worsening gonadal dysgenesis and infertility and can provide a rationale for earlier orchiopexy or SSC-preserving strategies. Study funding/competing interest(s)The authors declare no conflicts of interest and received no funding for this study. Data Availability StatementThe data underlying this article cannot be shared publicly due to ethical and legal restrictions related to the use of human tissue specimens, which may compromise donor privacy and confidentiality. Data are available from the corresponding author upon reasonable request and subject to institutional and ethical approvals.

Purpose: Lymphatic system plays a critical role in fluid regulation and immune response. The conjunctiva of the ocular surface is endowed with a rich lymphatic network, and it provides an ideal site to study lymphatic structure and function. The purpose of this study is to investigate potential morphological and functional changes of conjunctival lymphatics with aging, a time-dependent biological deterioration process. Methods: Young and aged fluorescently labeled transgenic mice of Prox-1, the master control gene for lymphatic determination, were used in the study. For morphological assessment, conjunctival lymphatic vessels were examined in vivo by our advanced live imaging system. For functional analysis, lymphatic drainage efficiencies were measured by fluorescently labeled tracer injection. Results: Compared to young mice, both vascular branching points and intraluminal valves were significantly reduced in conjunctival lymphatic vessels of aged mice. Moreover, lymphatic functional deterioration and drainage deficiencies, such as fluid leakage and reflux, were also detected in the aged condition. Conclusions: Conjunctival lymphatic system undergoes morphological as well as functional changes with aging. Further investigation into this phenomenon may provide novel insights into lymphatic and age-related diseases inside and outside the eye.

Microcomputed tomography (micro-CT) is a useful tool that can be utilised for 3D structural characterisation and volumetric quantification of small biological specimens. Its potential application is particularly valuable within the field of cardiac development, where phenotypic profiling at the whole organ, cell, and molecular level is often most informative within the same sample. Consequently, this study sought to develop a multimodal imaging protocol to enable 3D phenotypic characterisation of embryonic avian hearts (iodine-based contrast X-ray imaging) prior to immunohistochemistry-based cell and molecular analysis. Micro-CT parameters were tested to establish an optimal protocol for 3D analysis of embryonic cardiac specimens across multiple developmental timepoints. Optimised parameters provided reliable and reproducible 3D analysis of cardiac macrostructures. Sodium thiosulphate treatment of X-ray imaged hearts effectively reversed the iodine-based contrast stain whilst maintaining antigen availability of nuclear, membranous, and cytoplasmic targets in traditional downstream imaging studies. Together, this study demonstrates a robust and highly efficient multimodal imaging strategy to comprehensively characterise cardiac morphology in avian embryos and may serve as a versatile foundation for a broad range of bioimaging applications within the wider scientific community.

Precise knock-in of fluorescent reporters is a powerful tool for studying the dynamic cellular and molecular processes of embryogenesis. However, conventional CRISPR-Cas9 knock-in of large inserts, such as full-length fluorescent proteins, is inefficient. This has limited its application in many emerging model systems, including sea urchins. Here, we overcome this barrier using a transgenic Lytechinus pictus line that constitutively and ubiquitously expresses a large fragment of mNeonGreen (mNG3K1-10). In this line, fluorescence is only reconstituted when CRISPR-mediated knock-in delivers mNG211, the 11th beta strand of the fluorescent protein, to complement the constitutively expressed fragment. Because this strategy requires integrating only the short 11th-strand, together with short homology arms (~130 nt total), by homology directed repair, it circumvents the size constraints that limit conventional full-length reporter knock-ins using CRISPR. Using this approach, we achieved integration efficiencies of 14-22%, roughly an order of magnitude higher than those obtained with full-length fluorescent protein knock-ins. This provides a streamlined, scalable method for endogenous protein visualization in echinoderm embryos and a valuable resource for studying gene function, morphogenesis, and toxicant response in this classic developmental model.

The African clawed frog Xenopus laevis is a widely utilized model organism in biomedical research; however, significant challenges in experimental reproducibility and colony management remain. A major obstacle lies in the reliable identification of individual animals, since frogs are generally housed in large groups and are difficult to distinguish due to their high morphological similarity. Conventional methods, including toe clipping and microchipping, are invasive and cause distress, emphasizing the need for non-invasive methods for accurate documentation and welfare monitoring. In this study, we introduce XIBBIT (Xenopus Image-Based Biometric-pattern Identification Tool), a web-based application integrating computer vision and machine learning to identify individual Xenopus laevis based on their dorsal patterning. By exploiting these natural biometric signatures, the platform achieves reliable identification with up to 95.7% accuracy within three image captures under real life conditions. In addition to identification, XIBBIT provides a centralized colony management system. It archives individual data, including health records and experimental histories, with customizable fields. To demonstrate XIBBITs capabilities, we used the application to track egg quality across repeated egg-laying events, revealing that egg quality is a repeatable, individual-specific trait in Xenopus laevis. Furthermore, we find seasonal effects on egg laying performance with the lowest performance during late-spring and summer months. Ultimately, XIBBIT provides an effective, time-efficient, and non-invasive solution to the problem of individual Xenopus laevis identification, facilitating both experimental reproducibility and high animal welfare standards.

Bergmann glia-like progenitors (BGLPs) are transient astroglial progenitors in the postnatal cerebellum, but how their lineage potential changes during development remains incompletely understood. Our previous electroporation-based study suggested that P0 BGLPs possess broader lineage potential than P6 BGLPs. Here, we performed recombination-based lineage tracing by cerebellar surface application of tamoxifen to Ai9/+; GlastCreERT2/+ mice and temporally analyzed the progeny of BGLPs labeled at P0, P3, P6, and P8. We found that BGLPs undergo progressive lineage restriction during postnatal development. P0 BGLPs gave rise to Bergmann glial cells (BGs), inner granule cell layer astrocytes (IGL astrocytes), white matter astrocytes (WM astrocytes), and molecular layer inhibitory neurons (ML-INs), confirming our previous electroporation-based findings. In contrast, P3 BGLPs generated BGs, IGL astrocytes, and WM astrocytes, whereas P6 BGLPs generated BGs and IGL astrocytes, and P8 BGLPs generated predominantly BGs. Thus, BGLP lineage output was progressively restricted from four progeny categories at P0 to a predominantly BG-restricted output by P8, suggesting that BGLPs dynamically adjust their cellular output during postnatal cerebellar maturation. Additional temporal analyses suggested that ML-INs are unlikely to be generated directly from P0 BGLPs, but may arise indirectly through astrocyte-like progenitors (AsLPs) and inhibitory neuron progenitors (INPs). These findings identify postnatal BGLPs as a useful in vivo model for studying progressive lineage restriction and stage-specific cellular supply during cerebellar development.

Phenylketonuria (PKU) is a genetic metabolic disorder caused by the lack of functional phenylalanine hydroxylase (PAH). Elevated levels of phenylalanine (Phe) are known to be neurotoxic; however, the molecular mechanisms underlying Phe's effects remain elusive. This study investigates the impact of PKU on proteostasis, redox balance, and metabolism in BTBR PAHenu2 mice, a severe disease animal model. Combined proteomics and metabolomics revealed impaired redox homeostasis in the brain and disrupted mitochondrial energy metabolism (ATP and TCA intermediates). The dysregulation was further supported by decreased levels of ATP, reduced glutathione (GSH), cysteine, and reduced catalase activity. Western blot analyses revealed substantial remodeling of protein degradation systems: the 19S regulatory (Rpt1) subunit and 26S proteasome content and activity were significantly increased, and ubiquitinated protein levels were elevated, indicating protein turnover and activation of the ubiquitin-proteasome system. Autophagy was also activated, as evidenced by a reduced LC3-II/LC3-I ratio, decreased p62 levels, unchanged ATG5 levels, and increased HSPA8 protein expression. By contrast, UPR markers remained stable despite an increase in the oxidized-to-reduced PDI ratio, suggesting a localized shift without activation of a full ER stress response. In parallel, systemic alterations were assessed in whole blood. Indeed, GSH, cysteine, ATP and ADP were decreased in PKU, whereas NADPH increased. These changes were accompanied by reduced activities of GSH reductase and GSH peroxidase, thereby confirming metabolic and redox disruption. Collectively, these findings indicate that PKU is associated with activation of protein degradation pathways as an adaptive response to cellular stress combined with redox imbalance and energy dysregulation.

Pyruvate dehydrogenase complex (PDHc) deficiency (PDCD) is a primary mitochondrial disorder characterized by neurodevelopmental disability, altered intermediary metabolism and early mortality. Dichloroacetate (DCA), a pyruvate analogue, is a well-described PDHc activator that remains under clinical investigation for treatment of PDCD. Here, we studied the in vivo efficacy of a 5-point log concentration range of DCA on animal health and metabolism in C. elegans with feeding RNA interference (RNAi) expression knockdown of either PDHA-1 or DLD-1 homologues at graded degrees to model variable disease severity. These worm models recapitulate phenotypic features of PDCD observed in human patients, including reduced survival, delayed growth, locomotor impairment, and elevated lactate and/or pyruvate tissue levels. DCA treatment appeared well-tolerated, with no gross morphologic toxicity seen at doses up to 25 mM. Significantly improved health, survival, tissue lactate levels, and mitochondrial physiology were observed at 25 mM in pdha-1(RNAi) knockdown animals. DCA treatment in dld-1(RNAi) C. elegans models (undiluted, 1:20 dilution, and 1:100 dilution) showed significant therapeutic benefits on survival, neuromuscular function and metabolic phenotypes primarily in the moderate (1:20) and/or mild (1:100) dld-1(RNAi) deficiency strains, but not in full-dose dld-1(RNAi). Importantly, linear growth, neuromuscular activity, and mitochondrial physiology were significantly improved with DCA treatment even in the most severe dld-1(RNAi) undiluted model. Overall, preclinical modeling provides objective evidence of DCA therapeutic efficacy in C. elegans expression knockdown strains for two well-conserved homologues of PDHA1 and DLD that represent distinct genetic etiologies of PDHc deficiency, with demonstrated beneficial effects on survival, healthspan, tissue lactate, and mitochondrial physiology. These data further confirm that DCA's therapeutic effect correlates with PDHc disease phenotype severity in dld-1(RNAi) animals.