Higher Vertebrates Research Articles

Methylmercury Toxicity During Heart Development: A Combined Analysis of Morphological and Functional Parameters.

The heart of higher vertebrates develops early as a tubular structure, which requires cellular and molecular events for proliferation, differentiation and apoptosis for growth, and individualization of cardiac chambers. Exposure to different stressors can cause disturbances in the normal development and functionality of the cardiovascular system. This study aimed to characterize the impact of methylmercury (MeHg) on heart development, specifically related to tissue morphology and parameters of vascular integrity and contractility, also focusing on cell cycle and apoptosis, using Gallus domesticus embryos as a model. The results showed morphological alterations, reduction in the thickness of the ventricular walls, and trabeculae changes in the hearts of embryos exposed to 0.1µg MeHg/50 µL saline solution. These impacts were associated with increased contents of proteins related to cell cycle arrest and reduced cardiomyocyte proliferation. In addition, the contents of endothelial mediators for contractility and vascular integrity were imbalanced. The quantity and morphology of mitochondria of cardiomyocytes were injured. Together, these negative measurements impacted the reduction of heartbeats. In general, the parameters identified here demonstrate the relevance of combined molecular cellular tissue and physiological diagnosis for a better understanding of the cardiotoxicity of MeHg duringdevelopment.

Effect of novel therapeutic medicine swertiamarin from Enicostema axillare in zebrafish infected with Salmonella typhi.

Herbal treatments have been practiced by humans over centuries and therefore possess time-proven safety. However, it is crucial to evaluate the toxic effects of herbal medicine to confirm their safety, particularly when developing therapeutic drugs. Use of laboratory animals such as mice, rat, and rabbits was considered as gold standard in herbal toxicity assessments. However, in the last few decades, the ethical consideration of using higher vertebrates for toxicity testing has become more controversial. As a possible alternative model involving lower vertebrates such as zebra fish were introduced. Hence in the present study, swertiamain compound isolated from E. axillare was assessed for it antimicrobial activity in zebra fish larvae againt S. typhi. The cumulative mortality rate and bacterial localization in zebra fish larvae were studied. Biochemical markers assays were performed to find the preventive role of the compound during the typhoid infection. The results showed that zebra fish can be successfully used as a model to study typhoid infection and the anti-bacterial compound swertiamarin used in this study clears the bacterial load and pathogenic symptoms to a great extent.

MLL regulates the actin cytoskeleton and cell migration by stabilising Rho GTPases via the expression of RhoGDI1.

Attainment of proper cell shape and the regulation of cell migration are essential processes in the development of an organism. The mixed lineage leukemia (MLL or KMT2A) protein, a histone 3 lysine 4 (H3K4) methyltransferase, plays a critical role in cell-fate decisions during skeletal development and haematopoiesis in higher vertebrates. Rho GTPases - RhoA, Rac1 and CDC42 - are small G proteins that regulate various key cellular processes, such as actin cytoskeleton formation, the maintenance of cell shape and cell migration. Here, we report that MLL regulates the homeostasis of these small Rho GTPases. Loss of MLL resulted in an abnormal cell shape and a disrupted actin cytoskeleton, which lead to diminished cell spreading and migration. MLL depletion affected the stability and activity of Rho GTPases in a SET domain-dependent manner, but these Rho GTPases were not direct transcriptional targets of MLL. Instead, MLL regulated the transcript levels of their chaperone protein RhoGDI1 (also known as ARHGDIA). Using MDA-MB-231, a triple-negative breast cancer cell line with high RhoGDI1 expression, we show that MLL depletion or inhibition by small molecules reduces tumour progression in nude mice. Our studies highlight the central regulatory role of MLL in Rho/Rac/CDC42 signalling pathways. This article has an associated First Person interview with the first author of the paper.

Evolution of surrogate light chain in tetrapods and the relationship between lengths of CDR H3 and VpreB tails.

In the mammalian immune system, the surrogate light chain (SLC) shapes the antibody repertoire during B cell development by serving as a checkpoint for production of functional heavy chains (HC). Structural studies indicate that tail regions of VpreB contact and cover the third complementarity-determining region of the HC (CDR H3). However, some species, particularly bovines, have CDR H3 regions that may not be compatible with this HC-SLC interaction model. With immense structural and genetic diversity in antibody repertoires across species, we evaluated the genetic origins and sequence features of surrogate light chain components. We examined tetrapod genomes for evidence of conserved gene synteny to determine the evolutionary origin of VpreB1, VpreB2, and IGLL1, as well as VpreB3 and pre-T cell receptor alpha (PTCRA) genes. We found the genes for the SLC components (VpreB1, VpreB2, and IGLL1) only in eutherian mammals. However, genes for PTCRA occurred in all amniote groups and genes for VpreB3 occurred in all tetrapod groups, and these genes were highly conserved. Additionally, we found evidence of a new VpreB gene in non-mammalian tetrapods that is similar to the VpreB2 gene of eutherian mammals, suggesting VpreB2 may have appeared earlier in tetrapod evolution and may be a precursor to traditional VpreB2 genes in higher vertebrates. Among eutherian mammals, sequence conservation between VpreB1 and VpreB2 was low for all groups except rabbits and rodents, where VpreB2 was nearly identical to VpreB1 and did not share conserved synteny with VpreB2 of other species. VpreB2 of rabbits and rodents likely represents a duplicated variant of VpreB1 and is distinct from the VpreB2 of other mammals. Thus, rabbits and rodents have two variants of VpreB1 (VpreB1-1 and VpreB1-2) but no VpreB2. Sequence analysis of VpreB tail regions indicated differences in sequence content, charge, and length; where repertoire data was available, we observed a significant relationship between VpreB2 tail length and maximum DH length. We posit that SLC components co-evolved with immunoglobulin HC to accommodate the repertoire – particularly CDR H3 length and structure, and perhaps highly unusual HC (like ultralong HC of cattle) may bypass this developmental checkpoint altogether.

Domain Evolution of Vertebrate Blood Coagulation Cascade Proteins

Vertebrate blood coagulation is controlled by a cascade containing more than 20 proteins. The cascade proteins are found in the blood in their zymogen forms and when the cascade is triggered by tissue damage, zymogens are activated and in turn activate their downstream proteins by serine protease activity. In this study, we examined proteomes of 21 chordates, of which 18 are vertebrates, to reveal the modular evolution of the blood coagulation cascade. Additionally, two Arthropoda species were used to compare domain arrangements of the proteins belonging to the hemolymph clotting and the blood coagulation cascades. Within the vertebrate coagulation protein set, almost half of the studied proteins are shared with jawless vertebrates. Domain similarity analyses revealed that there are multiple possible evolutionary trajectories for each coagulation protein. During the evolution of higher vertebrate clades, gene and genome duplications led to the formation of other coagulation cascade proteins.

C-MAF coordinates enterocyte zonation and nutrient uptake transcriptional programs.

Small intestinal villi are structural and functional units present in higher vertebrates and uniquely adapted to nutrient absorption. Villus enterocytes are organized in transcriptional "zones" dedicated to specialized tasks such as absorption of specific nutrients. We report that the transcription factor c-MAF is expressed in differentiated lower and mid-villus enterocytes and is a target of BMP signaling. Maf inactivation perturbed the villus zonation program by increasing carbohydrate-related transcripts while suppressing transcripts linked to amino-acid and lipid absorption. The formation of cytoplasmic lipid droplets, shuttling dietary fat to chylomicrons, was impaired upon Maf loss indicating its role in dietary lipid handling. Maf inactivation under homeostatic conditions expanded tuft cells and led to compensatory gut lengthening, preventing weight loss. However, delayed Maf-/- enterocyte maturation impaired weight recovery after acute intestinal injury, resulting in reduced survival. Our results identify c-MAF as a regulator of the intestinal villus zonation program, while highlighting the importance of coordination between stem/progenitor and differentiation programs for intestinal regeneration.

Potential role of human papillomavirus proteins associated with the development of cancer.

Papillomaviruses are viruses with double-stranded DNA that are epitheliotropic and non-enveloped that infects cutaneous epithelial and mucosal cells in a species-specific way in several higher vertebrate species and cause cellular growth."There are around 100 different human papillomaviruses (HPVs)", as "more than 150 HPV types have been isolated and fully sequenced". We classify the probability of cancer development following viral infection with each HPV genotype into two types: "low-risk" and "high-risk." As a result, HPV diagnosis is a critical component of HPV genotype identification and characterization. Based on its activities, we may classify the HPV genome into three regions: the long control region (LCR) or the non-coding upstream regulatory region (URR), the late (L) region, and the early (E) region. Functional proteins are mostly static things that are not inflexible; they have undergone both local and global movements at various times and time ranges. The structural differences between HPV16 and 18 discovered by molecular modeling of the E6 oncoprotein were associated with their carcinogenic characteristics. Similarly, the E6 protein has two sets of C-X-X-C motifs that play significant roles in transformation, transcriptional activation, interactions, and immortalization with other proteins of cells in the host environment. Here, we review the literature regarding the protein mechanisms associated with HPV and how they cause cancer. Unless otherwise noted, it described all protein activities in terms of HPV proteins. The term "papillomaviruses" refers to groups of papillomavirus proteins that have a characteristic in common. HPV proteins can study the genetic influences on pathogenicity and the therapeutic applications of genomics. The future study provides a potential advancement in HPV infections and malignant illnesses to improve preventive and treatment strategies. Patients have been able to conquer this condition using a range of therapies and vaccines that were projected to be effective and robust enough to put an end to the ailment completely. In cancer prevention strategies, HPV vaccination is one of the most effective. It is safe, efficient, and long-lasting.

Unique Features of River Lamprey (Lampetra fluviatilis) Myogenesis.

The river lamprey (L. fluviatilis) is a representative of the ancestral jawless vertebrate group. We performed a histological analysis of trunk muscle fiber differentiation during embryonal, larval, and adult musculature development in this previously unstudied species. Investigation using light, transmission electron (TEM), and confocal microscopy revealed that embryonal and larval musculature differs from adult muscle mass. Here, we present the morphological analysis of L. fluviatilis myogenesis, from unsegmented mesoderm through somite formation, and their differentiation into multinucleated muscle lamellae. Our analysis also revealed the presence of myogenic factors LfPax3/7 and Myf5 in the dermomyotome. In the next stages of development, two types of muscle lamellae can be distinguished: central surrounded by parietal. This pattern is maintained until adulthood, when parietal muscle fibers surround the central muscles on both sides. The two types show different morphological characteristics. Although lampreys are phylogenetically distant from jawed vertebrates, somite morphology, especially dermomyotome function, shows similarity. Here we demonstrate that somitogenesis is a conservative process among all vertebrates. We conclude that river lamprey myogenesis shares features with both ancestral and higher vertebrates.

Local mRNA translation and cytoskeletal reorganization: Mechanisms that tune neuronal responses.

Neurons are highly polarized cells with significantly long axonal and dendritic extensions that can reach distances up to hundreds of centimeters away from the cell bodies in higher vertebrates. Their successful formation, maintenance, and proper function highly depend on the coordination of intricate molecular networks that allow axons and dendrites to quickly process information, and respond to a continuous and diverse cascade of environmental stimuli, often without enough time for communication with the soma. Two seemingly unrelated processes, essential for these rapid responses, and thus neuronal homeostasis and plasticity, are local mRNA translation and cytoskeletal reorganization. The axonal cytoskeleton is characterized by high stability and great plasticity; two contradictory attributes that emerge from the powerful cytoskeletal rearrangement dynamics. Cytoskeletal reorganization is crucial during nervous system development and in adulthood, ensuring the establishment of proper neuronal shape and polarity, as well as regulating intracellular transport and synaptic functions. Local mRNA translation is another mechanism with a well-established role in the developing and adult nervous system. It is pivotal for axonal guidance and arborization, synaptic formation, and function and seems to be a key player in processes activated after neuronal damage. Perturbations in the regulatory pathways of local translation and cytoskeletal reorganization contribute to various pathologies with diverse clinical manifestations, ranging from intellectual disabilities (ID) to autism spectrum disorders (ASD) and schizophrenia (SCZ). Despite the fact that both processes are essential for the orchestration of pathways critical for proper axonal and dendritic function, the interplay between them remains elusive. Here we review our current knowledge on the molecular mechanisms and specific interaction networks that regulate and potentially coordinate these interconnected processes.

Salvia miltiorrhiza polysaccharide activated macrophages and improved the disease resistance of sturgeon against Aeromonas hydrophila

The use of plant polysaccharides in aquaculture is recognized as a healthy strategy to enhance disease resistance and reduce medication use. Salvia miltiorrhiza polysaccharide (SMP) can regulate the immune function of higher vertebrates. However, the effects of SMP on fish have not been fully investigated. In this study, the ability of SMP to activate the macrophages of Siberian sturgeon (Acipenser bareii) was analyzed in vitro. The effects of SMP on immune cell activity of hybrid sturgeon (A. baerii ♀ × Acipenser schrenckii ♂) and resistance to Aeromonas hydrophila were further detected in vivo. The in vitro results showed that SMP up-regulated phagocytosis, respiratory burst, inducible nitric oxide synthase activity, nitric oxide (NO) concentration, and cytokine mRNA expression of macrophages. The in vivo results showed that dietary supplementation with SMP enhanced the respiratory burst of macrophages and proliferative activity of lymphocytes. Dietary supplementation with SMP increased serum concentrations of lysozyme and NO, and improved the survival rate of hybrid sturgeon challenged with A. hydrophila. Collectively, these results suggest that SMP can improve the immune function and disease resistance of sturgeon. This study provides a theoretical basis for the application of SMP for healthy farming of sturgeon.

Pyroptosis in host defence against bacterial infection

Pyroptosis, a regulated form of pro-inflammatory cell death, is characterised by cell lysis and by the release of cytokines, damage- and pathogen-associated molecular patterns. It plays an important role during bacterial infection, where it can promote an inflammatory response and eliminate the replicative niche of intracellular pathogens. Recent work, using a variety of bacterial pathogens, has illuminated the versatility of pyroptosis, revealing unexpected and important concepts underlying host defence. In this Review, we overview the molecular mechanisms underlying pyroptosis and discuss their role in host defence, from the single cell to the whole organism. We focus on recent studies using three cellular microbiology paradigms - Mycobacterium tuberculosis, Salmonella Typhimurium and Shigella flexneri - that have transformed the field of pyroptosis. We compare insights discovered in tissue culture, zebrafish and mouse models, highlighting the advantages and disadvantages of using these complementary infection models to investigate pyroptosis and for modelling human infection. Moving forward, we propose that in-depth knowledge of pyroptosis obtained from complementary infection models can better inform future studies using higher vertebrates, including humans, and help develop innovative host-directed therapies to combat bacterial infection.

Development of ribs and intercostal muscles in the chicken embryo.

In the thorax of higher vertebrates, ribs and intercostal muscles play a decisive role in stability and respiratory movements of the body wall. They are derivatives of the somites, the ribs originating in the sclerotome and the intercostal muscles originating in the myotome. During thorax development, ribs and intercostal muscles extend into the lateral plate mesoderm and eventually contact the sternum during ventral closure. Here, we give a detailed description of the morphogenesis of ribs and thoracic muscles in the chicken embryo (Gallus gallus). Using Alcian blue staining as well as Sox9 and Desmin whole‐mount immunohistochemistry, we monitor synchronously the development of rib cartilage and intercostal muscle anlagen. We show that the muscle anlagen precede the rib anlagen during ventrolateral extension, which is in line with the inductive role of the myotome in rib differentiation. Our studies furthermore reveal the temporary formation of a previously unknown eighth rib in the chicken embryonic thorax.

Imaging Intron Evolution

Intron evolution may be readily imaged through the combined use of the “dot plot” function of the NCBI BLAST, aligning two sequences at a time, and the Vertebrate “Multiz” alignment and conservation tool of the UCSC Genome Browser. With the NCBI BLAST, an ideal alignment of two highly conserved sequences generates a diagonal straight line in the plot from the lower left corner to the upper right corner. Gaps in this line correspond to non-conserved sections. In addition, the dot plot of the alignment of a sequence with the same sequence after the removal of the Transposable Elements (TEs) can be observed along the diagonal gaps that correspond to the sites of TE insertion. The UCSC Genome Browser can graph, along the entire sequence of a single gene, the level of overall conservation in vertebrates. This level can be compared with the conservation level of the gene in one or more selected vertebrate species. As an example, we show the graphic analysis of the intron conservation in two genes: the mitochondrial solute carrier 21 (SLC25A21) and the growth hormone receptor (GHR), whose coding sequences are conserved through vertebrates, while their introns show dramatic changes in nucleotide composition and even length. In the SLC25A21, a few short but significant nucleotide sequences are conserved in zebrafish, Xenopus and humans, and the rate of conservation steadily increases from chicken/human to mouse/human alignments. In the GHR, a less conserved gene, the earlier indication of intron conservation is a small signal in chicken/human alignment. The UCSC tool may simultaneously display the conservation level of a gene in different vertebrates, with reference to the level of overall conservation in Vertebrates. It is shown that, at least in SLC25A21, the sites of higher conservation are not always coincident in chicken and zebrafish nor are the sites of higher vertebrate conservation.

Immune Modulation Ability of Hepcidin from Teleost Fish.

Simple SummaryAntimicrobial peptides are part of the fish defense system, which can directly eliminate pathogenic microorganisms and, at the same time, regulate the immune response against them. This study evaluated the immunomodulatory effects of the antimicrobial peptide hepcidin in both juvenile fish and fish leukocyte cells. The results showed that hepcidin increased the expression of TNF-α, IL-1β, and IL-10 cytokines in leukocyte cells from trout. Moreover, the mRNA expressions of the same cytokines were up-regulated in different immune tissue of sea bass, confirming in vitro results. This study provides new insights into immunomodulatory function complementary to hepcidin’s previously established antimicrobial activity modulating the pro- and anti-inflammatory responses in teleost fish.Antimicrobial peptides (AMP) play an essential role in the innate immune system, modulating the defense response. In a previous study, we demonstrated the antimicrobial activity of synthetic hepcidin (hep20) from rainbow trout (Oncorhynchus mykiss), and its protective effect in European sea bass (Dicentrarchus labrax) challenged with Vibrio anguillarum. Additionally, we described the uptake and distribution of hep20 in different tissues and leukocyte cells. Interestingly, various AMPs characterized in high vertebrates, called host defense peptides (HDPs), also possess immunomodulation activity. For that reason, the present study explores the immunomodulatory abilities of hep20 through in vitro and in vivo studies. First, a monocyte/macrophage RTS-11 cell line from rainbow trout was used to evaluate hep20 effects on pro- and anti-inflammatory cytokines in fish leukocyte cells. Next, the European sea bass juveniles were used to determine if hep20 can regulate the expression of cytokines in fish immune tissues. The results show that hep20 was uptake inner to RTS-11 cells and was able to induce the expression of IL-10, IL-1β, and TNFα at transcriptional and protein levels. Then, the European sea bass juveniles were given intraperitoneal injections of the peptide. At 1, 3, 7, 14, and 21 days post-injection (dpi), IL-10, IL -1β, and TNFα mRNA were quantified in the anterior gut, spleen, and head kidney. The hep20 was able to up-regulate cytokine gene expression in these tissues, mainly in the head kidney. Furthermore, the evaluated cytokines showed a cyclical tendency of higher to lesser expression. Finally, a bioinformatics analysis showed that the structure adopted by hep20 is similar to the γ-core domain described for cysteine-stabilized AMP, defined as immunomodulatory and antimicrobial, which could explain the ability of hep20 to regulate the cytokine expression. This study provides new insights into immunomodulatory function complementary to the previously established antimicrobial activity of hep20, suggesting a role as an HDP in teleost fish. These facts are likely to be associated with molecular functions underpinning the protective effect of fish hepcidin against pathogens.

Cytokine Receptor-Like Factor 3 (CRLF3) Contributes to Early Zebrafish Hematopoiesis.

Cytokine receptor-like factor 3 (CRLF3) is an ancient protein conserved across metazoans that contains an archetypal cytokine receptor homology domain (CHD). This domain is found in cytokine receptors present in bilateria, including higher vertebrates, that play key roles in a variety of developmental and homeostatic processes, particularly relating to blood and immune cells. However, understanding of CRLF3 itself remains very limited. This study aimed to investigate this evolutionarily significant protein by studying its embryonic expression and function in early development, particularly of blood and immune cells, using zebrafish as a model. Expression of crlf3 was identified in mesoderm-derived tissues in early zebrafish embryos, including the somitic mesoderm and both anterior and posterior lateral plate mesoderm. Later expression was observed in the thymus, brain, retina and exocrine pancreas. Zebrafish crlf3 mutants generated by genome editing technology exhibited a significant reduction in primitive hematopoiesis and early definitive hematopoiesis, with decreased early progenitors impacting on multiple lineages. No other obvious phenotypes were observed in the crlf3 mutants.

Zebrafish Larvae Behavior Models as a Tool for Drug Screenings and Pre-Clinical Trials: A Review.

To discover new molecules or review the biological activity and toxicity of therapeutic substances, drug development, and research relies on robust biological systems to obtain reliable results. Phenotype-based screenings can transpose the organism’s compensatory pathways by adopting multi-target strategies for treating complex diseases, and zebrafish emerged as an important model for biomedical research and drug screenings. Zebrafish’s clear correlation between neuro-anatomical and physiological features and behavior is very similar to that verified in mammals, enabling the construction of reliable and relevant experimental models for neurological disorders research. Zebrafish presents highly conserved physiological pathways that are found in higher vertebrates, including mammals, along with a robust behavioral repertoire. Moreover, it is very sensitive to pharmacological/environmental manipulations, and these behavioral phenotypes are detected in both larvae and adults. These advantages align with the 3Rs concept and qualify the zebrafish as a powerful tool for drug screenings and pre-clinical trials. This review highlights important behavioral domains studied in zebrafish larvae and their neurotransmitter systems and summarizes currently used techniques to evaluate and quantify zebrafish larvae behavior in laboratory studies.

Possible Event-Related Potential Correlates of Voluntary Attention and Reflexive Attention in the Emei Music Frog.

Simple SummaryWe investigated auditory event-related potentials (ERP) related to auditory attention in music frogs. Our objective was to explore whether ERP components related to voluntary attention and reflexive attention exist in frogs. We found that the amplitudes of stimulus preceding negativity (SPN, related to voluntary attention and under up-down control) evoked by silence replacement in the telencephalon were the largest when the sequence of acoustic stimuli could be predicted, while the N1 amplitudes (related to reflexive attention and under bottom-up control) evoked in the mesencephalon were the largest when the sequence of acoustic stimuli could not be predicted. This suggests that human-like ERP components related to voluntary attention and reflexive attention exist in the lower vertebrates also.Attention, referring to selective processing of task-related information, is central to cognition. It has been proposed that voluntary attention (driven by current goals or tasks and under top-down control) and reflexive attention (driven by stimulus salience and under bottom-up control) struggle to control the focus of attention with interaction in a push–pull fashion for everyday perception in higher vertebrates. However, how auditory attention engages in auditory perception in lower vertebrates remains unclear. In this study, each component of auditory event-related potentials (ERP) related to attention was measured for the telencephalon, diencephalon and mesencephalon in the Emei music frog (Nidirana daunchina), during the broadcasting of acoustic stimuli invoking voluntary attention (using binary playback paradigm with silence replacement) and reflexive attention (using equiprobably random playback paradigm), respectively. Results showed that (1) when the sequence of acoustic stimuli could be predicted, the amplitudes of stimulus preceding negativity (SPN) evoked by silence replacement in the forebrain were significantly greater than that in the mesencephalon, suggesting voluntary attention may engage in auditory perception in this species because of the correlation between the SPN component and top-down control such as expectation and/or prediction; (2) alternately, when the sequence of acoustic stimuli could not be predicted, the N1 amplitudes evoked in the mesencephalon were significantly greater than those in other brain areas, implying that reflexive attention may be involved in auditory signal processing because the N1 components relate to selective attention; and (3) both SPN and N1 components could be evoked by the predicted stimuli, suggesting auditory perception of the music frogs might invoke the two kind of attention resources simultaneously. The present results show that human-like ERP components related to voluntary attention and reflexive attention exist in the lower vertebrates also.

First ovum-in-ovo pathological titanosaurid egg throws light on the reproductive biology of sauropod dinosaurs

Pathologic eggs have been documented in the amniote eggs of birds, turtles, and dinosaurs. These eggs occur either in the form of one egg within another egg, a condition known as ovum-in-ovo or multi-shelled eggs showing additional pathological eggshell layer/s besides the primary shell layer. Though multi-shelled eggs and eggshells were previously recorded only in reptiles and ovum-in-ovo eggs in birds, now it has been shown that multi-shelled egg pathology occurs in birds as well. However, no ovum-in-ovo egg has been reported in dinosaurs or for that matter in other reptiles. Here we describe an ovum-in-ovo pathological egg from a titanosaurid dinosaur nest from the Upper Cretaceous Lameta Formation of western Central India which makes it the first report of this pathology in dinosaurs. Birds possess a specialized uterus while other amniotes have a generalized uterus. However, alligators and crocodiles retain a specialized uterus like birds along with a reptilian mode of egg-laying. The discovery of ovum-in-ovo egg from a titanosaurid dinosaur nest suggests that their oviduct morphology was similar to that of birds opening up the possibility for sequential laying of eggs in this group of sauropod dinosaurs. This new find underscores that the ovum-in-ovo pathology is not unique to birds and sauropods share a reproductive behavior very similar to that of other archosaurs.

Regulation of Stanniocalcin Secretion by Calcium and PTHrP in Gilthead Seabream (Sparus aurata).

Simple SummaryCalcium regulation in body fluids is a fundamental process in vertebrates, which is exerted by a plethora of hormones. Stanniocalcin (STC) is a hypocalcemic hormone ubiquitously expressed in tetrapods; in bony fishes, it is produced mainly by specific glands called the corpuscles of Stannius. The present study described an ELISA method for the analysis of fish STC. Moreover, it also develops a methodology for ex vivo cultures of Stannius corpuscles of gilthead seabream (Sparus aurata). The results show a direct control of the production of STC by one calcium and two PTHrP (parathyroid-related protein, a hypercalcemic hormone) receptors. This study highlights the tight control of circulating calcium in vertebrates and shows the complexity of the processes involved.Calcium balance is of paramount importance for vertebrates. In fish, the endocrine modulators of calcium homeostasis include the stanniocalcin (STC), and some members of the parathyroid hormone (PTH) family, such as the PTH-related protein (PTHrP), acting as antagonists. STC is ubiquitously expressed in higher vertebrates. In turn, bony fish exhibit specific STC-producing glands named the corpuscles of Stannius (CS). Previous studies pointed to a calcium-sensing receptor (CaSR) involvement in the secretion of STC, but little is known of the involvement of other putative regulators. The CS provides a unique model to deepen the study of STC secretion. We developed an ex vivo assay to culture CS of fish and a competitive ELISA method to measure STC concentrations. As expected, STC released from the CS responds to CaSR stimulation by calcium, calcimimetics, and calcilytic drugs. Moreover, we uncover the presence (by PCR) of two PTHrP receptors in the CS, e.g., PTH1R and PTH3R. Thus, ex vivo incubations revealed a dose-response inhibition of STC secretion in response to PTHrP at basal Ca2+ concentrations. This inhibition is achieved through specific and reversible second messenger pathways (transmembrane adenylyl cyclases and phospholipase C), as the use of specific inhibitors highlights. Together, these results provide evidence for endocrine modulation between two antagonist hormones, STC and PTHrP.

Structural and Functional Aspects of the Spleen in Molly Fish Poecilia sphenops (Valenciennes, 1846): Synergistic Interactions of Stem Cells, Neurons, and Immune Cells.

Simple SummaryFish are free-living organisms who are dependent on their innate immune system for survival. This system is fundamental for the defense mechanisms against invading pathogens. The head kidney, spleen, and thymus are the main immune organs in fish, albeit with slightly different roles between species. This study aims to highlight the cellular components of the spleen in the molly fish (Poecilia sphenops) by providing some background on their potential role in the immune system of fish. The splenic parenchyma of molly fish is comprised mainly of red pulp intermingled with clusters of white pulp cells. The main cells identified in this study included: erythrocytes, neurons, dendritic cells, stem cells, epithelial reticular cells, lymphocytes, ellipsoids, and granular leukocytes (eosinophils, basophils, neutrophils), and macrophages. The current study demonstrated a variety of immune cell types with specific functions in the spleen of molly fish. The strong positive immunoreactivity of the cellular constituents of the spleen for APG-5, IL-1β, NF-κB, and TGF-β suggests its critical role in immunity.In fish, the spleen is the prime secondary lymphoid organ. It has a role in the induction of adaptive immune responses, in addition to its significance in the elimination of immune complexes. This study was conducted on 18 randomly obtained adult molly fish (Poecilia sphenops) of both sexes using histological, immunohistochemical, and ultrastructural studies to highlight the cellular components of the spleen and their potential role in the immune system. The spleen of molly fish was characterized by the presence of well-distinct melanomacrophage centers, and other basic structures present in higher vertebrates including red and white pulps, blood vessels, and ellipsoids. Some mitotic cells could also be identified in the red pulp. Mast cells with characteristic metachromatic granules could be seen among the splenic cells. Rodlet cells were randomly distributed in the spleen and were also observed around the ellipsoids. The white pulp of the spleen expressed APG5. The expressions were well distinct in the melanomacrophages, leukocytes, and macrophages. Myostatin was expressed in leukocytes and epithelial reticular cells. IL-1β showed immunoreactivity in monocytes and macrophages around the ellipsoids. NF-κB and TGF-β were expressed in macrophages and epithelial reticular cells. Nrf2 expression was detected in stem cells and rodlet cells. Sox-9 had a higher expression in epithelial reticular cells and stem cells. The high frequency of immune cells in the spleen confirmed its role in the regulation of both innate and adaptive immunity, cell proliferation, and apoptosis.