José Antonio de Mera-Rodríguez

The avian retina presents an elegant and highly accessible model for investigating the spatial and temporal dynamics of neurogenesis within the central nervous system. In recent years, the microtubule cytoskeleton has emerged not merely as a structural scaffold, but as a dynamic regulator of the transition of a morphologically homogeneous pool of multipotent progenitor cells to postmitotic neurons. Doublecortin (DCX) is a microtubule‐associated protein broadly recognized for its fundamental roles in neuronal migration, differentiation, and structural plasticity within the developing nervous system. While its roles in the brain are well‐documented, the precise spatiotemporal distribution of DCX in the developing retina of birds remains poorly understood. In this study we investigated the expression pattern of DCX during embryonic development of the retina of one altricial (zebra finch, Taeniopygia guttata ) and two precocial (chicken, Gallus gallus ; Japanese quail, Coturnix coturnix ) bird species using immunohistochemical techniques. Our results demonstrated that the onset of DCX expression coincided chronotopographically with the onset of neurogenesis in the developing retina. In these early stages, DCX expression is tightly colocalized with the neuronal marker class III β‐tubulin. As retinal histogenesis progressed to more advanced stages, DCX expression persisted and segregated into specific subsets of ganglion, amacrine, and horizontal cells. Furthermore, prominent immunoreactivity was maintained within the ciliary marginal zone (CMZ), highlighting its association with late‐stage retinal progenitors and ongoing neurogenesis. These findings reveal a conserved spatiotemporal expression pattern of DCX across diverse avian species. Furthermore, it suggests that some elements of the cytoskeleton and its associated regulatory proteins form a functional continuous biological continuum: first acting as drivers of neurogenesis and migration and, subsequently, transitioning into structural stabilizers that lock in and maintain the complex morphology of fully differentiated, mature retinal neurons.

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.

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.

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–St20). 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 senescencein vivoin 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.

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.

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. 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. 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 sub‐populations of post‐mitotic neurons, which are non‐proliferative cells, even at early stages of cell differentiation.