Neural crest cells are multipotent, migratory stem-like cells essential for vertebrate development that contribute broadly to many tissues including the craniofacial skeleton, peripheral nervous system, and pigment-producing cells. Their development progresses through phases of induction, specification, delamination, migration, and differentiation. Importantly, advancement through these phases requires detecting and responding to extracellular cues through the plasma membrane. While the gene regulatory networks and the signals that govern neural crest development are well characterized, plasma membrane lipid composition as a regulatory layer controlling these transitions remains underexplored. Despite technical limitations in quantitative lipid measurements from vertebrate embryos, genetic and molecular studies have inferred roles of key lipid metabolites. Here we review how membrane lipid metabolism and composition regulate neural crest behavior, focusing primarily on sphingolipids, cholesterol, and lipid raft domains. We examine how morphogen signaling is modified and organized by lipids, how modulating lipid content alters the biophysical state of the neural crest plasma membrane, and the deleterious effects of dysregulated lipid metabolism in neurocristopathies. Drawing parallels to other developmental and disease contexts, we highlight membrane lipid metabolism as a critical frontier in neural crest biology with broad implications for understanding developmental mechanisms and congenital disorders.
BACKGROUND:The ability to replace lost or damaged neurons following an injury is termed reactive neurogenesis. Although reactive neurogenesis has been reported in several lizard species, the molecular mechanisms underlying this response remain largely unknown. Here, we investigate ontogenetic, injury-, and regeneration-associated proteomic changes to the forebrain of subadult leopard geckos (Eublepharis macularius), focusing on an area of the cerebral hemispheres that includes the medial and dorsomedial cortices, septum, and neighboring tissues. RESULTS:Among control (untreated) geckos, the proteome of the forebrain changes during a one-month period, providing evidence of an ontogenetically driven proteomic shift. To initiate reactive neurogenesis, we administered the neurotoxin 3-acetylpyridine (3-AP). Using liquid chromatography-tandem mass spectrometry, we found that 3-AP induces differential expression of proteins associated with cell death, immune activation, and neurogenesis. However, by 30 days post-injury, the proteomic profile of the forebrain was returning toward that of age-matched controls. CONCLUSIONS:Our findings demonstrate that structural regeneration of the gecko forebrain is associated with the differential expression of proteins involved in brain development and repair in mammals and provides a resource for future evolutionary studies of neurogenesis.
BACKGROUND:The continuously growing mouse incisor serves as a model for stem cell-driven tooth renewal. Although mice possess maxillary and mandibular incisors, most studies have focused on the mandible. Potential differences in structure and renewal between these serially homologous organs remain insufficiently characterized. RESULTS:Mandibular incisors are longer and contain greater enamel volume, mineral density, and mineral content than maxillary incisors, whereas maxillary incisors are more curved and display a larger cross-sectional area. Mineral density increases along the apical-to-incisal axis in both teeth, although mandibular incisors exhibit greater mineral deposition and retention. The maxillary incisor contains a larger pool of cycling progenitors with proliferation dynamics comparable to those of the mandibular incisor. Although ameloblast differentiation initiates at a similar position in both incisors, the mandibular incisor contains a ~20% longer secretory-stage ameloblast region. Consistent with this observation, gene expression analysis reveals earlier Igfbpl1 expression onset in the mandible. CONCLUSIONS:Homologous mouse incisors differ in progenitor pool size, ameloblast maturation, and enamel formation. Increased enamel output in the mandible is associated with a prolonged secretory phase, whereas the maxilla contains an expanded progenitor pool. These findings support the maxillary incisor as a complementary model for studying mechanisms that regulate tooth renewal.
Abstract Background Developmental control of jaw size is crucial to prevent birth defects and facilitate evolutionary adaptation. We have shown that jaw size is established by neural crest mesenchyme (NCM), which are progenitor cells that migrate into the mandibular primordia and produce the jaws. NCM relies on multiple signaling pathways including Sonic Hedgehog (SHH) to mediate interactions with mandibular epithelium and promote jaw outgrowth. We investigated if NCM‐mediated regulation of the SHH pathway underlies species‐specific evolution of jaw size. Results We analyze expression of SHH pathway members over time and find that Growth Arrest‐Specific 1 (GAS1), which is a SHH co‐receptor, is expressed 20–75‐fold higher in mandibular primordia of duck relative to those of quail. We generate quail‐duck chimeras and demonstrate Gas1 expression is NCM‐regulated. Gain‐ and loss‐of‐function experiments reveal species‐specific sensitivity to SHH signaling, especially for Gas1 . Gas1 overexpression and knockdown in NCM alters cell number and jaw size, and differentially affects genes involved in the SHH and WNT pathways, the cell cycle, and others. We also uncover intriguing differences in the Gas1 promoter and coding sequence between duck and quail. Conclusions Our work suggests changes to Gas1 expression and function may modulate jaw size during development, disease, and evolution.
BACKGROUND:Fate maps relate progenitor cell positions to later fates and locations of their progeny, revealing early embryonic organization. Previous zebrafish fate maps identified the origin of germ layers and derivative cell fates, but ventral gastrula progenitor domains were not fully resolved. In particular, the neural crest, a multipotent cell type central to vertebrate development and evolution, was not characterized in the zebrafish fate map. RESULTS:We generated a region-based fate map to resolve the origin of the neural crest and overall organization of the ventral gastrula. This fate map demonstrates that cranial neural crest and placodes arise from a broadly overlapping progenitor domain. Notably, trunk neural crest and neural tube always arise together, while cranial neural crest and neural tube can arise from segregated progenitor pools. A separate, far-ventral progenitor domain produces somites, spinal cord, neural crest, and epidermis in the posterior tail. CONCLUSIONS:We situate the neural crest in the fate map and clarify the early embryonic organization of the zebrafish ventral gastrula. Our fate map establishes multiple mixed-fate domains in the ventral gastrula and their contributions to the overall body plan. This fate map provides information for further investigation of early vertebrate development and neural crest biology.
BACKGROUND:Testicular development is a highly orchestrated process essential for male fertility, but its temporal genetic regulation remains incompletely resolved in many mammalian models. This study re-analyses rat testis transcriptomes across 15 stages from embryonic day 12 to postnatal week 16 using bulk RNA-sequencing data from a multispecies organ development atlas, generating a continuous, testis-focused temporal framework, and pinpointing key developmental transition windows and regulatory modules. RESULTS:Of 23,748 annotated genes, 17,717 were differentially expressed (DEGs), and hierarchical plus co-expression analyses identified four principal expression modules and five clusters capturing spermatogenic, morphogenetic, immune, and neurone-like programs. Four major transcriptional transition windows, embryonic day 14, embryonic day 19, postnatal week 2, and postnatal week 6, were defined, with the largest remodeling between 2 and 6 weeks coinciding with the onset of robust spermatogenesis and marking a particularly vulnerable window for environmental or toxicant-induced perturbation. Stage-specific spermatogenesis-associated gene sets at embryonic day 19 (n = 28), postnatal week 2 (n = 61), and week 6 (n = 735) showed minimal overlap, supporting sequential regulatory waves driving germ cell commitment, early meiosis, and terminal differentiation. Target enrichment analyses highlighted five conserved microRNAs (rno-miR-151-5p, rno-miR-29b-3p, rno-miR-384-5p, rno-miR-500-5p, rno-miR-672-5p) whose predicted and validated targets form modules related to extracellular matrix remodeling, apoptosis and cell-cycle control, ion-channel signaling, and neuronal-like pathways, with high sequence conservation to human homologs. Immune-related genes and blood-testis barrier components displayed coordinated expression dynamics, and a conserved core of 487 spermatogenesis genes showed strong cross-mammalian orthology, indicating a deeply shared transcriptional backbone on which species-specific regulatory nuances are layered. CONCLUSIONS:This multistage transcriptomic analysis delineates major developmental transitions, co-expression modules, and regulatory miRNA-mRNA networks that orchestrate rat testis maturation and highlight a critical P2W-P6W transition window. The resource complements existing multiorgan and single-cell datasets by providing a testis-focused temporal framework and identifies conserved gene sets and regulatory candidates of direct relevance to male infertility, toxicology, and cross-species translational studies.
BACKGROUND:Muscle-tendon integration is a crucial step in the morphogenesis of the vertebrate locomotion system. As the limb muscle and tendon progenitor cells derive from distinct embryonic origin, their integration requires precise mutual positioning. This process has been described mainly by tissue sectioning and in situ hybridization, which are not well suited for understanding the three-dimensional (3D) organization. To address these limitations, this study applied tissue clearing and light-sheet microscopy to visualize the muscle-tendon integration process in 3D in mouse embryonic limbs. RESULTS:By combining CUBIC reagents, immunofluorescence, and fluorescent reporter mice, we were able to resolve the 3D arrangement of forelimb muscles and tendons in mouse embryos from E11.5 to E15.0. Interestingly, we found that a subpopulation of Scleraxis+ tendon lineage cells remains within the interstitial space of myofibers even after muscle-tendon integration is established, implying the existence of previously undescribed cellular heterogeneity within the Scleraxis+ cell population at this stage. Moreover, our approach successfully captured the muscle-tendon morphologies altered by genetic perturbations, such as Scleraxis-lineage ablation and Myomaker knockout. CONCLUSIONS:In summary, this tissue clearing and light-sheet microscopy approach provides a versatile method for analyzing dynamically changing embryonic muscle-tendon morphologies and will facilitate deeper understanding of the muscle-tendon integration process.
The olfactory placode (OP) generates a broad array of chemosensory neurons in the nasal region, including olfactory sensory neurons, vomeronasal sensory neurons, neurons of the septal organ, and Grueneberg ganglion. During invagination, the OP also generates migratory neuronal populations, including gonadotropin-releasing hormone-1 (GnRH-1) neurons, somatostatin-expressing neurons, and Prokineticin Receptor 2 (Prokr2) pioneer/terminal nerve (TN) neurons. Despite extensive research, the genetic lineage and molecular identity of many OP-derived neurons remain unclear. GnRH-1 neurons are vital for reproductive functions and chemosensory processing but are genetically distinct from olfactory and vomeronasal sensory neurons. The regulator Goofy/Gfy is expressed in placode-derived chemosensory neurons. Our study aimed to determine whether its expression is restricted to these neurons or shared among OP derivatives. The 123Cre mouse expresses Cre under the Gfy1 promoter. Cre tracing analyses across embryonic stages showed widespread 123Cre recombination in chemosensory neurons but not in migrating GnRH-1. However, at late development and postnatal stages, some Gfy-traced cells appear in the basal forebrain, including a subset of GnRH-positive neurons. These findings demonstrate heterogeneity among migratory nasal neurons and suggest that, besides GnRH-1, other nasal-origin neurons also populate the forebrain. Postnatal studies of vomeronasal neurons revealed distinct developmental trajectories for the two main VSN subtypes.
BACKGROUND:Sexual dimorphism in pelvic morphology has been confirmed at the onset of primary ossification. Since pelvic tilt influences spinal alignment and bipedal locomotion, determining the initial emergence of sex-specific tilt differences is crucial. Herein, we measured pelvic tilt and lumbosacral inclination angles (LSIAs) using magnetic resonance imaging in 72 human fetuses (crown-rump length [CRL], 50-225 mm; 34 males [M] and 38 females [F]). RESULTS:The cranial-caudal axis was defined from the first to third sacral vertebrae, and sex differences were assessed using multiple regression analysis including sex, CRL subgroup, and their interaction. Females exhibited greater posterior iliac tilt than males (anterior superior iliac spine-posterior superior iliac spine line: least-squares mean; [F] -19.6° vs. [M] -17.0°; p = .038). The LSIA (L1/S5-sacral promontory) was larger in males ([F] 166.3° vs. [M] 170.2°; p = .033), indicating more dorsally positioned sacra in females. Principal component analysis identified two major components representing os coxae rotation and sacral inclination to the lumbar spine, indicating independent developmental patterns. CONCLUSIONS:The findings demonstrate that pelvic sexual dimorphism includes tilt orientation from the onset of ossification, reflecting sex-specific developmental trajectories during the prenatal period.
BACKGROUND:Advances in reproductive and cellular biotechnology have significantly enhanced the ability to study, conserve, and manipulate species within the Bovinae family, including domestic cattle and the endangered European bison (wisent). Central to the success of such technologies, including in vitro fertilization, somatic cell nuclear transfer, and the culture of pluripotent stem cells (PSCs), is the chromosome stability of cultured cells and embryos. RESULTS:Here, we present the design and application of a specific centromere Cot-1 DNA probe for fluorescence in situ hybridization (FISH). We demonstrate its utility in assessing chromosomal stability of bovine and wisent somatic cells and in vitro-produced embryos. The probe shows high specificity for Bovinae cells and can be used on both metaphase plates and interphase nuclei, enabling rapid cytogenetic analysis even in slow-dividing or senescent cells. CONCLUSIONS:The newly generated Cot-1 DNA-based FISH probe provides a rapid and reliable method for species-specific cytogenetic monitoring in Bovinae. Furthermore, it allows accurate monitoring of chromosomal stability and quality control in PSC systems reliant on feeder layers and offers a streamlined approach for monitoring embryo chromosomal integrity in vitro. Broader application of this method may improve outcomes in reproductive technologies, conservation programs, and the generation of stable cell lines for biotechnological and agricultural use.
BACKGROUND:The zebrafish heart regenerates upon injury. During injury response, fibroblasts and endothelial cells accumulate at the site of damage, and cardiomyocyte cell cycle reentry allows cardiac muscle regrowth. It is relevant to understand how the different cell types communicate with each other to coordinate regeneration. RESULTS:We present an in silico meta-analysis of ligand-receptor (LR) interactions among periostin b+ fibroblasts, kdlr+ endothelial cells, sox10-derived and rest of ventricular cardiomyocytes, at 7 days post-injury. Using bulk RNA-seq data sets from fluorophore-activated cell-sorted populations, we selected for differentially expressed genes encoding LR pairs. Human-centric interaction data from the OmniPath database were adapted to zebrafish data through ortholog mapping to reconstruct a comprehensive interactome. We observed that fibroblasts and, to a lesser extent, endothelial cells emerged as signaling hubs, while cardiomyocytes primarily acted as signal recipients. Network analysis, combining PageRank, expression change, and literature-based novelty, revealed both known and novel candidate genes in regeneration, and allowed pathway enrichment analysis. An interactive web tool enables exploration of the ranked interaction data set, providing a systematic resource to guide future functional studies. CONCLUSIONS:This study provides a systematic and unbiased map of regenerative signaling in the zebrafish heart, establishing a resource to guide functional investigations.
BACKGROUND:Craniofacial development is a complex developmental process that involves formation of the skeleton, muscle, and tendons. Defects in craniofacial development result in common hereditary disorders. Among genetic factors regulating craniofacial development, dysregulated hedgehog signaling is associated with craniofacial skeletal defects such as orofacial clefting and holoprosencephaly. Here, we characterize craniofacial phenotypes associated with two hedgehog signaling co-receptors, cdon and boc, in zebrafish. Genetic pedigree analyses have previously linked both cdon and boc to microform holoprosencephaly, and mutations of Cdon and Boc in mouse result in craniofacial phenotypes. However, a detailed analysis of craniofacial phenotypes associated with cdon and boc in zebrafish has not been completed. RESULTS:Our studies show that cdon and boc act redundantly to promote craniofacial cartilage, tendon, and muscle development in zebrafish. Using RNA-seq and HCR in situ hybridization, we show that mutations of cdon and boc result in misregulation of chondrogenesis gene expression including Indian hedgehog ligand, tendon-associated thrombospondin genes, and FOX transcription factors. CONCLUSIONS:Our data are consistent with a model whereby cdon and boc together modify hedgehog activity in the head and establish a foundation for using zebrafish to further understand the role of cdon and boc in craniofacial hereditary disorders such as holoprosencephaly.
BACKGROUND:Multiple studies have supported the hypothesis that incremental growth lines in crocodilian dentin are analogous with circadian von Ebner's lines in mammals; however, variation in these growth lines has been largely unstudied. It is also unknown whether accentuated dentin striations in crocodilians may represent a supradian periodicity analogous to Andresen lines in mammalian dentin. Therefore, the objective of this study is to histologically assess variation in von Ebner's lines and accentuated growth lines among crocodilians. RESULTS:The data do not support the hypothesis that crocodilians express supradian periodicity in accentuated striations of dentin. DSR is significantly correlated with total length and body mass. DSR variance also shows a significant relationship with body size. CONCLUSIONS:Body size seems to be the major factor governing DSR in crocodilians; this relationship is expressed both intra- and interspecifically in this dataset, warranting further study of intraspecific variation to assess the impact of ontogeny on DSR within this group. Accentuated striations may not be useful as markers of periodicity in crocodilians, but may be useful indicators of stress or other physiological events in life history analyses.
BACKGROUND:In the developing brain, neuronal migration is one of the critical steps in the establishment of an accurate neural network. When neurons reach their final destination, they form layered structures or nuclei. Precerebellar neurons (PCNs) form several nuclei in the pons and medulla and project mossy and climbing fibers to the cerebellum. PCNs originate from the lower rhombic lip in the dorsal hindbrain and migrate tangentially toward their destinations. When PCNs reach their presumptive nucleus-forming region, they change their migration direction from tangential to radial to form the precerebellar nuclei. Although various molecules have been shown to control PCN migration and nucleogenesis, the mechanisms underlying the development of the precerebellar system remain largely unknown. RESULTS:We investigated the possible roles of two members of the neurotrophin family, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), in PCN development in mouse embryos. We found that Bdnf and Ntf3, which encode BDNF and NT-3, respectively, are expressed in their nucleus-forming regions in the pons and medulla, whereas their receptor genes are expressed in migrating PCNs. Disrupting these genes singly or in combination led to abnormal nucleogenesis of PCNs. CONCLUSION:Our findings indicate that neurotrophin signaling is required for accurate formation of the precerebellar nuclei.
BACKGROUND:During early tooth development, cranial neural crest cells migrating to the oral region form dental mesenchyme. To date, the molecular mechanism by which dental mesenchymal cells further migrate and differentiate into odontoblasts remains largely unknown. We hypothesize that the dental epithelium, being the only source of Shh, a chemotactic peptide, attracts future odontoblasts expressing the Shh receptor Ptch1. RESULTS:Changes in the spatiotemporal distribution of cells expressing Shh, Ptch1, and Hip1 and of nestin protein throughout early tooth development were evaluated via in situ hybridization and immunohistochemistry, respectively. Dental mesenchymal cells were found to be separated into three putative cell population types based on spatiotemporal gene expression patterns: presumptive Ptch1(-)/Hip1(-) non-odontoblastic cells forming the initial dental papilla cell cluster; presumptive Ptch1(+)/Hip1(-) odontoblast lineage cells; and presumptive Ptch1(+)/Hip1(+) cells possibly forming dental follicle and surrounding mesenchyme. Ptch1(+)/Hip1(-) cells seemed to invade the developing dental papilla actively at embryonic day 15.5 (E15.5), resulting in an abundant distribution of the cells by the early bell stage at E16.5. The distribution pattern of presumptive Ptch1(+)/Hip1(-) cells partially overlapped with that of nestin-expressing cells. CONCLUSION:Presumptive Ptch1(+)/Hip1(-) dental mesenchymal cells may migrate to the cusp region of the dental papilla, leading to odontoblast differentiation.
The prevalence of maternal obesity is continuously increasing worldwide, making maternal overweight and obesity one of the most important early life risk factors that can affect the neurodevelopment of offspring. Data from large epidemiological studies indicate that higher prepregnancy BMI, as well as excessive weight gain during pregnancy, are associated with an increased risk of autism spectrum disorder, attention deficit/hyperactivity disorder, internalizing symptoms, cognitive impairment, cerebral palsy, and other neurodevelopmental outcomes. Preclinical studies have also provided a clearer understanding of the biological mechanisms, suggesting that maternal obesity can induce oxidative stress, metabolic inflammation, and placental dysfunction, which impair nutrient and oxygen delivery during critical periods of brain development. Other proposed pathways include activation of microglia and astroglia, dysbiosis of the gut microbiota with alterations in microbial metabolites, epigenetic rearrangements of DNA and histones, and disruption of serotonergic and dopaminergic signaling. Collectively, these biological alterations affect processes such as synaptogenesis, neuroimmune maturation, and neuroplasticity, and sex-specific and even multigenerational effects have been reported in animal studies. Although the presence of familial factors and socioeconomic conditions makes it difficult to draw causal conclusions in human studies, the body of evidence supports the existence of a real biological vulnerability. Various interventions, including improved maternal nutrition, n-3 fatty acid intake, antioxidant strategies, and prenatal physical activity, have shown promising potential to improve metabolic and inflammatory status during pregnancy. Understanding these intertwined mechanisms highlights the importance of early prevention and implementation of targeted maternal health strategies to achieve the best neurodevelopmental outcomes in offspring.
BACKGROUND:The midgut forms tertiary loops in the extraembryonic coelom, where biomechanical factors are thought to influence this process. The number of loops may stabilize once the midgut returns to the abdominal cavity. We therefore examined how midgut length and diameter affect the number of tertiary loops before and after intestinal return. RESULTS:Magnetic resonance images from 50 human embryo and fetus specimens and serial tissue sections from six fetuses were analyzed. The midgut was divided into four segments, and the length of each segment and the tube diameter at a representative central loop were measured and subjected to regression analysis. In the extraembryonic coelom, loop number increased linearly with midgut length, whereas diameter showed no independent association. In the abdominal cavity, greater length was associated with more loops, whereas greater diameter tended to be associated with fewer loops, particularly in Segment-2 and Segment-4. This pattern may be related to the apparent attenuation of further loop increase after return to the abdominal cavity during later fetal development. CONCLUSIONS:The effects of midgut length and diameter on tertiary loop formation differ between the extraembryonic coelom and the abdominal cavity, highlighting the importance of midgut position in loop formation.
BACKGROUND:Human mutations in INVERSIN are associated with nephronophthisis, variable penetrance of situs inversus and congenital heart disease. Inversin has been shown to localize to cilia and many of the patient phenotypes are attributed to disrupted cilia function. We set out to characterize the invssa12246 allele in zebrafish to investigate its utility in understanding patient disease. RESULTS:The left-right axis is established correctly in invssa12246 mutants, kidneys appear to develop normally without any cysts and cilia appear normal, however mutants are significantly shorter. In post-embryonic stages, invssa12246 mutants display significant growth delay and signs of retinal mis-patterning together with spinal deformities reminiscent of idiopathic scoliosis. The allele is lethal in the juvenile stage. CONCLUSIONS:We show that invssa12246 allele has a distinct phenotype from other models where inversin function is disrupted, uncovering novel roles in post-embryonic development.
