Dynein axonemal assembly factors (DNAAFs) play crucial roles in the formation and function of motile cilia, and their dysfunction often results in primary ciliary dyskinesia (PCD). We report the spatio-temporal expression patterns of dnaaf5 and dnaaf9 mRNA in zebrafish embryos, providing insight into their possible functions during development. We show that dnaaf5 and dnaaf9 mRNAs are expressed in motile ciliated tissues, such as the Kupffer's vesicle, pronephros, floor plate, brain and olfactory placode. The dnaaf5 and dnaaf9 crispants develop ciliopathic defects during zebrafish development. These data suggest that dnaaf5 and dnaaf9 may regulate motile cilia biogenesis and function in zebrafish. Our findings suggest functional redundancy and divergence among dynein arm assembly factors in vertebrates.
Failure to properly regenerate and maintain tissues is a root cause of disease and aging. One goal of regenerative biology is to unveil mechanisms by which simpler model organisms successfully regenerate so that we can better understand how to control the cellular and molecular switches in human tissues. Planarians have become a favored model system for these studies because of their amazing capacity to fully and rapidly regenerate following amputation, but also for their response to other stressors such as starvation. Planarians undergo proportional degrowth when starved, a unique process that balances stem cell proliferation and apoptotic cell death, autophagy-mediated cell turnover, and energy sensing. We chose a pharmacological approach to study autophagy in planarians by using chloroquine, an autophagy inhibitor. We found that head regression and tissue lysis were induced by chloroquine in whole planarians and that starved planarians were more sensitive than their fed counterparts. While chloroquine treatment did not prevent tissue regeneration following injury, it caused regeneration delays and disruptions in photoreceptor replacement. Finally, we show that an anterior-posterior gradient of chloroquine sensitivity exists in the planarian with prepharyngeal tissues exhibiting significantly low sensitivity. Together, our results extend our understanding of autophagic flux in planarian tissue regeneration and maintenance and establish a model by which an autophagic gradient exists in this highly regenerative system.
At birth, the gastric epithelium is immature and progressively matures during postnatal development to establish adult tissue architecture and function. Although this process has been investigated at functional, proteomic and transcriptomic levels, the precise temporal and spatial dynamics by which individual epithelial lineages initiate differentiation, exit proliferative states and establish adult homeostasis remain incompletely understood. Here, we performed a comprehensive immunofluorescence analysis of the mouse stomach from mid-gestation through adulthood, examining fetal markers, lineage-specific functional epithelial markers, and the proliferation marker KI67. By integrating functional epithelial marker expression with proliferative status, we delineated distinct maturation trajectories for pit, parietal, and chief cell lineages. Notably, parietal and chief cells exhibited a robust proliferative phase from late gestation to early postnatal stages, followed by a marked decline in proliferative activity, whereas pit cells showed the emergence of proliferative progenitors primarily during later postnatal stages. Moreover, we identified region-specific differences in the timing of maturation between the corpus and the antrum. Together, our study establishes a spatiotemporal in situ atlas of gastric epithelial maturation and provides a framework for understanding lineage- and region- specific mechanisms governing postnatal gastric development.
Most major organs, like muscles, bones, vessels and kidneys, develop from the mesoderm, one of three germ cell layers in triploblastic organisms. Sialic acids significantly affect embryonic development by regulating cell division, migration and death through signaling pathways and cell adhesion, which support morphogenesis. Loss of early biosynthetic enzymes reduces embryonic viability and leads to complex phenotypes, while the loss of terminal enzymes primarily results in tissue-specific defects in mesoderm-derived organs. Key sialylated glycoproteins involved in the developmental processes of mesoderm and mesoderm-derived organs have been identified across various species as major effectors. These enzymes and glycoproteins are of significant interest and are discussed in the present review.
Although the nonbilaterian Hydra has a simple body architecture, its signaling systems are as complex as those of Bilateria. We here add data to the fibroblast growth factor signaling system which has previously been identified in Hydra as essential for bud detachment. The localized expression patterns of its, now fifteen, FGFs indicate additional, yet unidentified, functions in sometimes very small subsets of cells at the body termini and/or during budding or in testes. Presence of a typical signal sequence (prediction >93%) in only 30% of the Hydra FGFs suggests mechanisms of action (and/or secretion) other than the canonical ones. A knockdown approach using siRNA revealed a potential role for Hv-FGF-c in tentacle morphogenesis. Our results document a high complexity of FGF signaling sites in Hydra and open the field for a detailed analysis of their functions.
The skull of amniotes is categorized into three conditions based on skeletal arrangement in the temporal region: anapsid, synapsid and diapsid. Mammals (class Mammalia), a descendent lineage of the clade Synapsida, possess the synapsid skull, which is characterized by a single lower temporal arch that ventrally borders the lower temporal fenestra. Although we previously suggested, based on the data from placental mammals, that the reduction in the expression domain of the upstream osteogenic genes Msx2 and Runx2 in the embryonic temporal mesenchyme might have played a role in the evolution of synapsid skulls, the molecular basis of synapsid skull evolution is still largely unknown. In this study, we investigated expression patterns of four osteogenic genes (two upstream genes Msx2 and Runx2 and two downstream genes Sp7 and Sparc) in the embryonic and neonatal temporal region of the gray short-tailed opossum Monodelphis domestica, the most commonly used experimental marsupial model, in order to more thoroughly understand the molecular basis of development of synapsid skulls unique to mammals. We found that M. domestica embryos and neonates display very restricted expressions of Msx2 and/or Runx2 in the dermal bone precursors in the temporal region, as two placental species do (the house mouse Mus musculus and the greater horseshoe bat Rhinolophus ferrumequinum). Spatially restricted expression of Msx2 and Runx2 in the embryonic temporal region may be a foundation for creating the "advanced" synapsid skull shared by all mammals where only three dermal bones configure the temporal region.
Following publication of this article, the authors identified typographical errors in the primer sequences listed in Table 1 in the Materials and Methods section. The corrected table is published.
Carnitine palmitoyltransferase 1 (CPT1) is a key regulatory enzyme in fatty acid metabolism, responsible for the translocation of long-chain fatty acids into the mitochondria for β-oxidation in diverse biological contexts. Recent studies implicated the critical role of cpt1 genes during zebrafish development and heart regeneration; however, a comprehensive characterization of their spatiotemporal expression dynamics remains lacking. Here, we systematically analyzed the expression profiles of four cpt1 paralogs (cpt1aa, cpt1ab, cpt1b, and cpt1a2b) during zebrafish embryogenesis and the expression of cpt1ab and cpt1b during zebrafish heart regeneration. Our results reveal that these paralogs exhibit distinct spatiotemporal expression patterns during zygotic development. While cpt1aa and cpt1ab share high sequence conservation (77%), their expression patterns diverge substantially. Conversely, cpt1ab and cpt1b display convergent cardiac and somitic expression despite lower sequence similarity (53%). Following ventricular ablation, cpt1b expression transiently ceased then recovered during regeneration, whereas cpt1ab remained unchanged. These findings shed light on the evolutionary conservation and functional divergence of cpt1 paralogs, which establish a critical foundation for elucidating paralog-specific roles in fatty acid metabolism during vertebrate development and regeneration.
Interactions between ephrins and their Eph receptors regulate a broad range of cellular processes, including attraction, repulsion, adhesion and migration, all of which play crucial roles in tissue remodeling and homeostasis. While several ephrin ligands and Eph receptors are known to be expressed in the developing kidney, their specific roles, particularly during nephrogenesis, remain poorly understood. The development of the Xenopus pronephros provides an accessible and relatively simple model for studying vertebrate nephrogenesis. Through a comprehensive gene expression analysis of all ephrin ligands and Eph receptors present in Xenopus genomes, we have identified members of the Eph-ephrin signaling pathway that may contribute to pronephric development. Among them, efna3, which encodes an ephrin ligand, is strongly expressed in the ventral region of the pronephric anlage and later in the intermediate and distal segments of the developing tubule. This expression pattern is strikingly complementary to that of epha4 and epha7, which are expressed in the region forming the proximal tubule. This suggests a potential role for efna3-epha4/epha7 signaling in establishing a boundary between these domains during pronephros development.
How the dorsal thalamus of amniotes (reptiles, birds, and mammals) is organized remains an important but incompletely answered question. Identification of meaningful subdivisions would greatly aid in its understanding. Because the dorsal thalamus is more simply organized during development, studies have examined this structure during embryogenesis. Most reports using this approach have examined the developing dorsal thalamus in mammals and birds. Only rarely has the development of the dorsal thalamus been investigated in reptiles. Regardless, any approach to identify subdivisions, the presumed building blocks of the dorsal thalamus, should include representatives of all three classes of vertebrates. To fill this gap in knowledge, the development of the dorsal thalamus was investigated in Alligator mississippiensis, a member of the reptilian group most closely related to birds. As the first detailed study of its kind, cytoarchitecture and calretinin expression were used to examine dorsal thalamus development. Three subdivisions, termed tiers, and the individual nuclei originating from each tier, were identified. These three tiers were similar to the subdivisions found in birds and, to a limited extent, in mammals. Taken together, these early subdivisions may represent the common building blocks of the dorsal thalamus and provide clues to understand how evolution has sculpted this structure in amniotes.
The development of cranial nerves, including the trigeminal nerve, and the formation of neuromuscular junctions (NMJs) are crucial processes for craniofacial motor function. Mllt11/Af1q/Tcf7c (hereafter Mllt11), a novel type of cytoskeletal-interacting protein, has been implicated in neuronal migration and neuritogenesis during central nervous system development. However, its role in peripheral nerve development and NMJ formation remains poorly understood. This study investigates the function of Mllt11 during trigeminal ganglion development and its impact on motor innervation of the masseter muscle. We report Mllt11 expression in the developing trigeminal ganglia, suggesting a potential role in cranial nerve development. Using a conditional knockout mouse model to delete Mllt11 in Wnt1-expressing neural crest cells, we assessed trigeminal ganglion development and innervation of the masseter muscle in the jaw. Surprisingly, we found that Mllt11 loss does not affect the initial formation of the trigeminal ganglion but disrupts its placodal vs. neural crest cellular composition. Furthermore, we showed that conditional inactivation of Mllt11 using Wnt1Cre2 led to a reduction of neurofilament density and NMJs within the masseter muscle, along with a reduction of Phox2b+ branchiomotor neurons in rhombomere 2, indicating altered trigeminal motor innervation. This was due to the surprising finding that the Wnt1Cre2/+ mouse driver promoted aberrant recombination and reporter gene expression within branchiomotor neuron pools in rhombomere 2, as well as targeting neural crest cell populations. Our findings show that Mllt11 regulates the cellular composition of the trigeminal ganglion and is essential for proper trigeminal motor innervation in the masseter muscle.
The transcription factor GATA4 is found in Sertoli and Leydig cells, whereas SOX9 is exclusive to Sertoli cells, being both factors essential for the normal development of murine and human fetal testis. In turn, the steroidogenic acute regulatory protein (STAR) is specifically expressed in Leydig cells. Nevertheless, the function of STAR, GATA4 and SOX9 in peripubertal, adolescent and adult testes in Klinefelter syndrome and azoospermic patients remains poorly understood. To characterize the developmental expression of STAR, GATA4 and SOX9 in human testicular somatic cells, we performed immunofluorescence using fetal, peripubertal, adolescent and adult testes. Our findings demonstrate that STAR is absent in early fetal stages, but present in Leydig cells from 12 weeks of gestation, as well as in peripubertal, adolescent and adult Klinefelter patients, in the adult testis with idiopathic azoospermia and in men showing normal spermatogenesis. GATA4 was expressed in both Sertoli and Leydig cells during all the studied developmental stages and in peripubertal, adolescent and adult patients with and without spermatogenesis. SOX9 was mainly expressed in Sertoli cells in fetal, peripubertal, adolescent and adult Sertoli cell patients. In patients with Klinefelter syndrome as well as in men with or without spermatogenesis SOX9 was also found in Leydig cells. Our findings support the premise that STAR is a key steroidogenic protein for androgen development in the fetal testis, that GATA4 regulates Sertoli and Leydig cells during testis development and that SOX9 regulates the development of Sertoli cells and is present in the Leydig cells of patients with azoospermia.
The enhancer threshold is defined as the minimum concentration of transcription factors (TFs) required to elicit an enhancer response in a given time and space. Here, evidence is presented that the enhancer threshold is relative to promoter strength in the early Drosophila embryo. The apparently inactive even-skipped (eve) minimal stripe element (MSE), in which a single Hunchback (Hb)-binding site is deleted, is functionally complemented by the hsp70 promoter in transgenic embryos. Forced pause release of RNA polymerase II (Pol II) and transcription bubble assays show that both eve and heat shock protein 70 (hsp70) promoters exhibit paused Pol II. However, bioinformatics analyses and transient transfection assays indicate that the strength of the hsp70 promoter is much stronger than that of the eve promoter. Consistently, inactive MSE function is also restored by promoters stronger than the eve promoter. It is conceivable that the functional complementarity between enhancer and promoter strengths defines the enhancer threshold, thus determining whether a genomic locus acts as an enhancer for a particular promoter.
Toll-Like Receptor 7 (TLR7) is recognized for its role in immune responses, particularly in detecting viral RNA. However, emerging evidence suggests that TLR7 may also contribute to ocular development. In this study, we assessed the expression pattern of TLR7 in various CD-1 mouse eye compartments during critical developmental stages, from embryonic day 12 to 16, as well as in adult tissues such as the cornea, pigmented epithelium, neural retina and lens. Our findings reveal a region-specific and time-dependent expression of TLR7, suggesting that it may play a role in the morphogenetic processes that shape the eye during intrauterine development.
Based on observations of in vivo morphogenesis, differentiation is expected to be regulated by mechanical cues. However, the detail mechanisms remain largely unknown. A previous study using human pluripotent stem cells (hPSCs) demonstrated that neural plate border (NPB) specification was enhanced by mechanical force. However, it is unknown whether mechanical force is also involved in the specification of the preplacodal ectoderm (PPE), which is derived from the NPB. Here, we verified the validity of the PPE induction method in stretch chambers, and conducted the stretching stimuli experiments. When repetitive stretching stimuli were applied from Day 2 to 10 or Day 2 to 7, expression of the PPE marker SIX1 was increased. However, this increase was not observed when the stimuli were applied from Day 5 to 10, suggesting there is a critical period of sensitivity to mechanical forces. Immunofluorescent staining revealed lower active β-catenin signals in the cell sheet in the stretched samples compared to those in the controls, suggesting a negative correlation between stretching stimuli and Wnt signaling. Our finding suggests that mechanical force is important in PPE differentiation.
Recent studies suggest that tail regeneration in lizards begins with a tumor-like stage usually termed regenerative blastema. Oncogenes and tumor suppressors are activated in blastema cells, resulting in a balanced cell proliferation that does not turn the blastema into a tumor. This outgrowth elongates forming new tissues and tail. We previously showed that physiological extracts from regenerating lizard tissues inhibit the growth of cancer cells in vitro within 2-4 days of administration, demonstrating that the growing lizard blastema contains regulatory molecules which can also influence human cancer cells. The molecules responsible for this inhibition were not identified in that initial study. In the present experimental study, after specific extractions of RNAs and/or proteins from the regenerating tail of lizard, we have confirmed the inhibition of breast cancer cell vitality in vitro within 2-3 days from their addition to the culture medium. Proteolysis or heat denaturation of proteins abolished the inhibitory effect. RNA delivered to breast cancer cells in vitro through lipid vesicles (liposomes) showed the highest inhibition of cancer cells vitality. Cell degeneration, detected by microscopy, revealed that RNA is more effective than proteins extracted from regenerating tissues. The present observations further suggest that RNAs coding for known tumor suppressor proteins, and non-coding RNAs that are highly expressed in the regenerating tail, may be key inhibitors (tumor suppressors) of blastema and cancer cell proliferation. The evolution of a mechanism for the self-remission of tumor growth in lizards remains uncertain, but continuing study of this reptile may help uncover natural mechanisms for tumor growth inhibition.
The Spanish Society for Developmental Biology (SEBD) organized its 18th meeting in October 2024 (hereafter SEBD2024), coinciding with the society's 30th anniversary and serving as the stage for its celebrations. This article provides an overview of the event, including the speakers, scientific sessions and the different activities related to the anniversary.
This study aims to analyze the pathways and the placental brain axis genes of gestational diabetes mellitus (GDM) affecting offspring neurodevelopment. Differentially expressed genes (DEGs) were identified through transcriptome sequencing of placental tissues. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed on DEGs. A protein-protein interaction (PPI) network was constructed and annotated using the STRING online software. The expression of neurodevelopment-related genes was analyzed by qPCR. Hubgenes were analyzed using Cytoscape 3.7.1 software. The correlation between Hubgenes and placental brain axis genes was analyzed through literatures alignment. The pathways of GDM affecting offspring neural development were predicted using the KEGG database. The placental transcriptome revealed that there were 404 DEGs between GDM and Normal groups. Among these DEGs, 125 were upregulated and 279 were downregulated. GO analysis indicated that DEGs were mainly involved in intracellular calcium activated chloride channel activity, anion channel activity, G protein-coupled peptide receptors, etc. Additionally, KEGG analysis revealed that DEGs were predominantly involved in neuroactive ligand receptor interaction pathways. STRING online software analysis revealed that the DLGAP1, NXNL2, SCG2, SLC18A2, LYNX1, GRM1, DLGAP1, BIRC7 genes were associated with neurodevelopment. PCR validation of these 8 genes was consistent with transcriptome results (P<0.05). Literatures alignment showed that DLGAP1, GRM1 and SLC18A2 are placental brain axis genes that influence offspring neurodevelopment. The placental brain axis genes DLGAP1, GRM1, SLC18A2 have been found to influence GDM offspring neurodevelopment through the regulation of the Gq/PLC/PKC pathway.
This study investigated the role of cyclooxygenase-2 (COX2) in angiogenesis during zebrafish embryogenesis by inhibiting COX2 activity with etoricoxib. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed the successful penetration of etoricoxib into zebrafish embryos, leading to selective inhibition of COX2 without affecting COX1 activity. COX2 inhibition caused a significant reduction in prostaglandin E2 levels throughout development. Phenotypically, treated embryos exhibited pericardial edema, bradycardia, and defective vascular development, including delays in intersegmental vessel (ISV) sprouting, incomplete dorsal longitudinal anastomotic vessel (DLAV) formation by 48 hpf, and impaired vascular networks by 72 hpf. Confocal imaging and AngioTool analysis revealed reduced vessel length, area and increased lacunarity. Molecular analysis showed significant downregulation of vascular endothelial growth factor A (vegfa), kdr, pi3k and akt transcripts, as well as reduced VEGFA, EP4 and Akt protein levels, disrupting VEGFA-PI3K-Akt signaling. Additionally, reduced expression of ephrinb and prox1 affected arterial and venous identity formation. These results demonstrate that COX2 is essential for proper angiogenesis during zebrafish development, and its inhibition leads to significant vascular defects, underscoring COX2's crucial role in regulating VEGFA-mediated angiogenesis.
The Transient Receptor Potential superfamily of proteins (TRPs) form cation channels that are abundant in animal sensory systems. Amongst TRPs, the Melastatin-related family (TRPMs) is composed of members that respond to temperature, pH, sex hormones, and various other stimuli. Some TRPMs exhibit enriched expression in the gonads of vertebrate and invertebrate species, but their contributions to germline development remain to be determined. We identified twenty-one potential TRPMs in the planarian flatworm Schmidtea mediterranea and analyzed their anatomical distribution of expression by whole-mount in situ hybridization. Enriched expression of two TRPMs (Smed-TRPM-c and Smed-TRPM-l) was detected in testis, whereas eight TRPM genes had detectable expression in patterns representative of neuronal and/or sensory cell types. Functional analysis of TRPM homologs by RNA-interference (RNAi) revealed that disruption of normal levels of Smed-TRPM-c expression impaired sperm development, indicating a role for this receptor in supporting spermatogenesis. Smed-TRPM-l RNAi alone did not result in a detectable phenotype, but it did increase sperm development deficiencies when combined with Smed-TRPM-c RNAi. Fluorescence in situ hybridization revealed expression of Smed-TRPM-c in early spermatogenic cells within testes, suggesting cell-autonomous regulatory functions in germ cells for this gene. In addition, Smed-TRPM-c RNAi resulted in reduced numbers of presumptive germline stem cell clusters in asexual planarians, suggesting that Smed-TRPM-c supports the establishment, maintenance, and/or expansion of spermatogonial germline stem cells. While further research is needed to identify the factors that trigger Smed-TRPM-c activity, these findings reveal one of the few known examples for TRPM function in the direct regulation of sperm development.
