Journal of Developmental Biology

The generation of mature gametes and competent embryos in vitro from pluripotent stem cells has been successfully achieved in a few species, mainly in mice, with recent advances in humans and scarce preliminary reports in other domestic species. These biotechnologies are very attractive as they facilitate the understanding of developmental mechanisms and stages that are generally inaccessible during early embryogenesis, thus enabling advanced reproductive technologies and contributing to the generation of animals of high genetic merit in a short period. Studies on the production of in vitro embryos in pigs and cattle are currently used as study models for humans since they present more similar characteristics when compared to rodents in both the initial embryo development and adult life. This review discusses the most relevant biotechnologies used in veterinary medicine, focusing on the generation of germ-cell-like cells in vitro through the acquisition of totipotent status and the production of embryos in vitro from pluripotent stem cells, thus highlighting the main uses of pluripotent stem cells in livestock species and reproductive medicine. Full article

Background: Little is known about morphogenetic changes in the umbilical cord during the maturation process. Extracellular matrix remodeling, angiogenesis, progenitor activity, and immunomodulation are represented by specific markers; therefore, the aim of this study was to determine the expression of matrix metalloproteinase-2 (MMP2), tissue inhibitor of metalloproteinases-2 (TIMP2), CD34, vascular endothelial growth factor (VEGF), and human β-defensin 2 (HBD2) in preterm and full-term human umbilical cord tissue. Methods: Samples of umbilical cord tissue were obtained from 17 patients and divided into two groups: very preterm and moderate preterm birth umbilical cords; late preterm birth and full-term birth umbilical cords. Routine histology examination was conducted. Marker-positive cells were detected using the immunohistochemistry method. The number of positive structures was counted semi-quantitatively using microscopy. Statistical analysis was carried out using the SPSS Statistics 29 program. Results: Extraembryonic mesenchyme cells are the most active cell producers, expressing MMP2, TIMP2, VEGF, and HBD2 at notable levels in preterm and full-term umbilical cord tissue. Statistically significant differences in the expression of CD34, MMP2, and TIMP2 between the two patient groups were found. The expression of VEGF was similar in both patient groups, with the highest number of VEGF-positive cells seen in the extraembryonic mesenchyme. The expression of HBD2 was the highest in the extraembryonic mesenchyme and the amniotic epithelium, where mostly moderate numbers of HBD2-positive cells were detected. Conclusions: Extracellular matrix remodeling in preterm and term umbilical cords is strongly regulated, and tissue factors MMP2 and TIMP2 take part in this process. The expression of VEGF is not affected by the umbilical cord’s age; however, individual patient factors can affect the production of VEGF. Numerous CD34-positive cells in the endothelium of the umbilical arteries suggest a significant role of progenitor cells in very preterm and moderate preterm birth umbilical cords. Antimicrobial activity provided by HBD2 is essential and constant in preterm and full-term umbilical cords. Full article

The planar cell polarity (PCP) system is essential for positioning cells in 3D networks to establish the proper morphogenesis, structure, and function of organs during embryonic development. The PCP system uses inter- and intracellular feedback interactions between components of the core PCP, characterized by coordinated planar polarization and asymmetric distribution of cell populations inside the cells. PCP signaling connects the anterior–posterior to left–right embryonic plane polarity through the polarization of cilia in the Kupffer’s vesicle/node in vertebrates. Experimental investigations on various genetic ablation-based models demonstrated the functions of PCP in planar polarization and associated genetic disorders. This review paper aims to provide a comprehensive overview of PCP signaling history, core components of the PCP signaling pathway, molecular mechanisms underlying PCP signaling, interactions with other signaling pathways, and the role of PCP in organ and embryonic development. Moreover, we will delve into the negative feedback regulation of PCP to maintain polarity, human genetic disorders associated with PCP defects, as well as challenges associated with PCP. Full article

During their biosynthesis, Sonic hedgehog (Shh) morphogens are covalently modified by cholesterol at the C-terminus and palmitate at the N-terminus. Although both lipids initially anchor Shh to the plasma membrane of producing cells, it later translocates to the extracellular compartment to direct developmental fates in cells expressing the Patched (Ptch) receptor. Possible release mechanisms for dually lipidated Hh/Shh into the extracellular compartment are currently under intense debate. In this paper, we describe the serum-dependent conversion of the dually lipidated cellular precursor into a soluble cholesteroylated variant (Shh^C) during its release. Although Shh^C is formed in a Dispatched- and Scube2-dependent manner, suggesting the physiological relevance of the protein, the depalmitoylation of Shh^C during release is inconsistent with the previously postulated function of N-palmitate in Ptch receptor binding and signaling. Therefore, we analyzed the potency of Shh^C to induce Ptch-controlled target cell transcription and differentiation in Hh-sensitive reporter cells and in the Drosophila eye. In both experimental systems, we found that Shh^C was highly bioactive despite the absence of the N-palmitate. We also found that the artificial removal of N-terminal peptides longer than eight amino acids inactivated the depalmitoylated soluble proteins in vitro and in the developing Drosophila eye. These results demonstrate that N-depalmitoylated Shh^C requires an N-peptide of a defined minimum length for its signaling function to Ptch. Full article

Gap junctional connection (GJC) in the cumulus–oocyte complex (COC) provides necessary support for message communication and nutrient transmission required for mammalian oocyte maturation. Cyclic adenosine monophosphate (cAMP) is not only a prerequisite for regulating oocyte meiosis, but also the key intercellular factor for affecting GJC function in COCs. However, there are no reports on whether cAMP regulates connexin 37 (Cx37) expression, one of the main connexin proteins, in sheep COCs. In this study, the expression of Cx37 protein and gene in immature sheep COC was detected using immunohistochemistry and PCR. Subsequently, the effect of cAMP on Cx37 expression in sheep COCs cultured in a gonadotropin-free culture system for 10 min or 60 min was evaluated using competitive ELISA, real-time fluorescent quantitative PCR (RT-qPCR), and Western blot. The results showed that the Cx37 protein was present in sheep oocytes and cumulus cells; the same results were found with respect to GJA4 gene expression. In the gonadotropin-free culture system, compared to the control, significantly higher levels of cAMP as well as Cx37 gene and protein expression were found in sheep COCs following treatment in vitro with Forskolin and IBMX (100 μM and 500 μM)) for 10 min (p < 0.05). Compared to the controls (at 10 or 60 min), cAMP levels in sheep COCs were significantly elevated as a result of Forskolin and IBMX treatment (p < 0.05). Following culturing in vitro for 10 min or 60 min, Forskolin and IBMX treatment can significantly promote Cx37 expression in sheep COCs (p < 0.05), a phenomenon which can be counteracted when the culture media is supplemented with RP-cAMP, a cAMP-specific competitive inhibitor operating through suppression of the protein kinase A (PKA). In summary, this study reports the preliminary regulatory mechanism of cAMP involved in Cx37 expression for the first time, and provides a novel explanation for the interaction between cAMP and GJC communication during sheep COC culturing in vitro. Full article

Developmental biology is intricately regulated by epigenetics and metabolism but the mechanisms are not completely understood. The situation becomes even more complicated during diseases where all three phenomena are dysregulated. A salient example is COVID-19, where the death toll exceeded 6.96 million in 4 years, while the virus continues to mutate into different variants and infect people. Early evidence during the pandemic showed that the host’s immune and inflammatory responses to COVID-19 (like the cytokine storm) impacted the host’s metabolism, causing damage to the host’s organs and overall physiology. The involvement of angiotensin-converting enzyme 2 (ACE2), the pivotal host receptor for the SARS-CoV-2 virus, was identified and linked to epigenetic abnormalities along with other contributing factors. Recently, studies have revealed stronger connections between epigenetics and metabolism in COVID-19 that impact development and accelerate aging. Patients manifest systemic toxicity, immune dysfunction and multi-organ failure. Single-cell multiomics and other state-of-the-art high-throughput studies are only just beginning to demonstrate the extent of dysregulation and damage. As epigenetics and metabolism directly impact development, there is a crucial need for research implementing cutting-edge technology, next-generation sequencing, bioinformatics analysis, the identification of biomarkers and clinical trials to help with prevention and therapeutic interventions against similar threats in the future. Full article

The present brief manuscript summarizes the main points supporting recently proposed hypotheses explaining the different distributions of regenerative capacity among invertebrates and vertebrates. The new hypotheses are based on the evolution of regeneration from marine animals to the terrestrial animals derived from them. These speculations suggest that animals that were initially capable of broad regeneration in the sea underwent epigenetic modifications during terrestrial adaptation that determined the loss of their regenerative abilities in sub-aerial conditions. These changes derived from the requirements of life on land that include variable dry and UV-exposed conditions. Terrestrial conditions do not allow for organ regeneration, especially in arthropods and amniotes. Nematodes, the other main metazoan group unable of regeneration, instead evolved eutely (a fixed number of body cells), a process which is incompatible with regeneration. All these changes involved gene loss, modification and new gene interactions within the genomes of terrestrial adapting animals that gave rise to sophisticated invertebrates and vertebrates adapted to living on land but with low cellular plasticity. Full article

Hyperplastic dental follicles (HDFs) represent odontogenic hamartomatous lesions originating from the pericoronal tissues and are often associated with impacted or embedded teeth. These lesions may occasionally feature unique calcifying bodies, known as calcifying whorled nodules (CWNs), characterized by stromal cells arranged in a whorled or spiral fashion. CWNs are typically observed in multiple calcifying hyperplastic dental follicles or regional odontodysplasia. In our study, we examined 40 cases of HDFs, including nine instances with characteristics of CWNs, referred to as calcifying hyperplastic dental follicles (CHDFs), which are infrequently accompanied by odontodysplasia. The median ages of the HDFs and CHDFs were 16 (ranging from 3 to 66) and 15 (ranging from 11 to 50) years, respectively. The lower third molars were the most frequently affected by HDSFs and CHDFs, followed by the upper canines. A histological examination was conducted on all 40 cases, with an immunohistochemical analysis performed on 21 of them. Among the cases with CWN, nine affected a single embedded tooth, with one exception. CWNs exhibited diverse calcifications featuring sparse or entirely deposited psammoma bodies, and some displayed dentinoid formation. Immunohistochemically, the stromal cells of HDFs were frequently positive for CD56 and nestin. By contrast, CWNs were negative for CD56 but positive for nestin as well as hairy and enhancer split 1 (HES1), with a few dentin sialoprotein (DSP)-positive calcified bodies. Our results revealed that hamartomatous CHDFs can impact multiple and single-embedded teeth. CWNs composed of nestin and HES1-positive ectomesenchymal cells demonstrated the potential to differentiate into odontoblasts and contribute to dentin matrix formation under the influence of HES1. This study is the first report documenting odontoblastic differentiation in HDFs. The rare occurrence of HDFs and CHDFs contributes to limited comprehension. To prevent misdiagnosis, a better understanding of these conditions is necessary. Full article

A characteristic feature of repair processes in mammals is the formation of scar tissue at the site of injury, which is designed to quickly prevent contact between the internal environment of the organism and the external environment. Despite this general pattern, different organs differ in the degree of severity of scar changes in response to injury. One of the areas in which regeneration after wounding leads to the formation of a structure close to the original one is the abdominal skin of laboratory rats. Finding out the reasons for such a phenomenon is essential for the development of ways to stimulate full regeneration. The model of skin wound healing in the abdominal region of laboratory animals was reproduced in this work. It was found that the wound surface is completely epithelialized on the abdomen by 20 days, while on the back—by 30 days. The qPCR method revealed higher expression of marker genes of skin stem cells (Sox9, Lgr6, Gli1, Lrig1) in the intact skin of the abdomen compared to the back, which corresponded to a greater number of hairs with which stem cells are associated on the abdomen compared to the back. Considering that some stem cell populations are associated with hair, it can be suggested that one of the factors in faster regeneration of abdominal skin in the rat is the greater number of stem cells in this area. Full article

Hox genes encode transcription factors whose roles in patterning animal body plans during embryonic development are well-documented. Multiple studies demonstrate that Hox genes continue to act in adult cells, in normal differentiation, in regenerative processes, and, with abnormal expression, in diverse types of cancers. However, surprisingly little is known about the regulatory mechanisms that govern Hox gene expression in specific cell types, as they differentiate during late embryonic development, and in the adult organism. The murine Hoxc8 gene determines the identity of multiple skeletal elements in the lower thoracic and lumbar region and continues to play a role in the proliferation and differentiation of cells in cartilage as the skeleton matures. This study was undertaken to identify regulatory elements in the Hoxc8 gene that control transcriptional activity, specifically in cartilage-producing chondrocytes. We report that an enhancer comprising two 416 and 224 bps long interacting DNA elements produces reporter gene activity when assayed on a heterologous transcriptional promoter in transgenic mice. This enhancer is distinct in spatial, temporal, and molecular regulation from previously identified regulatory sequences in the Hoxc8 gene that control its expression in early development. The identification of a tissue-specific Hox gene regulatory element now allows mechanistic investigations into Hox transcription factor expression and function in differentiating cell types and adult tissues and to specifically target these cells during repair processes and regeneration. Full article

The epidermal differentiation complex (EDC) is a cluster of genes that encode protein components of the outermost layers of the epidermis in mammals, reptiles and birds. The development of the stratified epidermis from a single-layered ectoderm involves an embryo-specific superficial cell layer, the periderm. An additional layer, the subperiderm, develops in crocodilians and over scutate scales of birds. Here, we review the expression of EDC genes during embryonic development. Several EDC genes are expressed predominantly or exclusively in embryo-specific cell layers, whereas others are confined to the epidermal layers that are maintained in postnatal skin. The S100 fused-type proteins scaffoldin and trichohyalin are expressed in the avian and mammalian periderm, respectively. Scaffoldin forms the so-called periderm granules, which are histological markers of the periderm in birds. Epidermal differentiation cysteine-rich protein (EDCRP) and epidermal differentiation protein containing DPCC motifs (EDDM) are expressed in the avian subperiderm where they are supposed to undergo cross-linking via disulfide bonds. Furthermore, a histidine-rich epidermal differentiation protein and feather-type corneous beta-proteins, also known as beta-keratins, are expressed in the subperiderm. The accumulating evidence for roles of EDC genes in the development of the epidermis has implications on the evolutionary diversification of the skin in amniotes. Full article

It has been more than three decades since the discovery of multifunctional factors, the Non-POU-Domain-Containing Octamer-Binding Protein, NonO, and the Splicing Factor Proline- and Glutamine-Rich, SFPQ. Some of their functions, including their participation in transcriptional and posttranscriptional regulation as well as their contribution to paraspeckle subnuclear body organization, have been well documented. In this review, we focus on several other established roles of NonO and SFPQ, including their participation in the cell cycle, nonhomologous end-joining (NHEJ), homologous recombination (HR), telomere stability, childhood birth defects and cancer. In each of these contexts, the absence or malfunction of either or both NonO and SFPQ leads to either genome instability, tumor development or mental impairment. Full article

Infertility is a major problem in farm animals, which has a negative economic effect on farm industries. Infertility can be defined as the inability of animals to achieve a successful pregnancy. Early pregnancy is crucial to establish a successful pregnancy, and it is reported that 70–80% and 20–30% of total embryonic loss occur in cattle and pigs, respectively, during the first month of pregnancy. The advanced high-throughput proteomics techniques provide valuable tools for in-depth understanding of the implantation process in farm animals. In the present review, our goal was to compile, assess, and integrate the latest proteomic research on farm animals, specifically focused on female reproduction, which involves endometrial tissues, uterine fluids, oviductal fluids, and microRNAs. The series of studies has provided in-depth insights into the events of the implantation process by unfolding the molecular landscape of the uterine tract. The discussed data are related to pregnant vs. non-pregnant animals, pregnancy vs. oestrous cycle, different days of the early pregnancy phase, and animals with uterine infections affecting reproduction health. Some of the studies have utilized non-invasive methods and in vitro models to decipher the molecular events of embryo-maternal interaction. The proteomics data are valuable sources for discovering biomarkers for infertility in ruminants and new regulatory pathways governing embryo-uterine interaction, endometrium receptivity, and embryonic development. Here, we envisage that the identified protein signatures can serve as potential therapeutic targets and biomarkers to develop new therapeutics against pregnancy diseases. Full article

The COVID-19 pandemic generated interest in the medicinal applications of messenger RNA (mRNA). It is expected that mRNA will be applied, not only to vaccines, but also to regenerative medicine. The purity of mRNA is important for its medicinal applications. However, the current mRNA synthesis techniques exhibit problems, including the contamination of undesired 5′-uncapped mRNA and double-stranded RNA. Recently, our group developed a completely capped mRNA synthesis technology that contributes to the progress of mRNA research. The introduction of chemically modified nucleosides, such as N1-methylpseudouridine and 5-methylcytidine, has been reported by Karikó and Weissman, opening a path for the practical application of mRNA for vaccines and regenerative medicine. Yamanaka reported the production of induced pluripotent stem cells (iPSCs) by introducing four types of genes using a retrovirus vector. iPSCs are widely used for research on regenerative medicine and the preparation of disease models to screen new drug candidates. Among the Yamanaka factors, Klf4 and c-Myc are oncogenes, and there is a risk of tumor development if these are integrated into genomic DNA. Therefore, regenerative medicine using mRNA, which poses no risk of genome insertion, has attracted attention. In this review, the author summarizes techniques for synthesizing mRNA and its application in regenerative medicine. Full article

The aim of this study was to provide the first systematic description of human placental cytology appearances and to investigate syncytiotrophoblast nuclear organisation patterns using cytology techniques. Term placentas from normal pregnancies were sampled using fine-needle aspiration (FNA) and direct scrapes. Standard histological examination was also performed to exclude pathological changes in the placentas being studied. Both Papanicolaou-stained cytospin preparations and air-dried Giemsa slides from FNA provided high-quality material for cytological assessment with good cellularity. Among the key features of the cytology preparations were villous “microbiopsies” that allowed for the three-dimensional appreciation of villous branching patterns. Cytological appearances, including nuclear characteristics of villous cytotrophoblast and syncytiotrophoblast, were also well demonstrated. In microbiopsies and detached villous trophoblast sheets, complex patterns of syncytiotrophoblast nuclear organisation, not previously described cytologically, were observed, including irregular spacing of nuclei, syncytioplasm windows and linear nuclear arrangements. This study showed that placental cytology (a) provides technically excellent material for cytological evaluation, (b) confirms the presence of complex nuclear organisational patterns in the syncytiotrophoblast by eliminating the possibility of tangential sectioning artefact, (c) provides superior nuclear detail over standard histological sections and (d) may be an untapped research resource for the investigation of normal and pathological processes because of its ability to look at the placenta in a novel way and through its potential for both ex vivo and in vivo placental sampling. Full article

A properly organized subcellular composition is essential to cell function. The canonical organizing principle within eukaryotic cells involves membrane-bound organelles; yet, such structures do not fully explain cellular complexity. Furthermore, discrete non-membrane-bound structures have been known for over a century. Liquid–liquid phase separation (LLPS) has emerged as a ubiquitous mode of cellular organization without the need for formal lipid membranes, with an ever-expanding and diverse list of cellular functions that appear to be regulated by this process. In comparison to traditional organelles, LLPS can occur across wider spatial and temporal scales and involves more distinct protein and RNA complexes. In this review, we discuss the impacts of LLPS on the organization of stem cells and their function during development. Specifically, the roles of LLPS in developmental signaling pathways, chromatin organization, and gene expression will be detailed, as well as its impacts on essential processes of asymmetric cell division. We will also discuss how the dynamic and regulated nature of LLPS may afford stem cells an adaptable mode of organization throughout the developmental time to control cell fate. Finally, we will discuss how aberrant LLPS in these processes may contribute to developmental defects and disease. Full article

The karyosphere (karyosome) is a structure that forms in the oocyte nucleus—germinal vesicle (GV)—at the diplotene stage of meiotic prophase due to the assembly of all chromosomes in a limited portion of the GV. In some organisms, the karyosphere has an extrachromosomal external capsule, the marker protein of which is nuclear F-actin. Despite many years of theories about the formation of the karyosphere capsule (KC) in the GV of the common frog Rana temporaria, we present data that cast doubt on its existence, at least in this species. Specific extrachromosomal strands, which had been considered the main elements of the frog’s KC, do not form a continuous layer around the karyosphere and, according to immunogold labeling, do not contain structural proteins, such as actin and lamin B. At the same time, F-actin is indeed noticeably concentrated around the karyosphere, creating the illusion of a capsule at the light microscopy/fluorescence level. The barrier-to-autointegration factor (BAF) and one of its functional partners—LEMD2, an inner nuclear membrane protein—are not localized in the strands, suggesting that the strands are not functional counterparts of the nuclear envelope. The presence of characteristic strands in the GV of R. temporaria late oocytes may reflect an excess of SMC1 involved in the structural maintenance of diplotene oocyte chromosomes at the karyosphere stage, since SMC1 has been shown to be the most abundant protein in the strands. Other characteristic microstructures—the so-called annuli, very similar in ultrastructure to the nuclear pore complexes—do not contain nucleoporins Nup35 and Nup93, and, therefore, they cannot be considered autonomous pore complexes, as previously thought. Taken together, our data indicate that traditional ideas about the existence of the R. temporaria KC as a special structural compartment of the GV are to be revisited. Full article

Epidermal development is responsible for the formation of the outermost layer of the skin, the epidermis. The establishment of the epidermal barrier is a critical aspect of mammalian development. Proper formation of the epidermis, which is composed of stratified squamous epithelial cells, is essential for the survival of terrestrial vertebrates because it acts as a crucial protective barrier against external threats such as pathogens, toxins, and physical trauma. In mammals, epidermal development begins from the embryonic surface ectoderm, which gives rise to the basal layer of the epidermis. This layer undergoes a series of complex processes that lead to the formation of subsequent layers, including the stratum intermedium, stratum spinosum, stratum granulosum, and stratum corneum. The stratum corneum, which is the topmost layer of the epidermis, is formed by corneoptosis, a specialized form of cell death. This process involves the transformation of epidermal keratinocytes in the granular layer into flattened dead cells, which constitute the protective barrier. In this review, we focus on the intricate mechanisms that drive the development and establishment of the mammalian epidermis to gain insight into the complex processes that govern this vital biological system. Full article

This review focuses on SARS-CoV-2 infection in placental and fetal tissues. Viremia is rare in infected pregnant women, and the virus is seldom amplified from placental tissues. Definite and probable placental infection requires the demonstration of viral RNA or proteins using in situ hybridization (ISH) and immunohistochemistry (IHC). Small subsets (1.0–7.9%, median 2.8%) of placentas of SARS-CoV-2-positive women showed definite infection accompanied by a characteristic histopathology named SARS-CoV-2 placentitis (SP). The conventionally accepted histopathological criteria for SP include the triad of intervillositis, perivillous fibrin deposition, and trophoblast necrosis. SP was shown to be independent of the clinical severity of the infection, but associated with stillbirth in cases where destructive lesions affecting more than 75% of the placental tissue resulted in placental insufficiency and severe fetal hypoxic–ischemic injury. An association between maternal thrombophilia and SP was shown in a subset of cases, suggesting a synergy of the infection and deficient coagulation cascade as one of the mechanisms of the pathologic accumulation of fibrin in affected placentas. The virus was amplified from fetal tissues in approximately 40% of SP cases, but definite fetal involvement demonstrated using ISH or IHC is exceptionally rare. The placental pathology in SARS-CoV-2-positive women also includes chronic lesions associated with placental malperfusion in the absence of definite or probable placental infection. The direct viral causation of the vascular malperfusion of the placenta in COVID-19 is debatable, and common predispositions (hypertension, diabetes, and obesity) may play a role. Full article