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The retinal development of the gilthead seabream Sparus aurata has been analysed from late embryonic development to juvenile stages using classical histological and immunohistological methods. Five significant phases were established. Phases 1 and 2 comprise the late embryonic and hatching stages, respectively. The results indicate that during these early stages the retina is composed of a single neuroblastic layer that consists of undifferentiated retinal progenitor cells. Phase 3 (late prolarval stage) is characterized by the emergence of the retinal layers and the appearance of neurochemical profiles in differentiating photoreceptors, amacrine and ganglion cells. Phases 4 and 5 comprise the late larval and juvenile stages. In these stages, all the retinal cell types can be detected immunohistochemically. All the maturational events described are first detected in the central retina and, as development progresses, spread to the rest of the retina following a central-to-peripheral gradient. The results of this study suggest that S. aurata is an altricial teleost species that hatches with a morphologically undifferentiated retina. The most relevant processes involved in retinogenesis occur during the late prolarval stage (phase 3). [ABSTRACT FROM AUTHOR]

Obesity has reached global epidemic proportions, and even though its effects are well-documented, studying the interactions among all influencing factors is crucial for a better understanding of its physiopathology. In a high-fat-diet-induced obesity animal model using C57BL/6J mice, behavioural responses were assessed through a battery of tests, while stress biomarkers and systemic inflammatory cytokines were measured using an Enzyme-Linked ImmunoSorbent Assay and a Bio-Plex Multiplex System. The peritoneal macrophage microbicide capacity was analysed via flow cytometry, and crown-like structures (CLSs) in white adipose tissue (WAT) were evaluated through staining techniques. Results indicated that obese mice exhibited increased body weight, hyperglycaemia, and hyperlipidaemia after 18 weeks on a high-fat diet, as well as worse physical conditions, poorer coordination and balance, and anxiety-like behaviour. Differences in corticosterone and noradrenaline concentrations were also found in obese animals, revealing a stress response and noradrenergic dysregulation, along with a weakened innate immune response characterized by a lower microbicide capacity, and the presence of an underlying inflammation evidenced by more CLSs in WAT. Altogether, these findings indicate that obesity deteriorates the entire stress, inflammatory, metabolic, sensorimotor and anxiety-like behavioural axis. This demonstrates that jointly evaluating all these aspects allows for a deeper and better exploration of this disease and its associated comorbidities, emphasizing the need for individualized and context-specific strategies for its management., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

In this work we present a detailed study of the major events during retinal histogenesis of the cuttlefish Sepia officinalis from early embryos to newly hatched animals and juveniles. For this purpose, we carried out morphometric and histological analyses using light and scanning electron microscopy. From St19, the first embryonic stage analysed, to St23/24 the embryonic retina is composed of a pseudostratified epithelium showing abundant mitotic figures in the more internal surface. At St24 the first photoreceptor nuclei appear in the presumptive inner segment layer, while an incipient layer of apical processes of the future rhabdomeric layer become visible at St25. From this stage onwards, both the rhabdomeric layer and the inner segment layer increase in size until postnatal ages. In contrast, the width of the supporting cell layer progressively decreases from St25/26 until postnatal ages. S. officinalis embryos hatched in a morphologically advanced state, showing a differentiated retina even in the last stages of the embryonic period. However, features of immaturity are still observable in the retinal tissue during the first postnatal weeks of life, such as the existence of mitotic figures in the apical region of the supporting cell layer and migrating nuclei of differentiating photoreceptors crossing the basal membrane to reach their final location in the inner segment layer. Therefore, postnatal retinal neurogenesis is present in juvenile specimens of S. officinalis., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

It has been shown that senescent cells accumulate in transient structures of the embryo that normally degenerate during tissue development. A collection of biomarkers is generally accepted to define senescence in embryonic tissues. The histochemical detection of β-galactosidase activity at pH 6.0 (β-gal-pH6) is the most widely used assay for cellular senescence. Immunohistochemical detection of common mediators of senescence which block cell cycle progression, including p16, p21, p63, p15 or p27, has also been used to characterize senescent cells in the embryo. However, the reliability of this techniques has been discussed in recent publications because non-senescent cells are also labelled during development. Indeed, increased levels of senescent markers promote differentiation over apoptosis in developing neurons, suggesting that machinery used for the establishment of cellular senescence is also involved in neuronal maturation. Notably, it has recently been argued that a comparable state of cellular senescence might be adopted by terminally differentiated neurons. The developing sensory systems provide excellent models for studying if canonical markers of senescence are associated with terminal neuronal differentiation., (©The Author(s) 2023. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

The quail ( Coturnix coturnix , Linnaeus 1758), a notable model used in developmental biology, is a precocial bird species in which the processes of retinal cell differentiation and retinal histogenesis have been poorly studied. The purpose of the present research is to examine the retinogenesis in this bird species immunohistochemically and compare the results with those from previous studies in precocial and altricial birds. We found that the first PCNA-negative nuclei are detected at Stage (St) 21 in the vitreal region of the neuroblastic layer, coinciding topographically with the first αTubAc-/Tuj1-/Isl1-immunoreactive differentiating ganglion cells. At St28, the first Prox1-immunoreactive nuclei can be distinguished in the vitreal side of the neuroblastic layer (NbL), but also the first visinin-immunoreactive photoreceptors in the scleral surface. The inner plexiform layer (IPL) emerges at St32, and the outer plexiform layer (OPL) becomes visible at St35-the stage in which the first GS-immunoreactive Müller cells are distinguishable. Newly hatched animals show a well-developed stratified retina in which the PCNA-and pHisH3-immunoreactivies are absent. Therefore, retinal cell differentiation in the quail progresses in the stereotyped order conserved among vertebrates, in which ganglion cells initially appear and are followed by amacrine cells, horizontal cells, and photoreceptors. Müller glia are one of the last cell types to be born. Plexiform layers emerge following a vitreal-to-scleral gradient. Finally, our results suggest that there are no significant differences in the timing of different events involved in retinal maturation between the quail and the chicken, but the same events are delayed in an altricial bird species.

Macrophage accumulation in the adipose tissue and changes in their inflammatory phenotype is a hallmark of obesity-induced inflammation, notably forming inflammatory structures known as "crown-like structures (CLS)". Exercise can be a key strategy to improve inflammation-related complications, but it is crucial to consider that, although exercise generally exerts systemic and local anti-inflammatory effects, this depends on the basal inflammatory status and exercise modality. In this context, the "bioregulatory effect of exercise" implies to achieve the reduction or prevention of an excessive inflammatory response and also the preservation or stimulation of the innate response. In the present work, our aim was to evaluate the effect of regular exercise on adipose tissue inflammation in high-fat diet-induced obesity in mice, as reflected by macrophage infiltration and phenotype, and CLS formation, together with a potential role for the chemokine MCP-1 in this process. Results showed that obesity is associated with greater MCP-1 expression (p<0.05), macrophage accumulation (p<0.05), and CLS presence (p<0.001). Regular exercise reduced macrophage accumulation (p<0.05), MCP-1 expression (p<0.01), and CLS presence (p<0.05) in obese mice; while it increased macrophage and CLS presence (p<0.01), MCP-1 expression (p<0.05), and M2 polarization (p<0.05) in lean mice. MCP-1 was associated with the proliferation of CLS, showing the first image demonstrating a potential role of this chemokine in the development of these structures. Altogether, these results confirm, for the first time, the "bioregulatory effect of exercise" in the adipose tissue: reducing inflammation in individuals with an elevated inflammatory setpoint, but stimulating this response of the immune system in healthy individuals., (Copyright © 2023 International Society of Exercise and Immunology. All rights reserved.)

This study examines the anatomical development of the visual system of Atlantic bluefin tuna, Thunnus thynnus, during the first 15 days of life at histological level, with emphasis in the immunohistochemical characterization of different cell types. As an altricial fish species, the retina was not developed at hatching. The appearance of eye pigmentation and the transformation of the retina from an undifferentiated neuroblastic layer into a laminated structure occurred during the first two days of life. At 16 days after hatching (DAH), the ganglion cells were arranged in a single row in the central region of the retina and the outer segments of the photoreceptors were morphologically developed. Furthermore, at this age, all the retinal cell types were immunohistochemically characterized. The presence of ganglion cell axons was confirmed with the TUJ1 antibody and the existence of functional synapses in the plexiform layers with antibodies against SV2. Cone opsins were immunostained with antibodies against visinin and CERN-922 immunoreactive rods were also identified. Different subpopulations of amacrine cells were immunostained with antibodies against αTH and PV. Highly GS-immunoreactive Müller cells were also detected at this age. These observations suggested that the T. thynnus retina was fully functional at the end of the second week of life. Basic studies on early morphology of the visual system and larval behavior are necessary to support applied research on larval rearing. Furthermore, they may have implications for understanding larval ecology in the wild., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

During development of the vertebrate retina, mitotic activity is defined as apical when is located at the external surface of the neuroepithelium or as non-apical when is found in more internal regions. Apical mitoses give rise to all retinal cell types. Non-apical mitoses are linked to committed horizontal cell precursors that subsequently migrate vitreo-sclerally, reaching their final position in the outer surface of the inner nuclear layer, where they differentiate. Previous studies have suggested differences in the timing of retinal maturation between altricial and precocial bird species. In the present study we analyze qualitatively and quantitatively the mitotic activity in the developing retina of an altricial (zebra finch, Taeniopygia guttata ) and a precocial (Japanese quail, Coturnix coturnix ) bird species. We found that pHisH3-immunoreactive apical and non-apical mitoses were abundant in the T. guttata retina at the hatching stage. In contrast, pHisH3 immunoreactivity almost disappeared from the quail retina at the embryonic day 10 (E10). Furthermore, we also found that the onset of the appearance of non-apical mitoses occurred at later stages in the altricial bird species than in the precocial one. The disappearance of apical mitoses and the spatiotemporal distribution of non-apical mitoses followed central to peripheral and dorsal to ventral gradients, similar to gradients of cell differentiation described in the retina of birds. Therefore, these results suggest that retinal neurogenesis is active at the hatching stage in T. guttata , and that horizontal cell differentiation is delayed in the altricial bird species compared to the precocial one. Together, this study reveals important insights into the timing differences that regulate bird retinal maturation and provides a better understanding of the evolution of avian altriciality and precociality., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Álvarez-Hernán, de Mera-Rodríguez, Hernández-Núñez, Acedo, Marzal, Gañán, Martín-Partido, Rodríguez-León and Francisco-Morcillo.)

The histochemical detection of β-galactosidase enzymatic activity at pH 6.0 (β-gal-pH6) is a widely used biomarker of cellular senescence in aging tissues. This histochemical assay also detects the presence of programmed cell senescence during specific time windows in degenerating structures of vertebrate embryos. However, it has recently been shown that this enzymatic activity is also enhanced in subpopulations of differentiating neurons in the developing central nervous system in vertebrates. The present study addressed the histochemical detection of β-gal-pH6 enzymatic activity in the developing postnatal olfactory epithelium in the mouse. This activity was detected in the intermediate layer of the olfactory epithelium. As development progressed, the band of β-gal-pH6 labeling in this layer increased in width. Immunohistochemistry and lectin histochemistry showed the β-gal-pH6 staining to be strongly correlated with the immunolabeling of the olfactory marker protein (OMP) that identifies mature olfactory sensory neurons. The cell somata of a subpopulation of differentiated olfactory neurons that were recognized with the Dolichos biflorus agglutinin (DBA) were always located inside this band of β-gal-pH6 staining. However, the β-gal-pH6 histochemical signal was always absent from the apical region where the cytokeratin-8 positive supporting cells were located. Furthermore, no β-gal-pH6 staining was found in the basal region of the olfactory epithelium where PCNA/pHisH3 immunoreactive proliferating progenitor cells, GAP43 positive immature neurons, and cytokeratin-5 positive horizontal basal cells were located. Therefore, β-gal-pH6 seems to be linked to neuronal differentiation and cannot be regarded as a biomarker of cellular senescence during olfactory epithelium development in mice.

Planar cell polarity (PCP) is evolutionary conserved and play a critical role in proper tissue development and function. During central nervous system development, PCP proteins exhibit specific patterns of distribution and are indispensable for axonal growth, dendritogenesis, neuronal migration, and neuronal differentiation. The retina constitutes an excellent model in which to study molecular mechanisms involved in neural development. The analysis of the spatiotemporal expression of PCP proteins in this model constitutes an useful histological approach in order to identify possible roles of these proteins in retinogenesis. Immunohistochemical techniques revealed that Frz6, Celsr1, Vangl1, Pk1, Pk3, and Fat1 were present in emerging axons from recently differentiated ganglion cells in the chicken retina. Except for Vangl1, they were also asymmetrically distributed in differentiated amacrine cells. Pk1 and Pk3 were restricted in the outer nuclear layer to the outer segment of photoreceptors. Vangl1 was also located in the cell somata of Müller glia. Given these findings together, the distribution of PCP proteins in the developing chicken retina suggest essential roles in axonal guidance during early retinogenesis and a possible involvement in the establishment of cell asymmetry and maintenance of retinal cell phenotypes., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tight-junction (TJ) proteins are essential for establishing the barrier function between neighbor epithelial cells, but also for recognition of pathogens or cell migration. Establishing the expression pattern and localization of different TJ proteins will help to understand the development and physiology of the airway. Here we identify that the junctional adhesion molecule 3 ( Jam3 ) expression is restricted to multiciliated cells (MCCs) in the airway epithelium. In vitro , Jam3 expression varies along airway basal stem cell (BSC) differentiation and upon DAPT treatment or IL6 exposure. However, Jam3 is not required for BSC differentiation to specific cell types. In addition, we found that MCC lacking Jam3 display normal cilia morphology and cilia beating frequency with a delay in BB assembly/positioning in MCCs during differentiation. Remarkably, Jam3 in MCC is mostly localized to subapical organelles, which are negative for the apical recycling endosome marker Rab11 and positive for EEA1. Our data show that Jam3 expression is connected to mature MCC in the airway epithelium and suggest a Jam3 role unrelated to its known barrier function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mateos-Quiros, Garrido-Jimenez, Álvarez-Hernán, Diaz-Chamorro, Barrera-Lopez, Francisco-Morcillo, Roman, Centeno and Carvajal-Gonzalez.)

This study shows the distribution patterns of apoptotic cells and biomarkers of cellular senescence during the ontogeny of the retina in the zebra finch ( T. guttata ). Neurogenesis in this altricial bird species is intense in the retina at perinatal and post-hatching stages, as opposed to precocial bird species in which retinogenesis occurs entirely during the embryonic period. Various phases of programmed cell death (PCD) were distinguishable in the T. guttata visual system. These included areas of PCD in the central region of the neuroretina at the stages of optic cup morphogenesis, and in the sub-optic necrotic centers (St15-20). A small focus of early neural PCD was detected in the neuroblastic layer, dorsal to the optic nerve head, coinciding with the appearance of the first differentiated neuroblasts (St24-St25). There were sparse pyknotic bodies in the non-laminated retina between St26 and St37. An intense wave of neurotrophic PCD was detected in the laminated retina between St42 and P8, the last post-hatching stage included in the present study. PCD was absent from the photoreceptor layer. Phagocytic activity was also detected in Müller cells during the wave of neurotrophic PCD. With regard to the chronotopographical staining patterns of senescence biomarkers, there was strong parallelism between the SA- β -GAL signal and p21 immunoreactivity in both the undifferentiated and the laminated retina, coinciding in the cell body of differentiated neurons. In contrast, no correlation was found between SA- β -GAL activity and the distribution of TUNEL-positive cells in the developing tissue.

During vertebrate embryonic development, cellular senescence occurs at multiple locations. To date, it has been accepted that when there has been induction of senescence in an embryonic tissue, β-galactosidase activity is detectable at a pH as high as 6.0, and this has been extensively used as a marker of cellular senescence in vivo in both whole-mount and cryosections. Such senescence-associated β-galactosidase (SA-β-GAL) labeling appears enhanced in degenerating regions of the vertebrate embryo that are also affected by programmed cell death. In this sense, there is a strong SA-β-GAL signal which overlaps with the pattern of cell death in the interdigital tissue of the developing limbs, and indeed, many of the labeled cells detected go on to subsequently undergo apoptosis. However, it has been reported that β-GAL activity at pH 6.0 is also enhanced in healthy neurons, and some retinal neurons are strongly labeled with this histochemical technique when they begin to differentiate during early embryonic development. These labeled early post-mitotic neurons also express other senescence markers such as p21. Therefore, the reliability of this histochemical technique in studying senescence in cells such as neurons that undergo prolonged and irreversible cell-cycle arrest is questionable because it is also expressed in healthy post-mitotic cells. The identification of new biomarkers of cellular senescence would, in combination with established markers, increase the specificity and efficiency of detecting cellular senescence in embryonic and healthy mature tissues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 de Mera-Rodríguez, Álvarez-Hernán, Gañán, Martín-Partido, Rodríguez-León and Francisco-Morcillo.)

The visual system is affected by neurodegenerative diseases caused by the degeneration of specific retinal neurons, the leading cause of irreversible blindness in humans. Throughout vertebrate phylogeny, the retina has two kinds of specialized niches of constitutive neurogenesis: the retinal progenitors located in the circumferential marginal zone and Müller glia. The proliferative activity in the retinal progenitors located in the circumferential marginal zone in precocial birds such as the chicken, the commonest bird model used in developmental and regenerative studies, is very low. This region adds only a few retinal cells to the peripheral edge of the retina during several months after hatching, but does not seem to be involved in retinal regeneration. Müller cells in the chicken retina are not proliferative under physiological conditions, but after acute damage some of them undergo a reprogramming event, dedifferentiating into retinal stem cells and generating new retinal neurons. Therefore, regenerative response after injury occurs with low efficiency in the precocial avian retina. In contrast, it has recently been shown that neurogenesis is intense in the retina of altricial birds at hatching. In particular, abundant proliferative activity is detected both in the circumferential marginal zone and in the outer half of the inner nuclear layer. Therefore, stem cell niches are very active in the retina of altricial birds. Although more extensive research is needed to assess the potential of proliferating cells in the adult retina of altricial birds, it emerges as an attractive model for studying different aspects of neurogenesis and neural regeneration in vertebrates., Competing Interests: None

The bird retina offers an excellent model to investigate the mechanisms that coordinate the morphogenesis, histogenesis, and differentiation of neuron and glial cells. Although these developmental features have been intensively studied in the chicken (Gallus gallus, Linnaeus 1758), a precocial bird species, little is known about retinogenesis in altricial birds. The purpose of this study was to examine the differentiation of retinal cells in the altricial zebra finch (Taeniopygia guttata, Vieillot, 1817) and compare the results with those from previous studies in G. gallus. By using immunohistochemical techniques, the first differentiated TUJ1-/Isl1-positive neuroblasts were detected in the vitreal surface of the neuroblastic layer at later incubation times in T. guttata than in G. gallus (108 h vs 55 h). The immunoreactivity of these early differentiation markers coincided temporo-spatially with the appearance of the first PCNA-negative nuclei. Furthermore, the first visinin-positive photoreceptors (132 h vs 120 h) and the first Prox-1-immunoreactive neuroblasts (embryonic day 7.25 (E7.25) vs E6.5) were also detected at later embryonic stages in the retina of T. guttata than in the retina of G. gallus. At E13, one day before hatching, abundant PCNA- and pHisH3-immunoreactivities were detected in the T. guttata retina, while proliferation was almost absent in the G. gallus retina at perinatal stages. Therefore, these results suggest that cell differentiation in the retina is delayed in the altricial bird compared to precocial birds. Furthermore, the T. guttata retina was not completely developed at hatching, and abundant mitotically active precursor cells of retinal neurons were found, suggesting that retinal neurogenesis was intense at perinatal stages., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Senescence-associated β-galactosidase (SA-β-GAL) histochemistry is the most commonly used biomarker of cellular senescence. These SA-β-GAL-positive cells are senescent embryonic cells that are usually removed by apoptosis from the embryo, followed by macrophage-mediated clearance., Results: Some authors have proposed that SA-β-GAL activity in differentiated neurons from young and adult mammals cannot be uniquely attributed to cell senescence, whether in vivo or in vitro. Using the developing visual system of the chicken as a model, the present study found that SA-β-GAL detected in the developing retina corresponded to lysosomal β-galactosidase activity, and that SA-β-GAL activity did not correlate with the chronotopographical distribution of apoptotic cells. However, SA-β-GAL staining in the undifferentiated retina coincided with the appearance of early differentiating neurons. In the laminated retina, SA-β-GAL staining was concentrated in the ganglion, amacrine, and horizontal cell layers. The photoreceptors and pigment epithelial cells also exhibited SA-β-GAL activity throughout retinal development. We have also found that SA-β-GAL staining strongly correlated p21 immunoreactivity., Conclusion: In conclusion, the results clearly show that SA-β-GAL activity cannot be regarded as a specific marker of senescence during retinal development, and that it is mainly expressed in subpopulations of postmitotic neurons, which are nonproliferative cells, even at early stages of cell differentiation., (© 2019 Wiley Periodicals, Inc.)

Phenolics are involved in many of plants' biological functions. In particular, they play important roles in determining the quality of grape berries and the wine made from them, and can also act as antioxidants with beneficial effects for human health. Several enzymes are involved in the synthesis of phenolic compounds. Among them, stilbene synthase (STS) is a key to the biosynthesis of stilbenes, which are considered to be important secondary metabolites in plants. Other enzymes, such as polyphenol oxidase (PPO) and peroxidase (POD), are involved in the degradation of phenolics, and become activated during late stages of berry ripening. In the present study, Vitis vinifera L. berries were sampled at eight stages of development, from 10 days after anthesis to late harvest. The PPO and POD enzymatic activities were determined at each stage. The presence of STS, PPO, and POD proteins in the grape exocarp and mesocarp was detected immunohistochemically and histochemically. The amount and intensity of the immunohistochemical and histochemical signals correlate with the variations in enzyme activities throughout fruit development. Strong STS immunoreactivity was detected until the onset of ripening. Labeled tissue increased gradually from mesocarp to exocarp, showing an intense signal in epidermis. At subcellular level, STS was mainly detected in cytoplasm grains and cell walls. The amount of PPO immunoreactivity increased progressively until the end of ripening. The PPO signal was detected in hypodermal layers and, to a lesser extent, in mesocarp parenchyma cells, especially in cytoplasm grains and cell walls. Finally, POD activity was stronger at the onset of ripening, and the POD histochemical signal was mainly detected in the cell walls of both exocarp and mesocarp tissue.

Comparative developmental studies have shown that the retina of altricial fish and mammals is incompletely developed at birth, and that, during the first days of life, maturation proceeds rapidly. In contrast, precocial fish and mammals are born with fully differentiated retinas. Concerning birds, knowledge about retinal development is generally restricted to a single order of precocial birds, Galliformes, due to the fact that both the chicken and the Japanese quail are considered model systems. However, comparison of embryonic pre-hatchling retinal development between altricial and precocial birds has been poorly explored. The purpose of this study was to examine the morphogenesis and histogenesis of the retina in the altricial zebra finch (Taeniopygia guttata, Vieillot 1817) and compare the results with those from previous studies in the precocial chicken. Several maturational features (morphogenesis of the optic vesicle and optic cup, appearance of the first differentiated neurons, the period in which the non-apical cell divisions are observable, and the emergence of the plexiform layers) were found to occur at later stages in the zebra finch than in the chicken. At hatching, the retina of T. guttata showed the typical cytoarchitecture of the mature tissue, although features of immaturity were still observable, such as a ganglion cell layer containing many thick cells, very thin plexiform layers, and poorly developed photoreceptors. Moreover, abundant mitotic activity was detected in the entire retina, even in the regions where the layering was complete. The circumferential marginal zone was very prominent and showed abundant mitotic activity. The partially undifferentiated stage of maturation at hatching makes the T. guttata retina an appropriate model with which to study avian postnatal retinal neurogenesis., (© 2018 Anatomical Society.)

Müller cells are the predominant glial cell type in the retina of vertebrates. They play a wide variety of roles in both the developing and the mature retina that have been widely reported in the literature. However, less attention has been paid to their role in phagocytosis of cell debris under physiological, pathological or experimental conditions. Müller glia have been shown to phagocytose apoptotic cell bodies originated during development of the visual system. They also engulf foreign molecules that are injected into the eye, cone outer segments and injured photoreceptors. Phagocytosis of photoreceptor cell debris in the light-damaged teleost retina is primarily carried out by Müller cells. Once the microglial cells become activated and migrate to the photoreceptor cell layer, the phagocytic activity of Müller cells progressively decreases, suggesting a possible mechanism of communication between Müller cells and neighbouring microglia and photoreceptors. Additionally, it has been shown that phagocytic Müller cells acquire proliferating activity in the damaged teleost retina, suggesting that engulfment of apoptotic photoreceptor debris might stimulate the Müller glia to proliferate during the regenerative response. These findings highlight Müller glia phagocytosis as an underlying mechanism contributing to degeneration and regeneration under pathological conditions., (© 2017 Anatomical Society.)

In tetrapods the digit pattern has evolved to adapt to distinct locomotive strategies. The number of digits varies between species or even between hindlimb and forelimb within the same species. These facts illustrate the plasticity of embryonic limb autopods. Sox9 is a precocious marker of skeletal differentiation of limb mesenchymal cells. Its pattern of expression in the developing limb has been widely studied and reflects the activity of signaling cascades responsible for skeletogenesis. In this assay we stress previously overlooked differences in the pattern of expression of Sox9 in limbs of avian, mouse and turtle embryos which may reflect signaling differences associated with distinct limb skeletal morphologies observed in these species. Furthermore, we show that Sox9 gene expression is higher and maintained in the interdigital region in species with webbed digits in comparison with free digit animals.

The LIM-homeodomain transcription factor Islet-1 (Isl1) has been widely used as a marker of different subtypes of neurons in the developing and mature retina of vertebrates. During retinal neurogenesis, early Isl1 expression is detected in the nuclei of neuroblasts that give rise to ganglion, amacrine, bipolar, and horizontal cells. In the mature retina, Isl1 expression is restricted to the nuclei of ganglion cells, cholinergic amacrine cells, ON-bipolar cells, and subpopulations of horizontal cells. Recent studies have explored the functional mechanisms of Isl1 during specification and differentiation of these retinal cell types. Thus, conditional inactivation of Isl1 in the developing mouse retina disrupts retinal function, and also results in optic nerve hypoplasia, marked reductions in mature ganglion, amacrine, and bipolar cells, and a substantial increase in horizontal cells. Furthermore, conditional knockout shows delayed ganglion cell axon growth, ganglion cell axon guidance error, and ganglion cell nerve fiber defasciculation. These data together suggest a possible role for Isl1 in the early differentiation and maintenance of different vertebrate retinal cell types. This review examines whether the expression pattern of Isl1 during vertebrate retinal development is conserved across vertebrate species, and discusses current understanding of the developmental functions of Isl1 in retinogenesis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Programmed cell death (PCD), together with cell proliferation, cell migration, and cell differentiation, is an essential process during development of the vertebrate nervous system. The visual system has been an excellent model on which to investigate the mechanisms involved in ontogenetic cell death. Several phases of PCD have been reported to occur during visual system ontogeny. During these phases, comparative analyses demonstrate that dying cells show similar but not identical spatiotemporally restricted patterns in different vertebrates. Additionally, the chronotopographical coincidence of PCD with the entry of specialized phagocytes in some regions of the developing vertebrate visual system suggests that factors released from degenerating cells are involved in the cell migration of macrophages and microglial cells. Contradicting this hypothesis however, in many cases the cell corpses generated during degeneration are rapidly phagocytosed by neighboring cells, such as neuroepithelial cells or Müller cells. In this review, we describe the occurrence and the sites of PCD during the morphogenesis and differentiation of the retina and optic pathways of different vertebrates, and discuss the possible relationship between PCD and phagocytes during ontogeny., (Copyright © 2014 Wiley Periodicals, Inc.)

The LIM-homeodomain transcription factor Islet1 (Isl1) has been widely used as a marker of neuronal differentiation in the developing visual system of different classes of vertebrates, including mammals, birds, reptiles, and fish. In the present study, we analyzed the spatial and temporal distribution of Isl1-immunoreactive cells during Xenopus laevis retinal development and its relation to the formation of the retinal layers, and in combination with different markers of cell differentiation. The earliest Isl1 expression appeared at St29-30 in the cell nuclei of sparse differentiating neuroblasts located in the vitreal surface of the undifferentiated retina. At St35-36, abundant Isl1-positive cells accumulated at the vitreal surface of the neuroepithelium. As development proceeded and through the postmetamorphic juveniles, Isl1 expression was identified in subpopulations of ganglion cells and in subsets of amacrine, bipolar, and horizontal cells. These data together suggest a possible role for Isl1 in the early differentiation and maintenance of different retinal cell types, and Isl1 can serve as a specific molecular marker for the study of retinal cell specification in X. laevis.

The patterns of distribution of TUNEL-positive bodies and of lectin-positive phagocytes were investigated in the developing visual system of the small-spotted catshark Scyliorhinus canicula, from the optic vesicle stage to adulthood. During early stages of development, TUNEL-staining was mainly found in the protruding dorsal part of the optic cup and in the presumptive optic chiasm. Furthermore, TUNEL-positive bodies were also detected during detachment of the embryonic lens. Coinciding with the developmental period during which ganglion cells began to differentiate, an area of programmed cell death occurred in the distal optic stalk and in the retinal pigment epithelium that surrounds the optic nerve head. The topographical distribution of TUNEL-positive bodies in the differentiating retina recapitulated the sequence of maturation of the various layers and cell types following a vitreal-to-scleral gradient. Lectin-positive cells apparently entered the retina by the optic nerve head when the retinal layering was almost complete. As development proceeded, these labelled cells migrated parallel to the axon fascicles of the optic fiber layer and then reached more external layers by radial migration. In the mature retina, lectin-positive cells were confined to the optic fiber layer, ganglion cell layer and inner plexiform layer. No evident correlation was found between the chronotopographical pattern of distribution of TUNEL-positive bodies and the pattern of distribution of lectin-labelled macrophages/microglial cells during the shark's visual system ontogeny., (© 2013 Anatomical Society.)

Constant intense light causes apoptosis of photoreceptors in the retina of albino fish. However, very few studies have been performed on pigmented species. Tench (Tinca tinca) is a teleost inhabiting dimly lit environments that has a predominance of rods within the photoreceptor layer. To test the hypothesis that constant high intensity light can result in retinal damage in such pigmented epibenthonic teleost species, photodegeneration of the retina was investigated in the larvae and in juveniles of tench to assess whether any damage may also be dependent on fish age. We exposed both groups of animals to 5 days of constant darkness, followed by 4 days of constant 20,000 lx light, and then by 6 days of recovery in a 14 h light:10 h dark cycle. The results showed that the retina of the larvae group exhibited abundant photoreceptor cell apoptosis during the time of exposition to intense light, whereas that of juveniles was indifferent to it. Damaged retinas showed a strong TUNEL signal in photoreceptor nuclei, and occasionally a weak cytoplasmic TUNEL signal in Müller glia. Specific labelling of microglial cells with Griffonia simplicifolia lectin (GSL) histochemistry revealed that photoreceptor cell death alerts microglia in the degenerating retina, leading to local proliferation, migration towards the injured outer nuclear layer (ONL), and enhanced phagocytosis of photoreceptor debris. During the first days of intense light treatment, Müller cells phagocytosed dead photoreceptor cells but, once microglial cells became activated, there was a progressive increase in the phagocytic capacity of the microglia.

Here we present a detailed study of the major events in the retinal histogenesis in a slow-developing elasmobranch species, the small-spotted catshark, during embryonic, postnatal and adult stages using classical histological and immunohistological methods, providing a complete neurochemical characterization of retinal cells. We found that the retina of the small-spotted catshark was fully differentiated prior to birth. The major developmental events in retinal cell differentiation occurred during the second third of the embryonic period. Maturational features described in the present study were first detected in the central retina and, as development progressed, they spread to the rest of the retina following a central-to-peripheral gradient. While the formation of both plexiform layers occurs simultaneously in the retina of the most common fish models, in the small-spotted catshark retina the emergence of the outer plexiform layer was delayed with respect to the inner plexiform layer. According to the expression of the markers used, retinal cell differentiation followed a vitreal-to-scleral gradient, with the exception of Müller cells that were the last cell type generated during retinogenesis. This vitreal-to-scleral progression of neural differentiation seems to be specific to slow-developing fish species., (© 2012 The Authors. Journal of Anatomy © 2012 Anatomical Society.)

Here, we show a detailed chronotopographical analysis of cathepsin B and D expression during development of the mouse visual system. Both proteases were detected in large rounded/ameboid cells usually located in close relationship with prominent sites of extensive physiological cell death. In concordance with their morphological features and topographical distribution, we demonstrate that expressing cells corresponded with macrophages and microglial precursors. We found that as microglial precursors differentiated the expression of both cathepsins was down-regulated. Of interest, cathepsin B and D transcripts were never observed in degenerating cells. Our findings point to a role for cathepsin D and B in cell debris degradation after apoptotic processes rather than promoting cell death, as proposed for other developmental models. Additionally their pattern of expression suggests a role in the maturation of the microglial precursors., (Copyright © 2011 Wiley-Liss, Inc.)

We describe the major events in the retinogenesis in an altricial fish species, the Senegalese sole. The major developmental events in the sole retina occurred early after hatching (posthatching day 0, P0). Thus, (1) plexiform layers became recognizable at P1. (2) Proliferative activity disappeared from the central retina at P1, and, as development progressed, became restricted to cells located in the circumferential germinal zone, and to sparse cells dispersed throughout the inner nuclear layer and the outer nuclear layer. (3) Apoptotic cells were sparsely observed, randomly localized in all three nuclear layers of the early posthatching retina from P0 to P4. (4) The first synaptic vesicles were detected at P0 in early postmitotic ganglion cells. However, their appearance in the plexiform layers was delayed until P2. (5) The neurochemical development of most major retinal cell classes occurred between P0 and P5. Thus, although Isl1 immunoreactive ganglion cells were the first to become postmitotic in the vitreal surface of the central retina at P0, the first glutamine synthetase-expressing Müller cells appeared in the central retina by P5. The onset of expression for other retinal markers, such as rod opsin, calretinin, parvalbumin, a-tyrosine hydroxylase, and a-protein kinase C, occurred between P2 and P4. Our results suggest that the most relevant processes involved in Senegalese sole retinogenesis occur during the prolarval and early larval stages (P0–P5). Furthermore, we conclude that altricial fish species may constitute a convenient model organism to address the relationship between the structural and functional development of sensory organs with the acquisition of behavioral repertoires., (Copyright © 2010 Wiley-Liss, Inc., A Wiley Company.)

Here we present a detailed study of the major events in the retinal histogenesis in a freshwater epibenthonic fish species, the Tench (Tinca tinca, Linneo 1758) during embryonic, prolarval, larval, and juvenile stages, using classical histological and immunohistological methods, providing a complete neurochemical characterization of retinal cells. We find a morphologically undifferentiated retina during embryonic stages and even at the hatching stage (postnatal day 0, P0). However, the emergence of the different retinal layers occurs in the first postnatal day (P1). Proliferating PCNA-positive cells are found in the retina of all postnatal individuals included in the present study, located in the circumferential germinal zone (CGZ), and in sparse cells dispersed throughout the inner nuclear layer (INL) and the outer nuclear layer (ONL). All neurochemical markers used start to express between P0 and P2. Anti-opsin, -alpha-protein kinase C, -alpha-tyrosine hydroxylase, -glutamine synthetase antibodies stain selectively different subpopulations of photoreceptor, bipolar, amacrine, and Müller cells respectively. Parvalbumin immunoreactivity is detected in amacrine and displaced amacrine cells. Several subpopulations of calretinin-positive ganglion, amacrine, and bipolar cells are detected in tench retina. Islet1 expression is confined to the nuclei of subpopulations of ganglion, amacrine, bipolar, and horizontal cells. All the maturational events described are first detected in the central retina and, as development progresses, they spread to the rest of the retina following a central-to-peripheral gradient. Therefore, tench postnatal retinal differentiation is a remarkable process not observed in the more common models of teleosts used in developmental biology.

In spite of the numerous reports on the optic fiber distribution in the optic nerve and tract of vertebrates, there have been few studies of the visual pathway in reptiles. The arrangement of fibers in the optic nerve and tract of the turtle Mauremys leprosa was studied by placing a small granule of carbocyanine dye (DiI or DiA) in one of the four quadrants of the retina. The labeled fibers were traced through transverse sections of the retinofugal pathway with confocal microscopy. Retinal axons displayed a quadrant-specific order along the optic nerve. However, retinal ganglion cell axons were re-organized as they passed through the chiasmatic region of the optic pathway. In the optic tract, the nasal and temporal fibers remained intermingled, but there was segregation of dorsal from ventral fibers. This re-ordering is similar to that described in other vertebrates, suggesting the existence of an evolutionarily conserved mechanism.

Paraquat (PQ) (1, 1'-dimethyl-4, 4'-bipyridinium dichloride), a widely used herbicide, has been suggested as a potential etiologic factor for the development of Parkinson's disease (PD). In neurons from patients with PD display characteristics of autophagy, a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Low concentrations of paraquat have been recently found to induce autophagy in human neuroblastoma cells, and ultimately the neurons succumb to apoptotic death. Whereas caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by paraquat. These findings suggest a relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with paraquat and open a new line of investigation to advance our knowledge regarding the origin of PD.

We show the distribution of the neural and non-neural elements in the early development of the optic nerve in the freshwater turtle, Mauremys leprosa, using light and electron microscopy. The first optic axons invaded the ventral periphery of the optic stalk in close relationship to the radial neuroepithelial processes. Growth cones were thus exclusively located in the ventral margin. As development progressed, growth cones were present in ventral and dorsal regions, including the dorsal periphery, where they intermingled with mature axons. However, growth cones predominated in the ventral part and axonal profiles dorsally, reflecting a dorsal to ventral gradient of maturation. The size and morphology of growth cones depended on the developmental stage and the region of the optic nerve. At early stages, most growth cones were of irregular shape, showing abundant lamellipodia. At the following stages, they tended to be larger and more complex in the ventral third than in intermediate and dorsal portions, suggesting a differential behavior of the growth cones along the ventro-dorsal axis. The arrival of optic axons at the optic stalk involved the progressive transformation of neuroepithelial cells into glial cells. Simultaneously with the fiber invasion, an important number of cells died by apoptosis in the dorsal wall of the optic nerve. These findings are discussed in relation to the results described in the developing optic nerve of other vertebrates.

The inner ear is a complex sensorial structure with hearing and balance functions. A key aim of developmental biology is to understand the molecular and cellular mechanisms involved in the induction, patterning and innervation of the vertebrate inner ear. These developmental events could be mediated by the expression of regulating genes, such as the members of the family of Fibroblast Growth Factors (Fgfs). This work reports the detailed spatial and temporal patterns of Fgf19 expression in the developing inner ear from otic cup (stage 14) to 8 embryonic days (stage 34). In the earliest stages, Fgf19 and Fgf8 expressions determine two subdomains within the Fgf10-positive proneural-sensory territory. We show that, from the earliest stages, the Fgf19 expression was detected in the acoustic-vestibular ganglion and the macula utriculi. The Fgf19 gene was also strongly, but transiently, expressed in the macula lagena, whereas the macula neglecta never expressed this gene in the period analysed. The Fgf19 expression was also clearly observed in some borders of various sensory elements. These results could be useful from further investigations into the role of FGF19 in otic patterning.

The changes in the axon and growth cone numbers in the optic nerve of the freshwater turtle Mauremys leprosa were studied by electron microscopy from the embryonic day 14 (E14) to E80, when the animals normally hatch, and from the first postnatal day (P0) to adulthood (5 years on). At E16, the first axons appeared in the optic nerve and were added slowly until E21. From E21, the fibre number increased rapidly, peaking at E34 (570,000 fibres). Thereafter, the axon number decreased sharply, and from E47 declined steadily until reaching the mature number (about 330,000). These observations indicated that during development of the retina there was an overproduction and later elimination of retinal ganglion cells. Growth cones were first observed in the optic nerve at as early as E16. Their number increased rapidly until E21 and continued to be high through E23 and E26. After E26, the number declined steeply and by E40 the optic nerve was devoid of growth cones. These results indicated that differentiation of the retinal ganglion cells occurred during the first half of the embryonic life. To examine the correlation between the loss of the fibres from the optic nerve and loss of the parent retinal ganglion cells, retinal sections were processed with the TUNEL technique. Apoptotic nuclei were detected in the ganglion cell layer throughout the period of loss of the optic fibres. Our results showed that the time course of the numbers of the fibres in the developing turtle optic nerve was similar to those found in birds and mammals.

Here we show for the first time different aspects of the pattern of neurogenesis in the developing turtle retina by using different morphological and molecular clues. We show the chronotopographical fashion of occurrence of three major aspects of retinal development: (1) morphogenesis of the optic primordia and emergence of the different retinal layers, (2) the temporal progression of neurogenesis by the cessation of proliferative activity, and (3) the apparition and cellular localization of different antigens and neuroactive substances. Retinal cells were generated in a conserved temporal order with ganglion cells born first, followed by amacrine, photoreceptor, horizontal and bipolar/Müller cells. While eventually expressed in many types of retinal neurons, Islet1 was permanently expressed in differentiating and mature ganglion cells. Calbindin-immunoreactive elements were found in the ganglion cell layer and the inner nuclear layer. Interestingly, at later stages the amount of expressing cells in these layers was reduced dramatically. On the contrary, the number of calbindin-immunoreactive photoreceptors increased as development proceeded. In addition, calretinin expressing cells were prominent in the horizontal cell bodies, and their processes extending into the outer plexiform layer were also strongly labeled. Finally, the synthesis of gamma-aminobutyric acid (GABA) was detected in developing and matured horizontal and amacrine cells. All these maturational features began in the dorso-central area, in a region slightly displaced towards the temporal retina.

We compared the spatial and temporal patterns of distribution of macrophages, with patterns of naturally occurring cell death and optic fibre growth during early retina and optic nerve development, in the mouse. We used embryos between day 10 of embryogenesis (E10; before the first optic fibres are generated in the retina) and E13 (when the first optic fibres have crossed the chiasmatic anlage). The macrophages and optic axons were identified by immunocytochemistry, and the apoptotic cells were detected by the TUNEL technique, which specifically labels fragmented DNA. Cell death was observed in the retina and the optic stalk long before the first optic axons appeared in either region. Subsequently, specialized F4/80-positive phagocytes were detected in chronological and topographical coincidence with cell death, which disappeared progressively. As development proceeded, the pioneer ganglion cell axons reached the regions where the macrophages were located. As the number of optic fibres increased, the macrophages disappeared. Therefore, cell death, accompanied by macrophages, preceded the growth of fibres in the retina and the optic nerve. Moreover, these macrophages synthesized NGF and the optic axons were p75 neurotrophin receptor (p75(NTR))- and TrkA-positive. These findings suggest that macrophages may be involved in optic axon guidance and fasciculation.

Fibroblast growth factor 19 (FGF19) is a new member of the FGF family of growth factors. Here, we describe the localization of Fgf19 mRNA in the developing chick retina and lens in stages from the Hamburger and Hamilton stage 15 (HH15) to postnatal day 30 (P30). Fgf19 was expressed in a transient manner in postmitotic neuroblasts during the migration from the ventricular surface to their final location. Moreover, from HH31 (embryonic day 7, E7) on, a subset of lined up Fgf19 expressing cells was distributed in the outer region of the presumptive INL. These cells were Pax6 immunoreactive horizontal cells. During the last third of embryogenesis, Fgf19 expression in the retina was progressively down-regulated and was not detected at P30. Also, it was transiently expressed in the equatorial region of the lens.