MOXD1 is a lineage-specific gene and a tumor suppressor in neuroblastoma.

Neuroblastoma is a childhood developmental cancer; however, its embryonic origins remain poorly understood. Moreover, in-depth studies of early tumor-driving events are limited because of the lack of appropriate models. Herein, we analyzed RNA sequencing data obtained from human neuroblastoma samples and found that loss of expression of trunk neural crest-enriched gene MOXD1 associates with advanced disease and worse outcome. Further, by using single-cell RNA sequencing data of human neuroblastoma cells and fetal adrenal glands and creating in vivo models of zebrafish, chick, and mouse, we show that MOXD1 is a determinate of tumor development. In addition, we found that MOXD1 expression is highly conserved and restricted to mesenchymal neuroblastoma cells and Schwann cell precursors during healthy development. Our findings identify MOXD1 as a lineage-restricted tumor-suppressor gene in neuroblastoma, potentiating further stratification of these tumors and development of novel therapeutic interventions.

Histological and scanning electron microscope observations on the developing retina of the cuttlefish (Sepia officinalis Linnaeus, 1758).

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.

Retinal Development in a Precocial Bird Species, the Quail (Coturnix coturnix, Linnaeus 1758).

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.

Markers of senescence are often associated with neuronal differentiation in the developing sensory systems.

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 ß-galactosidase activity at pH 6.0 (ß-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.

Timing and Distribution of Mitotic Activity in the Retina During Precocial and Altricial Modes of Avian Development.

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.

Histogenesis and cell differentiation in the retina of Thunnus thynnus: A morphological and immunohistochemical study.

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.

Endogenous pH 6.0 ß-Galactosidase Activity Is Linked to Neuronal Differentiation in the Olfactory Epithelium.

The histochemical detection of ß-galactosidase enzymatic activity at pH 6.0 (ß-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 ß-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 ß-gal-pH6 labeling in this layer increased in width. Immunohistochemistry and lectin histochemistry showed the ß-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 ß-gal-pH6 staining. However, the ß-gal-pH6 histochemical signal was always absent from the apical region where the cytokeratin-8 positive supporting cells were located. Furthermore, no ß-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, ß-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.

Junctional Adhesion Molecule 3 Expression in the Mouse Airway Epithelium Is Linked to Multiciliated Cells.

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.

Distribution of planar cell polarity proteins in the developing avian retina.

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.

Analysis of Programmed Cell Death and Senescence Markers in the Developing Retina of an Altricial Bird Species.

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-ß-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-ß-GAL activity and the distribution of TUNEL-positive cells in the developing tissue.

Is Senescence-Associated ß-Galactosidase a Reliable in vivo Marker of Cellular Senescence During Embryonic Development?

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, ß-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 ß-galactosidase (SA-ß-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-ß-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 ß-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.

Development and postnatal neurogenesis in the retina: a comparison between altricial and precocial bird species.

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.

Retinal differentiation in an altricial bird species, Taeniopygia guttata: An immunohistochemical study.

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.

Senescence-associated ß-galactosidase activity in the developing avian retina.

BACKGROUND: Senescence-associated ß-galactosidase (SA-ß-GAL) histochemistry is the most commonly used biomarker of cellular senescence. These SA-ß-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-ß-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-ß-GAL detected in the developing retina corresponded to lysosomal ß-galactosidase activity, and that SA-ß-GAL activity did not correlate with the chronotopographical distribution of apoptotic cells. However, SA-ß-GAL staining in the undifferentiated retina coincided with the appearance of early differentiating neurons. In the laminated retina, SA-ß-GAL staining was concentrated in the ganglion, amacrine, and horizontal cell layers. The photoreceptors and pigment epithelial cells also exhibited SA-ß-GAL activity throughout retinal development. We have also found that SA-ß-GAL staining strongly correlated p21 immunoreactivity. CONCLUSION: In conclusion, the results clearly show that SA-ß-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.

Retinal histogenesis in an altricial avian species, the zebra finch (Taeniopygia guttata, Vieillot 1817).

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.

Expression and function of the LIM-homeodomain transcription factor Islet-1 in the developing and mature vertebrate retina.

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.

Islet-1 immunoreactivity in the developing retina of Xenopus laevis.

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.