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The study of hybridogenesis is expected to shed light on the role played by the so-called 'asexual reproduction' in vertebrate evolution, since hybridogenesis, gynogenesis and parthenogenesis are restricted to lower vertebrates. Hence, it seemed interesting to define the stage and time of germline differentiation in reproductively unstable organisms which, to accomplish functional gamete maturation, can change the normal approach to meiosis by means of unusual cytological steps, such as genome elimination and endoreduplication. The cytogenetic aspects of 'asexual' meiosis have already been thoroughly studied. At present, molecular investigations are in progress to characterise the single steps of germline differentiation in a hybridogenetic green frog system, and the results obtained so far can be considered promising, as shown in this work. Vasa, PL10 and Y-box homolog genes, first isolated in Rana (Pelophylax), are expressed during early phases of gametogenesis in R. (P.) ridibunda and R. (P.) lessonae (parental species), and R. (P.) esculenta (their natural hybrid), suggesting that gametogenesis, during the adult life, follows a unique genetic sequence in the two parental species as well as in hybridogenetic hybrids. Similar molecular investigations need to be carried out on larval stages prior to metamorphosis, when the genetic plasticity of gemline cells allows their commitment versus either the standard and/or hybridogenetic reproduction mode. [ABSTRACT FROM AUTHOR]

Y-box proteins are a highly conserved family of gene expression regulatory factors. During gametogenesis they may play a dual role, as both transcriptional activators of germ cell-specific genes and as translational repressors of stored maternal transcripts. We report the identification of RlYB2, a Y-box homolog gene specifically expressed in the germ cells of green frogs belonging to Rana esculenta complex, a model system characterized by a hybridogenetic gametogenesis. In developing germ cells of the hybrid R. esculenta, arisen by natural cross of the parental species R. ridibunda and R. lessonae, one set of the parental genomes is excluded and the remaining one, first endoreduplicates and then is transmitted to gametes. In situ hybridizations performed on gonadal tissues showed that the RlYB2 transcript was widely expressed in the ooplasm at early stages of oogenesis in both the parental species and hybrids. Interestingly, a hybridization signal, presumably related to RlYB2-like nascent transcripts, was observed in nuclei of stage II oocytes. The presence of RlYB2 mRNA during early oogenesis suggests that this gene may be involved in regulating the transcription and/or translation of maternal mRNAs in this special vertebrate model system. [ABSTRACT FROM AUTHOR]

Glaucoma is characterized by cupping of the optic disc, apoptotic degeneration of retinal ganglion cells (RGCs) and their axons, and thinning of the retinal nerve fiber layer, with patchy loss of vision. Elevated intraocular pressure (IOP) is a major risk factor for hypertensive glaucoma and the only modifiable one. There is a need to find novel compounds that counteract other risk factors contributing to RGC degeneration. The oil derived from the wild olive tree ( Olea europaea var. sylvestris ), also called Acebuche (ACE), shows powerful anti-inflammatory, antioxidant and retinoprotective effects. We evaluated whether ACE oil could counteract glaucoma-related detrimental effects. To this aim, we fed mice either a regular or an ACE oil-enriched diet and then induced IOP elevation through intraocular injection of methylcellulose. An ACE oil-enriched diet suppressed glaucoma-dependent retinal glia reactivity and inflammation. The redox status of the glaucomatous retinas was restored to a control-like situation, and ischemia was alleviated by an ACE oil-enriched diet. Notably, retinal apoptosis was suppressed in the glaucomatous animals fed ACE oil. Furthermore, as shown by electroretinogram analyses, RGC electrophysiological functions were almost completely preserved by the ACE oil-enriched diet. These ameliorative effects were IOP-independent and might depend on ACE oil's peculiar composition. Although additional studies are needed, nutritional supplementation with ACE oil might represent an adjuvant in the management of glaucoma.

Distress, or negative stress, is known to considerably increase the incidence of several diseases, including cancer. There is indeed evidence from pre-clinical models that distress causes a catecholaminergic overdrive that, mainly through the activation of β-adrenoceptors (β-ARs), results in cancer cell growth and cancer progression. In addition, clinical studies have evidenced a role of negative stress in cancer progression. Moreover, plenty of data demonstrates that β-blockers have positive effects in reducing the pro-tumorigenic activity of catecholamines, correlating with better outcomes in some type of cancers as evidenced by several clinical trials. Among β-ARs, β2-AR seems to be the main β-AR subtype involved in tumor development and progression. However, there are data indicating that also β1-AR and β3-AR may be involved in certain tumors. In this chapter, we will review current knowledge on the role of the three β-AR isoforms in carcinogenesis as well as in cancer growth and progression, with particular emphasis on recent studies that are opening new avenues in the use of β-ARs as therapeutic targets in treating tumors., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Thyroid hormone (TH) dyshomeostasis is associated with poor prognosis in acute and prolonged illness, but its role in diabetic retinopathy (DR) has never been investigated. Here, we characterized the TH system in the retinas of db/db mice and highlighted regulatory processes in MIO-M1 cells. In the db/db retinas, typical functional traits and molecular signatures of DR were paralleled by a tissue-restricted reduction of TH levels. A local condition of low T3 (LT3S) was also demonstrated, which was likely to be induced by deiodinase 3 (DIO3) upregulation, and by decreased expression of DIO2 and of TH receptors. Concurrently, T3-responsive genes, including mitochondrial markers and microRNAs (miR-133-3p, 338-3p and 29c-3p), were downregulated. In MIO-M1 cells, a feedback regulatory circuit was evidenced whereby miR-133-3p triggered the post-transcriptional repression of DIO3 in a T3-dependent manner, while high glucose (HG) led to DIO3 upregulation through a nuclear factor erythroid 2-related factor 2-hypoxia-inducible factor-1 pathway. Finally, an in vitro simulated condition of early LT3S and hyperglycemia correlated with reduced markers of both mitochondrial function and stress response, which was reverted by T3 replacement. Together, the data suggest that, in the early phases of DR, a DIO3-driven LT3S may be protective against retinal stress, while, in the chronic phase, it not only fails to limit HG-induced damage, but also increases cell vulnerability likely due to persistent mitochondrial dysfunction., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Cisplatin is a chemotherapeutic drug for the treatment of several solid tumors, whose use is limited by its nephrotoxicity, neurotoxicity, ototoxicity, and development of resistance. The toxicity is caused by DNA cross-linking, increase in reactive oxygen species and/or depletion of cell antioxidant defenses. The aim of the work was to study the effect of antioxidant compounds (Lisosan G, Taurisolo ® ) or hydrogen sulfide (H 2 S)-releasing compounds (erucin) in the auditory HEI-OC1 cell line treated with cisplatin. Cell viability was determined using the MTT assay. Caspase and sphingomyelinase activities were measured by fluorometric and colorimetric methods, respectively. Expression of transcription factors, apoptosis hallmarks and genes codifying for antioxidant response proteins were measured by Western blot and/or RT-qPCR. Lisosan G, Taurisolo ® and erucin did not show protective effects. Sodium hydrosulfide (NaHS), a donor of H 2 S, increased the viability of cisplatin-treated cells and the transcription of heme oxygenase 1, superoxide dismutase 2, NAD(P)H quinone dehydrogenase type 1 and the catalytic subunit of glutamate-cysteine ligase and decreased reactive oxygen species (ROS), the Bax/Bcl2 ratio, caspase-3, caspase-8 and acid sphingomyelinase activity. Therefore, NaHS might counteract the cytotoxic effect of cisplatin by increasing the antioxidant response and by reducing ROS levels and caspase and acid sphingomyelinase activity.

Nutraceuticals are natural substances whose anti-oxidant and anti-inflammatory properties may be used to treat retinal pathologies. Their efficacy is limited by poor bioavailability, which could be improved using nanocarriers. Lisosan G (LG), a fermented powder from whole grains, protects the retina from diabetic retinopathy (DR)-induced damage. For this study, we tested whether the encapsulation of LG in liposomes (LipoLG) may increase its protective effects. Diabetes was induced in mice via streptozotocin administration, and the mice were allowed to freely drink water or a water dispersion of two different doses of LG or of LipoLG. Electroretinographic recordings after 6 weeks showed that only the highest dose of LG could partially protect the retina from diabetes-induced functional deficits, while both doses of LipoLG were effective. An evaluation of molecular markers of oxidative stress, inflammation, apoptosis, vascular endothelial growth factor, and the blood-retinal barrier confirmed that the highest dose of LG only partially protected the retina from DR-induced changes, while virtually complete prevention was obtained with either dose of LipoLG. These data indicate that the efficacy of LG in contrasting DR is greatly enhanced by its encapsulation in liposomes and may lay the ground for new dietary supplements with improved therapeutic effects against DR.

Retinopathy of prematurity (ROP) is one of the main blinding diseases affecting preterm newborns and is classically considered a vascular disorder. The premature exposure to the extrauterine environment, which is hyperoxic in respect to the intrauterine environment, triggers a cascade of events leading to retinal ischemia which, in turn, makes the retina hypoxic thus setting off angiogenic processes. However, many children with a history of ROP show persistent vision impairment, and there is evidence of an association between ROP and neurosensory disabilities. This is not surprising given the strict relationship between neuronal function and an adequate blood supply. In the present work, we revised literature data evidencing to what extent ROP can be considered a neurodegenerative disease, also taking advantage from data obtained in preclinical models of ROP. The involvement of different retinal cell populations in triggering the neuronal damage in ROP was described along with the neurological outcomes associated to ROP. The situation of ROP in Italy was assessed as well.

A major player in the homeostatic response to hypoxia is the hypoxia-inducible factor (HIF)-1 that transactivates a number of genes involved in neovessel proliferation in response to low oxygen tension. In the retina, hypoxia overstimulates β-adrenoceptors (β-ARs) which play a key role in the formation of pathogenic blood vessels. Among β-ARs, β3-AR expression is increased in proliferating vessels in concomitance with increased levels of HIF-1α and vascular endothelial growth factor (VEGF). Whether, similarly to VEGF, hypoxia-induced β3-AR upregulation is driven by HIF-1 is still unknown. We used the mouse model of oxygen-induced retinopathy (OIR), an acknowledged model of retinal angiogenesis, to verify the hypothesis of β3-AR transcriptional regulation by HIF-1. Investigation of β3-AR regulation over OIR progression revealed that the expression profile of β3-AR depends on oxygen tension, similar to VEGF. The additional evidence that HIF-1α stabilization decouples β3-AR expression from oxygen levels further indicates that HIF-1 regulates the expression of the β3-AR gene in the retina. Bioinformatics predicted the presence of six HIF-1 binding sites (HBS #1-6) upstream and inside the mouse β3-AR gene. Among these, HBS #1 has been identified as the most suitable HBS for HIF-1 binding. Chromatin immunoprecipitation-qPCR demonstrated an effective binding of HIF-1 to HBS #1 indicating the existence of a physical interaction between HIF-1 and the β3-AR gene. The additional finding that β3-AR gene expression is concomitantly activated indicates the possibility that HIF-1 transactivates the β3-AR gene. Our results are indicative of β3-AR involvement in HIF-1-mediated response to hypoxia.

The natural alkaloid protopine (PRO) exhibits pharmacological properties including anticancer activity. We investigated the effects of PRO, alone and in combination with the chemotherapeutic gemcitabine (GEM), on human tumor cell lines and non-tumor human dermal fibroblasts (HDFs). We found that treatments with different PRO/GEM combinations were cytotoxic or cytoprotective, depending on concentration and cell type. PRO/GEM decreased viability in pancreatic cancer MIA PaCa-2 and PANC-1 cells, while it rescued the GEM-induced viability decline in HDFs and in tumor MCF-7 cells. Moreover, PRO/GEM decreased G1, S and G2/M phases, concomitantly with an increase of subG1 phase in MIA PaCa-2 and PANC-1 cells. Differently, PRO/GEM restored the normal progression of the cell cycle, altered by GEM, and decreased cell death in HDFs. PRO alone increased mitochondrial reactive oxygen species (ROS) in MIA PaCa-2, PANC-1 cells and HDFs, while PRO/GEM increased both intracellular and mitochondrial ROS in the three cell lines. These results indicate that specific combinations of PRO/GEM may be used to induce cytotoxic effects in pancreatic tumor MIA PaCa-2 and PANC-1 cells, but have cytoprotective or no effects in HDFs.

HMGA (high mobility group A) (HMGA1 and HMGA2) are small non-histone proteins that can bind DNA and modify chromatin state, thus modulating the accessibility of regulatory factors to the DNA and contributing to the overall panorama of gene expression tuning. In general, they are abundantly expressed during embryogenesis, but are downregulated in the adult differentiated tissues. In the present review, we summarize some aspects of their role during development, also dealing with relevant studies that have shed light on their functioning in cell biology and with emerging possible involvement of HMGA1 and HMGA2 in evolutionary biology.

Collagen prolyl 4-hydroxylases (c-P4Hs) are evolutionary conserved enzymes whose activity is essential for the correct folding of stable triple helical molecules of collagen and collagen-like proteins. They play crucial roles in embryo development, connective tissue functional organization, tumor growth and metastasis. Despite the important function of these enzymes, little is known about their expression during vertebrate development. In this study, we determine and compare the previously undescribed spatio-temporal expression patterns of the p4ha1 and p4ha2 genes, which encode the main subunits containing the enzyme active site, during Xenopus development. The two genes are maternally inherited and share expression in dorsal mesoderm, branchial arches and their derivatives, as well as in the central nervous system, although with distinct spatio-temporal patterns. A major co-expression domain for p4ha1 and p4ha2 is represented by the developing notochord, where these genes are transcribed from early neurula stage to stage 42 tadpole, thus paralleling the profile of collagen II production and suggesting a coordination between collagen synthesis and its post-translational modifications.

The natural alkaloid berberine has several pharmacological properties and recently received attention as a potential anticancer agent. In this work, we investigated the molecular mechanisms underlying the anti-tumor effect of berberine on glioblastoma U343 and pancreatic carcinoma MIA PaCa-2 cells. Human dermal fibroblasts (HDF) were used as non-cancer cells. We show that berberine differentially affects cell viability, displaying a higher cytotoxicity on the two cancer cell lines than on HDF. Berberine also affects cell cycle progression, senescence, caspase-3 activity, autophagy and migration in a cell-specific manner. In particular, in HDF it induces cell cycle arrest in G2 and senescence, but not autophagy; in the U343 cells, berberine leads to cell cycle arrest in G2 and induces both senescence and autophagy; in MIA PaCa-2 cells, the alkaloid induces arrest in G1, senescence, autophagy, it increases caspase-3 activity and impairs migration/invasion. As demonstrated by decreased citrate synthase activity, the three cell lines show mitochondrial dysfunction following berberine exposure. Finally, we observed that berberine modulates the expression profile of genes involved in different pathways of tumorigenesis in a cell line-specific manner. These findings have valuable implications for understanding the complex functional interactions between berberine and specific cell types.

Planarian flatworms possess extraordinary regenerative capability and body plasticity, which rely on a composite population of stem cells, the neoblasts. Despite impressive advances have been recently achieved in the knowledge of neoblast biology, few is still known about factors that are released by differentiated tissues and influence the neoblast fate. Extracellular matrix (ECM) is a fundamental component of the stem cell niche and its remodeling affects stem cell fate. Here we provide the characterization of the astacin gene family of metalloproteinases in planarians, good candidate enzymes for generating dynamicity in the ECM. Ten and eighteen astacin isoforms were identified in the planarian species Schmidtea mediterranea and Dugesia japonica, respectively. Besides the already characterized Smedolloid, in Schmidtea mediterranea are present eight astacins with a minimal structure (a signal peptide, an activation domain and a Zn-binding catalytic domain), that are colocalized in large cells organized in a peculiar, not yet morphologically characterized, two-ring-shaped structure located in the middle of the body. A single astacin, characterized by a ShK toxin domain in its C-terminal region, has been found to be produced in gastrodermal cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The natural alkaloid sanguinarine has remarkable therapeutic properties and has been used for centuries as a folk remedy. This compound exhibits interesting anticancer properties and is currently receiving attention as a potential chemotherapeutic agent. Nevertheless, limited information exists regarding its safety for developing organisms. Planarians are an animal model known for their extraordinary stem cell-based regenerative capabilities and are increasingly used for toxicological and pharmacological studies. Here, we report that sanguinarine, at micromolar concentrations, perturbs the regeneration process in the planarian Dugesia japonica. We show that sanguinarine exposure causes defects during anterior regeneration and visual system recovery, as well as anomalous remodelling of pre-existing structures. Investigating the effects of sanguinarine on stem cells, we found that sanguinarine perturbs the transcriptional profile of early and late stem cell progeny markers. Our results indicate that sanguinarine exposure alters cell dynamics and induces apoptosis without affecting cell proliferation. Finally, sanguinarine exposure influences the expression level of H + , K + -ATPase α subunit, a gene of the P-type-ATPase pump family which plays a crucial role during anterior regeneration in planaria. On the whole, our data reveal that sanguinarine perturbs multiple mechanisms which regulate regeneration dynamics and contribute to a better understanding of the safety profile of this alkaloid in developing organisms.

Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in neuronal and endothelial cell development. The expression of the pdzrn3 gene is developmentally regulated in various vertebrate tissues, including muscular, neural and vascular system. Little is known about its expression during kidney development, although genetic polymorphisms and alterations around the human pdzrn3 chromosomal region have been found to be associated with renal cell carcinomas and other kidney diseases. We investigated the pdzrn3 spatio-temporal expression pattern in Xenopus laevis embryos by in situ hybridization. We focused our study on the development of the pronephros, which is the embryonic amphibian kidney, functionally similar to the most primitive nephric structures of human kidney. To explore the role of pdzrn3 during renal morphogenesis, we performed loss-of-function experiments, through antisense morpholino injections and analysed the morphants using specific pronephric markers. Dynamic pdzrn3 expression was observed in embryonic tissues, such as somites, brain, eye, blood islands, heart, liver and pronephros. Loss of function experiments resulted in specific alterations of pronephros development. In particular, at early stages, pdzrn3 depletion was associated with a reduction of the pronephros anlagen and later, with perturbations of the tubulogenesis, including deformation of the proximal tubules. Rescue experiments, in which mRNA of the zebrafish pdzrn3 orthologue was injected together with the morpholino, allowed recovery of the kidney phenotypes. These results underline the importance of pdzrn3 expression for correct nephrogenesis.

We recently identified pfdn6a and tcp1α (also known as cct-α) as genes coregulated by the transcription factor Rx1. The proteins encoded by these genes belong to two interacting complexes (Prefoldin and "chaperonin containing t-complex polypeptide 1"), which promote the folding of actin and tubulin and have more recently been reported to be involved in a variety of additional functions including cell cycle control and transcription regulation. However, little is known about the expression and function of these two genes during vertebrate development. To assess whether pfdn6a and tcp1α display a general coordinated expression during Xenopus development, we determined, by RT-PCR and in situ hybridization, the spatio-temporal expression pattern of pfnd6a, which was not previously described, and compared it to that of tcp1α, extending the analysis to stages not previously investigated for this gene. We detected maternal transcripts of pfnd6a in the animal hemisphere at early blastula stage. During gastrulation, pfdn6a was expressed in the involuting mesoderm and subsequently in the anterior and dorsal neural plate. At tailbud and tadpole stages, pfdn6a RNA was mainly detected in the forebrain, midbrain, eye vesicle, otic vesicle, branchial arches, and developing pronephros. The pfnd6a expression pattern largely overlaps with that of tcp1α indicating a spatio-temporal transcriptional coregulation of these genes in the majority of their expression sites, which is suggestive of a possible involvement in the same developmental events.

Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin Repeat-rich Membrane Spanning) is a conserved scaffold protein that acts as a downstream substrate for protein kinase D and mediates multiple receptor signalling pathways. Despite the dissecting of the function of this protein in mammals, using both in vitro and in vivo studies, a detailed characterization of its gene expression during early phases of embryogenesis has not been described yet. Here, we have used Xenopus laevis as a vertebrate model system to analyze the gene expression and the protein localization of Kidins220/ARMS. We found its expression was dynamically regulated during development. Kidins220/ARMS mRNA was expressed from neurula to larval stage in different embryonic regions including the nervous system, eye, branchial arches, heart and somites. Similar to the transcript, the protein was present in multiple embryonic domains including the central nervous system, cranial nerves, motor nerves, intersomitic junctions, retinal ganglion cells, lens, otic vesicle, heart and branchial arches. In particular, in some regions such as the retina and somites, the protein displayed a differential localization pattern in stage 42 embryos when compared to the earlier examined stages. Taken together our results suggest that this multidomain protein is involved in distinct spatio-temporal differentiative events.

In many organisms, the specification of cell fate and the formation of embryonic axes depend on a proper distribution of maternal mRNAs during oogenesis. Asymmetrically localized determinants are required both for embryonic axes and germline determination in anuran amphibians. As a model system of these processes, we have used a species complex of the genus Pelophylax (Rana), characterized by a hybridogenetic reproduction that involves events of genome exclusion and endoreduplication during meiosis in both sexes. With the aim of characterizing the still largely unknown molecular events regulating Pelophylax gametogenesis, we have isolated in this animal model homologues of the deleted in AZoospermia-like (DAZl) and pumilio gene families (named RlDazl and RlPum1, respectively), which encode posttranscriptional regulators. Expression pattern analysis of these genes showed that RlDazl is exclusively expressed in gonadal tissues, whereas RlPum1 is expressed in both somatic tissues and gonads. In situ hybridization carried out on gonads revealed that the two transcripts were asymmetrically localized along the animal-vegetal (A-V) axis of oocytes. In particular, the RlDazl transcript progressively collected to the vegetal pole during oogenesis, whereas the RlPum1 mRNA was preferentially enriched at the animal hemisphere. In adult testes, RlDazl and RlPum1 were expressed in specific phases of spermatogenetic divisions as shown by immunostaining with anti-H3 phosphohistone antibody. Our results indicate that RlDazl and RlPum1 represent two early indicators of oocyte polarity in this hybridogenetic vertebrate model. Additionally, RlDazl share with vertebrate DAZ- like genes a germ cell-specific expression pattern., (Copyright © 2011 Wiley-Liss, Inc., A Wiley Company.)

RrS1-like sequences of water frogs (genus Pelophylax) display varied genomic organization, whereas the centromeric hybridization pattern reveals species-specific differences. Using fluorescent in situ hybridization, Pelophylax cf. bedriagae, Pelophylax kurtmuelleri, and Pelophylax ridibundus showed a hybridization signal at centromeres of chromosomes 1-5, but in P. kurtmuelleri the medium-small chromosome labeled was 10 rather than 8. Pelophylax cretensis had almost 16 of 26 centromeres labeled, as did Pelophylax lessonae from Poland when its chromosomes are hybridized with a homologous probe. When StuI-digested genomic DNA was hybridized with RrS1 probe, hybridization ladders for P. ridibundus from Poland have evenly spaced steps (about 100 bp) of uniform intensity from about 200 bp upward. Steps in hybridization ladders from circum-Aegean taxa vary in intensity: larger, odd-numbered steps are often fainter. A strong double band (800/900 bp) in Anatolian P. cf. bedriagae, emphasized by a weak 700 bp band, distinguishes them from P. kurtmuelleri from the Peloponnisos, in which the 900 bp band is almost absent. The ladder in P. cretensis lacks odd-numbered steps. A and B repeats, observed originally within the RrS1 satellite of P. ridibundus, occur also in the circum-Aegean frogs and in P. lessonae, Pelophylax epeiroticus, Pelophylax saharicus, and Pelophylax shqipericus. It is plausible that AB dimers or ABB trimers rather than A or B monomers correspond to functional/evolutionary units. The presence of regions similar to yeast CDEs and mammalian CENP-B boxes suggests a role for RrS1 sequences in centromere organization.

Germline cell fate decisions are primarily controlled at the post-transcriptional level with DEAD-box RNA helicases playing a crucial role in germline development. In this study, we report the identification of two DEAD-box vasa/PL10 orthologues (RlVlg and RlPL10) in a species complex of the genus Rana, characterized by hybridogenetic reproduction, an enigmatic process that involves the exclusion of an individual genome, and endoreduplication events. Both genes were expressed during the early stages of gametogenesis of R. ridibunda, R. lessonae, and their natural hybrid R. esculenta. RlVlg expression was germline specific. On the other hand, RlPL10 was also expressed in somatic tissues, although only at low levels. The two genes were expressed in different phases of mitotic and meiotic spermatogenetic divisions as demonstrated by immunostaining with an anti-H3 phosphohistone antibody. The data indicate that RlVlg and RlPL10 may represent useful markers for dissecting the molecular aspects of genome exclusion and endoreduplication of the hybridogenetic gametogenesis.

European water frog hybrids Rana esculenta (R. ridibundaxR. lessonae) reproduce hemiclonally, by hybridogenesis: in the germ line they exclude the genome of one parental species and produce haploid gametes with an unrecombined genome of the other parental species. In the widespread L-E population system, both sexes of hybrids (E) coexist with R. lessonae (L). They exclude the lessonae genome and produce ridibunda gametes. In the R-E system, hybrid males coexist with R. ridibunda (R); they exclude either their ridibunda or their lessonae genome and produce sperm with a lessonae or with a ridibunda genome or a mixture of both kinds of sperm. We examined 13 male offspring, 12 of which were from crosses between L-E system and R-E system frogs. All were somatically hybrid. With one exception, they excluded the lessonae genome in the germ line and subsequently endoreduplicated the ridibunda genome. Spermatogonial metaphases contained a haploid or a diploid number of ridibunda chromosomes, identified through in situ hybridization to a satellite DNA marker, and by spermatocyte I metaphases containing a haploid number of ridibunda bivalents. The exception, an F1 hybrid between L-E system R. lessonae and R-E system R. ridibunda, was not hybridogenetic, showed no genome exclusion, and evidenced a disturbed gametogenesis resulting from the combination of two heterospecific genomes. None of the hybridogenetic hybrids showed any cell lines excluding the ridibunda genome, the pattern most frequent in hybrids of the R-E system, unique to that system, and essential for its persistence. A particular combination of R-E system lessonae and R-E system ridibunda genomes seems necessary to induce the R-E system type of hemiclonal gametogenesis.

Here we clone the Xenopus 5-HT2B receptor cDNA and describe its spatio-temporal mRNA expression within the developing larval brain and visual system. Expression of the 5-HT2B transcripts is compared to that of 5-HT2C as well as proliferation and neurogenic markers. In developing brain and retina, 5-HT2B and 2C mRNAs are mainly expressed in proliferative regions. We suggest that these receptors may play a role in the larval secondary neurogenesis by mediating mitogenic effects of serotonin.

We report the isolation of Xdtx1, a Xenopus homologue of the Drosophila Deltex gene. Starting from tailbud stage, Xdtx1 transcripts are detected in the olfactory bulbs, pineal complex and along the neural tube according to an antero-posterior gradient showing a gap at the midbrain-hindbrain boundary. At tadpole stage, Xdtx1 expression is activated in the differentiating retina, where it is also found in the neuronal fibres of the outer and inner plexiform layers, while its expression in the pineal complex becomes restricted to the photosensitive frontal organ. Differently from other vertebrate Deltex homologues, Xdtx1 is exclusively expressed in regions undergoing neuronal differentiation as shown by complementarity with X-Notch-1 expression.

We isolated three Xenopus cDNA clones, Xcalp1, Xcalp2 and Xcalp3, which encode different forms of calpastatin mRNA. Compared to the canonical form of mammalian calpastatin, the predicted Xcalp3 protein contained a very long N-terminal domain L and an additional inhibitory domain. The other two deduced calpastatin proteins were truncated forms, both lacking domain L and containing four (Xcalp2) and two (Xcalp1) inhibitory domains, respectively. The presence of Xcalp1, Xcalp2 and Xcalp3 transcripts was detected by in situ hybridization in the notochord from the embryonic stage 20 to stage 36, afterwards the expression was only present in the growing tailbud. As shown by RT-PCR, the three calpastatin mRNAs were also expressed in the adult brain.

In a previous work we isolated a Xenopus 5-HT1A receptor gene and now report the characterization of this receptor. The HindIII-XbaI fragment of this gene was cloned into the pcDNA I NEO vector and stably transfected into eukaryotic cells (NIH-3T3). To determine the specific 5-HT1A receptor binding, [3H]8-OH-DPAT was used as radioligand. The selective 5-HT1A receptor agonist bound only a single class of saturable high-affinity binding sites with pharmacological characteristics similar to those of the mammalian 5-HT1A receptor. The effects of X5-HT1A receptor activation on cell growth were also investigated in stably transfected NIH-3T3 cells. The 5-HT1A agonist 8-OH-DPAT was found to increase DNA synthesis and accelerated cell growth., (Copyright 1999 Elsevier Science B.V.)

The aim of our work is to investigate the potential involvement of serotonin and its G-protein-coupled receptors in neural differentiation or other developmental processes in Xenopus laevis. By using a RT-PCR strategy, we isolated a cDNA fragment from X. laevis brain showing high amino-acid similarity with the mammalian 5-HT1A receptor. We used this fragment to isolate a cDNA clone containing a single ORF of 408 amino-acids with an overall amino-acid identity of 73% with the human and rat 5-HT1A receptor. This structural similarity suggests that this clone encodes the Xenopus homolog of the mammalian 5-HT1A receptor (X5-HT1A). In order to establish a possible role for this receptor in development, we analyzed the pattern of its gene expression during embryogenesis, larval stages and in adult brain by in situ hybridization. The first signal of mRNA expression appears in the rostral part of brain stem at stage 22, when the first neurons start differentiation [38,21]. In later stages of development, the cells expressing X5-HT1A transcripts appear to correspond to serotonergic neurons. By stage 41, X5-HT1A mRNA is also detected in the inner nuclear layer (INL) of the developing retina. This pattern of expression is maintained until stage 46, i.e. at the beginning of metamorphosis. In adult, additional brain areas express X5-HT1A mRNA, particularly in telencephalon, diencephalon and mesencephalon. On the whole, our data show that the X5-HT1A receptor mRNA is developmentally regulated, with expression first appearing in differentiating serotonergic neurons, where this receptor may mediate, through an autocrine regulatory pathway, the trophic action of serotonin on developing serotonergic system.

We have studied a family of long repetitive DNA sequences (Hsr1) interspersed in the large genome of the European plethodontid salamander Hydromantes. The sequence analysis of a 5-kb fragment (Hsr1A) of one member has revealed significant similarities with amino acidic domains of retroviruses and retrotransposons. The similarity of the reverse transcriptase domain and the gene organization identifies Hsr1A as a member of the gypsy/Ty3 class of retrotransposons. We hypothesize that Hsr1 sequences are vestiges of an invasion of the Hydromantes genome that occurred early in the evolutionary history of these European plethodontids. About 10(6) Hsr1 sequences are present in the large Hydromantes genome. This is the highest number of copies so far discovered for retrotransposon-like elements in eukaryote organisms.

We have characterized a highly repetitive family, named Hy/Pol III, in the genome of the European salamanders Hydromantes (Plethodontidae). This family consists of short, tandemly repeated sequences organized in clusters, scattered through the genome as shown both by in situ hybridization to chromosomes and by Southern blot hybridization. The repeat unit is about 200 bp in length and it is a composite element since it contains a SINE-like retroposon with a tRNA structure, flanked by two short direct repeats. The whole element itself is bordered by two other direct repeats. The sequence data suggest that two elements, presumably derived from polymerase III transcripts, have been inserted one into the other, giving rise to the observed composite structure. During evolution the Hy/Pol III family was then amplified by tandem duplication at the DNA level. The inferred relationships between Hy/Pol III members from three representative species of the European Hydromantes suggests that a subfamily structure characterizes the evolutionary history of this family.