Transgenic reporters offer valuable opportunities to investigate the genetic and cellular basis of embryonic development in live organisms. While the specificity of transgenic reporters is essential for their effectiveness in developmental studies, many existing transgenic muscle reporters used for craniofacial muscle development in zebrafish also label trunk skeletal and cardiac muscles, as well as other head tissues. Here, we report the identification of a new transgenic reporter marking craniofacial muscles, Tg(nkx2.3:Alcama-GFP), in zebrafish. This transgenic reporter exhibits Alcama-GFP expression in growing muscle fibers at embryonic stages and in grown muscle fibers at larval stages. However, endogenous nkx2.3 transcripts are barely detected in growing craniofacial muscle fibers by in situ hybridization, and nkx2.3 mutants display fairly normal ventral jaw muscles adjacent to the lower jaw cartilages and extraocular muscles adjacent to the eye. Although an essential role for Nkx2.3 in craniofacial muscle formation remains to be determined, the Tg(nkx2.3:Alcama-GFP) craniofacial muscle reporter provides better opportunities for the precise analysis of craniofacial muscle development at the cellular and genomic levels.
The uterus is an essential organ for fetal development in most mammals. Uterine glands, highly conserved structures in the mammalian uterus, play critical roles in the establishment and maintenance of pregnancy and have been implicated in the pathogenesis of uterine diseases, including endometrial cancer and endometriosis. Previous studies have shown that Retinol dehydrogenase 1 (Rdh1) is specifically expressed in the glandular epithelium (GE) from the onset of gland formation through adulthood. In this study, to develop a GE-specific Cre driver line, we generated Rdh1-iCre mice by introducing an improved Cre recombinase (iCre) into the Rdh1 locus using the CRISPR/Cas9 system. To evaluate the utility of this model, Rdh1-iCre mice were crossed with ROSA26-H2B-mCherry reporter mice, and Cre-dependent reporter expression was analyzed. Robust mCherry fluorescence was observed throughout the uterine glands at 2 weeks after birth, coinciding with the active elongation and branching of the GE. These results demonstrate that the Rdh1-iCre mouse line is a valuable and highly efficient tool for investigating the physiological roles of uterine glands during development and pregnancy, as well as their contribution to the progression of GE-derived uterine diseases.
Follicular thyroid cells in vertebrates are considered to have evolved from the endostyle of invertebrate chordates based on the shared features of the concentration of iodine and expression of thyroid-related genes, including Nkx2-1, FoxE, and TPO. We previously demonstrated using gene knockout (KO) experiments on the ascidian Ciona robusta that the thyroid-related transcription factors, Nkx2-1 and FoxE, were associated with endostyle development, and also that Nkx2-1 regulated the expression of TPO via FoxE, even though the expression of Pax2/5/8 homologs was not affected. In the present study, which involved detailed observations of Ciona Nkx2-1-KO and FoxE-KO specimens, we found that ectopic bulged structures neighboring the hypoplastic endostyle occasionally emerged. In situ hybridization revealed that the ectopic bulged structures expressed TPO and Pax2/5/8 homologs. Further KO experiments on Ciona Pax2/5/8 homologs revealed no effects on endostyle development or TPO expression. These results suggest that, in addition to the established "Nkx2-1 -> FoxE-FoxE -> TPO" pathway, the ventral pharynx offers an insight into a potential relationship between Pax2/5/8 homologs and TPO. Accordingly, ascidians may show that the ventral pharyngeal endoderm possessed multiple gene regulatory networks for TPO expression prior to the evolutionary divergence leading to the vertebrate follicular thyroid.
Epigenetic regulation is indispensable for embryonic development, yet the specific roles of histone deacetylases (HDACs)-a core family of epigenetic regulatory enzymes-in orchestrating craniofacial morphogenesis remain incompletely elucidated. C57BL/6 mouse embryos spanning embryonic Day 12.5 (E12.5) to E16.5 were analyzed. Histological staining (hematoxylin-eosin and Masson staining) was used to characterize morphological development of the tongue, palate, and mandible. Immunohistochemistry (IHC) was performed to map the spatiotemporal expression patterns of HDAC1-11. HDAC1 expression was scarcely detectable throughout the entire E12.5-E16.5 period. In contrast, HDAC2 showed robust expression in the mandible and tongue from E13.5 to E15.5. Notably, HDAC2 also exhibited strong positive staining in palatal epithelial cells at E14.5. By E16.5, HDAC1-10 expression became barely detectable in tongue, palate, and mandible, whereas HDAC11 retained intense expression specifically in the mandible at this late maturation stage. HDAC family members exhibit divergent spatiotemporal expression profiles during craniofacial development, with distinct subtype-specific patterns that align with key stages of tongue, palate and mandible morphogenesis. These findings establish a comprehensive spatiotemporal expression atlas of HDAC1-11 in developing craniofacial organs, providing essential foundational data to guide future investigations into the epigenetic regulation of craniofacial development.
The human neuroblastoma cell line SH-SY5Y is widely used in vitro as a model due to its ability to acquire neuron-like properties. Conventional differentiation protocols often rely on retinoic acid (RA), which can lead to limited stability of neuronal-like phenotypes over time. In this study, we evaluated a differentiation approach combining RA with a laminin-rich extracellular matrix (LrECM). This approach accelerated differentiation, with neuron-like morphology and neurite outgrowth observed as early as 4 days and sustained features for up to 10 days. Immunofluorescence analysis indicated increased NeuN expression, and Western blot confirmed the sustained presence of β3-tubulin during differentiation. Furthermore, a trend toward enhanced acetylcholinesterase (AChE) activity suggested partial cholinergic features. Altogether, these findings indicate that incorporating LrECM supports the stability of RA-induced neuron-like characteristics in SH-SY5Y cells and may provide a useful alternative for studies of neuronal-phenotype differentiation.
Here we describe the optimisation of a modular, generally applicable and rapid method to make multimerised signaling-molecule reporters for detecting different retinoic acid concentrations. The technical plasmid design allows for the easy exchange of reporters, promoters, signal-responsive elements as well as for the multimerization of entire cassettes of these modular units. These constructs are ideal for pronuclear injection resulting in random integration into genomic DNA so avoiding the use of embryonic stem cells for generating transgenic lines or embryos. The use of insulator elements was shown to isolate reporter cassettes from integration-specific influences and from adjacent cassettes. Such insulators were found to be beneficial in transient transfections of primary cells, where DNA would not have integrated, as seen by more accurate ligand concentration responsiveness. The choice of promoter is important as weak promoters in in vitro experiments did not function in transgenic embryos. Notably, the core binding site and surrounding nucleotides could be used to modulate retinoic acid responsiveness in vitro. However, in vivo, the construct not only reflected retinoic acid concentrations but also affected tissue specificity. Increasing the number of retinoic acid receptor binding sites from 4 to 12 copies increased in vitro RA sensitivity, but had a lesser effect on in vivo sensitivity. We observed robust expression in the primitive streak of presomitic embryos and in the somites, neural tube and presomitic mesoderm of 3-10-somite embryos. Notably, use of only two copies of the binding site resulted in construct-inherent variability in cell type specificity and RA concentration sensitivity. This was conclusively demonstrated using dual receptors, which inherently control for confounding integration or copy-number dependent effects. In conclusion, the use of dual reporters with insulators is a useful approach for concurrently measuring concentration and context dependent responses to one signaling pathway and also lends itself for examining the interaction of two signaling pathways within one embryo.
TWIST1 encodes a highly conserved basic helix-loop-helix transcription factor essential for embryonic development from Drosophila to humans. TWIST1 activity is regulated by post-translational modifications, including phosphorylation during development and cancer metastasis. Recent cancer studies identified acetylation of lysines K73 and K76 as a novel regulatory modification that shifts TWIST1 from a repressive to an activating state during epithelial-to-mesenchymal transition (EMT). However, the developmental and in vivo functions of TWIST1 acetylation remain unknown. To investigate the physiological role, we generated the first acetyl-deficient Twist1K73,76R K73,76R and acetyl-mimic Twist1K73,76Q K73,76Q mouse models using CRISPR/Cas9-mediated genome editing. Targeted sequencing confirmed substitutions, and founders were backcrossed onto a C57BL/6J genetic background. Phenotypic analysis revealed that Twist1 acetyl-deficient mice exhibited highly penetrant craniofacial abnormalities, including severe mandibular hypoplasia, mandibular bone fusion, replacement of premaxillary, maxillary, and palatine bones with cartilage, ectopic cartilage-like structures, ocular malformations, and reduced skull mineralization. In contrast, acetyl-mimic mutant embryos displayed milder craniofacial defects characterized by reduced mandibular condylar processes and palatine bones, along with decreased skull mineralization. These findings demonstrate that TWIST1 acetylation at K73 and K76 plays a crucial role in normal craniofacial skeletogenesis, supporting prior cancer studies showing reduced oncogenic activity following loss of TWIST1 acetylation.
Alternative splicing is a key post-transcriptional process that generates multiple mRNAs from a single pre-mRNA through the coordinated action of the spliceosome and associated splicing factors. SMNDC1 (survival motor neuron domain containing 1) has been identified as an integral component of the spliceosome complex. Previous studies have shown that SMNDC1 is essential for splicing catalysis in vitro and plays a regulatory role in intron retention in cancer. However, the phylogenetic relationships and expression patterns of SMNDC1 across the animal kingdom have not been systematically investigated. In this study, we conducted a comprehensive phylogenetic analysis of SMNDC1 genes in animals. A total of 72 SMNDC1 genes were identified from 66 animal species. Bioinformatic analyses revealed that the gene structure and protein domains of SMNDC1 are highly conserved, with only a few species possessing gene duplications. Notably, the human SMNDC1 gene exhibits elevated expression levels in multiple cancer types, including breast, colon, and rectal cancers, suggesting its potential role in tumor development and its value as a diagnostic or therapeutic target. Overall, our findings provide a systematic overview of the SMNDC1 gene family in animals and establish a foundation for future studies exploring its molecular functions and evolutionary significance.
Optimizing transient expression systems in fish embryos is crucial for rapid gene function analysis. Here, we established an efficient system in medaka (Oryzias latipes) embryos by evaluating nucleic acid type and injection site. Our results revealed that injecting the elongation factor 1αA (ef1αA) promoter-driven plasmid into the yolk yielded the highest expression on day 1 post-fertilization. Using this optimized system, we investigated cytosolic 5'-nucleotidase 1a (nt5c1a), which is involved in the metabolism of inosine monophosphate (IMP), an umami flavor compound. In silico analysis revealed that medaka had two nt5c1a paralogs: nt5c1aa and nt5c1ab. While nt5c1ab retains conserved substrate-recognition sequences and exhibits significant IMP degradation activity, nt5c1aa has lost these functions. Structural analysis using AlphaFold revealed that the Nt5c1aa L305P mutation causes local conformational changes near the substrate-binding site, potentially altering substrate orientation without disrupting the overall protein fold. Our expression system demonstrated that this single L305P substitution partially restored IMP-degrading activity in Nt5c1aa, confirming that residue 305 is a key determinant of its functional divergence. Our findings provide a robust foundation for molecular breeding to enhance umami flavor in farmed fish. Specifically, the targeted manipulation of these nt5c1a paralogs could facilitate developing breeds with maximized IMP accumulation in muscle tissues.
The directionality of blood flow is regulated by heart valves, among other things. While the heart valves in vertebrates are multicellular and complex, the valve in the Drosophila heart consists of exactly two highly specialized cells. They arise during early larval development from two cardiomyocytes, those that form the boundary between the aorta and the posterior ventricle. Here, we show that the conserved PDGF signaling pathway is involved in the determination of heart valve cells. RNAi-mediated knockdown of the Pvr receptor and one of its ligands, Pvf2, leads to an inhibition of valve cell differentiation. In contrast, the simultaneous expression of the Pvr and Pvf2 in the entire heart tube leads to the formation of additional heart valve cells in ectopic positions. A single expression of the receptor or the ligand does not lead to heart valve formation.
The developing vertebrate forelimb expresses seven T-box transcription factors, with several in overlapping expression domains. All T-box transcription family members share similarity within their DNA binding domain, the T-domain. Outside of the T-domain, these factors share little similarity, allowing family members to have different transcriptional properties and binding partners. Several human T-box genes show haploinsufficiency in the limb, including Tbx5 and Tbx3 that, when mutated, cause Holt-Oram and ulnar-mammary syndrome, respectively. This dosage sensitivity combined with the shared T-domain leads to our hypothesis that when co-expressed a competition between T-box factors at target genes can occur. To test this, we ectopically expressed two exogenous T-box factors, T and Tbx6, in the developing forelimb mesenchyme to examine how artificially changing the relative levels of T-box proteins affects forelimb formation. Skeletal, apoptotic, and gene expression assays were used to characterize the resulting phenotypes. While ectopic T and Tbx6 both affected the size and shape of the forearm bones and ossification, they differentially affected digit formation: T caused loss of digits and Tbx6 led to phalange bone duplications and extra digit formation. These dissimilar phenotypes suggest that these transcriptional activators differentially affect pathways critical for regulating forelimb development.
The intersex (ix) plays a pivotal role in the sex determination pathway of Drosophila melanogaster. The Drosophila Ix protein interacts with female-specific Doublesex protein to regulate female sexual differentiation. In this study, we functionally characterized the ix homologue (Tcix) in the red flour beetle, Tribolium castaneum. The Tcix gene comprises of four exons and three introns, and encodes a protein of 166 amino acids that contains a conserved 'Mediator complex subunit 29' (Med-29) domain. The pre-mRNA of Tcix splices in a non-sex-specific manner and is expressed across all the developmental stages of T. castaneum. RNA interference-mediated knockdown of Tcix in adult beetles led to a significant reduction in its expression in both sexes. Knockdown males did not show any phenotypic changes, but knockdown females displayed compromised ovarian development, resulting in reduced fecundity and failure of eggs to hatch. Interestingly, the expression of vitellogenin-1 (Vg-1), a key gene involved in oocyte development, was significantly downregulated in these females. Further, parental RNAi of Tcix demonstrated the failure in the hatching of eggs, despite no significant effects on the number of eggs laid. The phylogenetic analysis of Tcix revealed its close clustering with other coleopterans, which supports its evolutionary conservation. Overall, our study establishes the critical role of Tcix in female fertility and embryonic development in T. castaneum.
The classical definition of metamorphosis is a post-embryonic transformation, such as from a tadpole to a froglet. However, recent studies suggest this process occurs to some degree in all vertebrates, as the underlying endocrine and molecular pathways are highly conserved. With the advent of high-throughput sequencing, transcriptomic data for non-model species has revealed that protein-coding genes represent only a small fraction of the genome. In contrast, most transcriptional output produces non-coding RNAs with vital regulatory functions. Among these, long non-coding RNAs are a diverse and important class known to regulate gene expression at multiple levels and across various biological contexts. Despite their established importance, the study of lncRNAs across the tree of life remains an open field, crucial for understanding their potential roles and evolutionary conservation. This work summarizes the roles of lncRNAs as regulatory molecules, their functions in development and metamorphosis, computational strategies for their characterization, and the challenges and opportunities of studying them in non-model species.
Notch1 receptor activation plays an important role in epithelial homeostasis and immunoregulation; however, its role in oral mucosal immunity remains unknown. Testing the impact of epithelial Notch1 activation in oral innate responses, including antimicrobial proteins like phospholipase A2 group IIA (PLA2-IIA), requires epithelial-specific Notch1 knockout mice. Creating Balb/c; K13Cre; Notch1Flox/Flox mice was initiated by crossing C57BL/6; K13Cre; Notch1Flox/Flox mice (naturally deficient for PLA2-IIA), expressing Cre recombinase under the control of the Krt13 (keratin 13) promoter (K13Cre) and Notch1Flox/Flox alleles to PLA2-IIA expressing Balb/c mice to generate Balb/c:C57BL/6; K13Cre; Notch1Flox heterozygotes and to ultimately create Balb/c; K13Cre; Notch1Flox/Flox (Notch1 deficient) and Balb/c; Notch1Flox/Flox WT littermate controls. While all first-generation non-K13Cre expressing mice had the expected Notch1Flox heterozygote Notch1 genotype, surprisingly, all first-generation K13Cre expressing mice had an apparent K13Cre; Notch1 homozygote genotype, which is impossible when crossing Notch1 and Notch1Flox/Flox homozygotes. We found that the absence of the floxed Notch1 PCR amplicon resulted from recombination between the two LoxP sites flanking the forward Notch1 PCR primer, and the recombined Notch1 allele was present in the germline of first-generation congenic mice. Finally, we show that this unexpected recombination is avoided by using female K13Cre; Notch1Flox donors and is a cautionary reminder that individual promoters are expressed during spermiogenesis.
Homeobox genes encode diverse proteins, including proteins with highly conserved DNA-binding domains, and are essential for organismal growth, cell differentiation, organ development, and diverse physiological processes associated with diseases. BARX1 encodes a transcription factor containing the BarH1-like homeodomain. This gene plays a significant role in the development of various tissues and is closely associated with the pathogenesis and progression of numerous diseases. In this review, we provide a comprehensive overview of the functional networks and mechanisms of action of the BARX1 gene across different tissue developmental processes and disease contexts.
The corneal endothelium is a monolayer of specialized cells that maintains stromal deturgescence and transparency, functions essential for vision. Despite its clinical importance, the developmental origins and homeostatic programs of the endothelium remain poorly understood, in part due to the lack of a lineage-specific genetic driver. To overcome this limitation, we generated a Col8a2P2A-CreERT2 knock-in mouse line that enables selective genetic manipulations of corneal endothelial cells. Cre activity was validated with reporter alleles and functional importance was assessed by conditional ablation of Col8a2+ cells in adulthood, with phenotypic outcomes evaluated by histology, immunofluorescence, and in vivo imaging. We found that Col8a2P2A-CreERT2 drives robust and specific recombination in corneal endothelial cells. Functional assays demonstrated that Col8a2+ cells contribute continuously to Descemet's membrane synthesis and are essential for maintaining endothelial integrity. Ablation disrupted endothelial density and barrier function, resulting in phenotypes resembling human endothelial dystrophies, including features of Fuchs' endothelial corneal dystrophy and posterior polymorphous corneal dystrophy. These findings identify Col8a2+ cells as indispensable regulators of endothelial development, homeostasis, and disease pathogenesis. The Col8a2P2A-CreERT2 line provides the first corneal endothelium-specific genetic driver, establishing a platform for mechanistic investigation and therapeutic discovery in endothelial disorders.
The receptor tyrosine kinase RET regulates the development of multiple neuronal populations through GFRα co-receptors and their cognate ligands. Recent work has shown that RET is also required for enteroendocrine cell (EEC) function; however, the specific GFRα receptor operating in EECs has remained unclear. Here, we identify GFRα3 as the predominant GFRα receptor expressed in EECs. To determine its cell-autonomous role, we generated a Gfrα3 conditional allele and inactivated the Gfrα3 gene specifically in the intestinal epithelium. These Gfrα3 IEcKO mice exhibited a selective reduction in the number of CCK+ EECs among multiple EEC subtypes. A comparative decrease in CCK+ EECs in Ret IEcKO mice suggests that RET/GFRα3 signaling is required for the development and/or maintenance of this EEC lineage. Although CCK+ EECs can indirectly influence glucose metabolism, Gfrα3 IEcKO mice showed no significant change in glucose tolerance under high-fat diet (HFD) conditions. In contrast, they exhibited modest lipid malabsorption on HFD. Together, these findings demonstrate that GFRα3 is implicated in the structure and function of CCK EECs and highlight the utility of the Gfrα3 conditional allele for dissecting the biology of GFRα3-expressing cells.
The transformation of the eye primordium into a monolayer eye epithelium with identical ommatidial units requires corporate and coordinated efforts of the retinal determination genes. During the process, the same set of retinal determination genes is utilized reiteratively to fulfill distinct functions concerning eye specification, eye patterning, cell proliferation, and cell survival. To achieve this sophisticated goal, instructions sent from the key developmental pathways and the dorsal-ventral patterning genes are incorporated, and connections are built among the retinal determination genes with temporal and spatial specificity. This facilitates the establishment of a multi-directional regulatory network named the retinal determination gene network (RDGN), which influences a wide range of retinal developmental processes. In this review, I will introduce the functional outputs of the RDGN and discuss its complexity by investigating the genetic, molecular, and biochemical interactions between the retinal determination genes. I will also clarify the fundamental roles of the key developmental pathways and the dorsal-ventral patterning genes in organizing RDGN activity. Lastly, advancements in transcriptome and ChIP sequencing technology, which facilitate the identification of the downstream effectors of the RDGN, will enable extensive characterization of the retinal determination mechanisms and allow creation of a more comprehensive picture about eye development.
