All animals manipulations have been performed in accordance with the National and European legislation (Spanish Royal Decree RD53/2013 and EU Directive 86/609/CEE as modified by 2003/65/CE, respectively). Experimental protocols were approved by the Bioethics Committee for Animal Experimentation of the University of Extremadura (Ref 264/2019). Fertilized T. guttata (n = 63) and C. coturnix (n = 45) eggs were placed within a rotating egg incubator designed for chicken eggs (Masallés S.A.) and were maintained at 37.5 ± 1°C and 80–90% humidity. Eggs were placed in a small padded dish on the rotating bars of the incubator. They roll within the dish preventing embryo adhesion to the shell. A total of 34 T. guttata embryos and 34 embryos of C. coturnix and 2 newly hatched animals were used in the present study (Table 1). The oldest embryos of T. guttata were kindly provided by Dr. Michiel Vellema, from the University of Utrech, Netherlands. Zebra finch embryos were staged according to Murray et al. (2013). The degree of development of the quail embryos was estimated according to the stages established by Ainsworth et al. (2010). Embryos were fixed with paraformaldehyde (PFA) 4% in phosphate-buffered solution (PBS) (0.1M, pH 7.4) overnight at 4°C. For immunohistochemical techniques, embryos were immersed in a cryoprotective solution (15% sucrose in PBS) overnight at 4°C, soaked in embedding medium and frozen for 5 min in isopentane cooled to −70°C by dry ice, and then stored at −80°C until use. Cryosections of 20 μm were obtained in a cryostat microtome (Leica CM 1900), thaw-mounted on SuperFrost Plus slides, air-dried and stored at −20°C.

Sections were subjected to an antigen retrieval process with citrate buffer (pH 6.0) at 90°C during 30 min. Sections were chilled at room temperature for 20 min. Slides were washed one time in 0.1% Triton-X-100 in PBS (PBS-T) and two times in 0.2% gelatin, 0.25% Triton-X-100 in PBS (PBS-G-T) and then pre-blocked in 0.2% gelatin, 0.25% Triton-X-100, Lys 0.1 M in PBS (PBS-G-T-L) for 1 h. Sections were incubated with the primary antibody over night at RT in a humidified chamber:

After incubation with the primary antibody, slides were washed several times in PBS-T and PBS-G-T and incubated with Alexa Fluor 488 goat anti-mouse IgG antibody (1:200, Molecular Probes, A11029), Alexa Fluor 488 goat anti-rabbit IgG antibody (1:200, Molecular Probes, A11008) and Alexa Fluor 594 goat anti-rabbit IgG antibody (1:200, Molecular Probes, A11037) during 2 h at RT in a humidified chamber in darkness. Sections were washed two times in PBS-T and one in PBS-G-T in darkness. The tissue was incubated for 10 min with DAPI at RT and then washed three times in PBS. Slides were mounted with Mowiol (MKBD8495V, SIGMA).

Immunofluorescence sections were observed with a bright field and epifluorescence Nikon Eclipse 80i microscope and photographed using an ultra-high-definition Nikon DXM1200F digital camera. Images were processed with Adobe Photoshop CS4.

The eyes of the embryos were sectioned parallel to the sagittal plane (Howland and Howland, 2008), allowing the dorsal (D) and ventral (V) retinal regions to be distinguished when the optic nerve was sectioned (Figure 1). Sections were stained with the anti-pHisH3 antibody in combination with anti-Prox1 and anti-Isl1 antibodies. The D and V regions were divided into three equally large sectors by measuring the length of the inner limiting membrane (ILM): dorso-central (Dc-near the optic nerve exit), dorso-centro-peripheral (Dcp), Dorso-peripheral [Dp-near the ciliary marginal zone (CMZ)], Ventro-central (Vc), Ventro-centro-peripheral (Vcp), and Ventro-peripheral (Vp) (Figure 1). We captured fluorescence images of the entire retina using a 20x objective. As development progresses, a higher number of 20x images were included in each sector. The length of the ILM and the surface area of the retina in digital micrographs was measured using the ImageJ free open-source software package (¹ accessed on 28 January 2021). The density profiles were expressed as the mean ± sem of the number of pHisH3-positive mitoses per square millimeter (mitosis/mm²). Statistical analyses and the graphs were performed with the R programming language. To analyze if there were significative differences, the Mann–Whitney U test was used. Differences between groups were considered as significant when P < 0.05 and highly significant when P < 0.001.

Toluidine blue-stained semi-thin sections in the undifferentiated quail retina showed abundant mitoses mainly localized in the scleral surface of the neuroblastic layer (NbL), but sparse non-apical mitoses were also observed. We also evaluated the specificity of primary antibodies in the quail retina by comparison with published examples of labeling results and immunoassays in other bird species, and we found similar expression patterns for pHisH3, Prox1, and Isl1 antibodies (see Supplementary Figure 1). None of the observed staining was due to non-specific binding of secondary antibody or autofluorescence in the fixed tissues because sections labeled with secondary antibodies alone were devoid of fluorescence. Therefore, the location of mitotic activity and the immunohistochemical patterns observed in the developing quail retina were similar to that described in the retina of other bird species.

During retinogenesis, the nucleus of the retinal progenitors oscillates from the apical to the basal surface in proliferative neuroepithelia. The movement of the nucleus is in phase with the cell cycle, and M phase always occurs at the apical surface of the neuroepithelium (Baye and Link, 2007). Retinal progenitors either divide symmetrically to generate other progenitors or divide asymmetrically to generate both a progenitor and a postmitotic migrating neuroblast (Xiang, 2013). However, not all retinal progenitor divisions are restricted to the apical surface of the neuroepithelium, and committed horizontal cell progenitors divide in non-apical regions in the retina of fish (Godinho et al., 2007; Weber et al., 2014) and birds (Edqvist and Hallböök, 2004; Boije et al., 2009, 2016). Therefore, the pattern of cessation of neurogenesis could be monitored by the disappearance of mitotic figures in the retinal tissue (Sharma and Ehinger, 1997; Barton and Levine, 2008; Bejarano-Escobar et al., 2012).

In the present study we have shown that mitotic activity is abundant in the altricial retina even at hatching stages (see above). However, mitotic activity almost disappears from the apical surface in the entire quail retina at E10. Similar results were found when monitoring the disappearance of non-apical cell divisions. By these embryonic stages, the quail retina is fully differentiated and the main retinal cell types could be characterized immunohistochemically (unpublished observations, Javier Francisco-Morcillo). Then, our findings are in line with previous studies showing that neurogenesis ceases at embryonic stages in the precocial bird retina, and hatchlings present a mature and functional visual system (Fischer and Reh, 2000; Álvarez-Hernán et al., 2021a).

It has been described that during ontogeny, many morphological features begin earlier in the precocial bird species than in the altricial ones (Murray et al., 2013). In the case of the developing visual system, the formation of the optic cup, the formation of the optic vesicle, the onset of differentiation of ganglion cells and photoreceptors, and the emergence of the plexiform layers occur earlier in G. gallus than in T. guttata (Álvarez-Hernán et al., 2018, 2020). Here we also showed that the onset of the appearance of non-apical mitoses in T. guttata take place at E6 (St30), while in C. coturnix non-apical mitoses were numerous at E5 (St27). Furthermore, the density of non-apical mitosis in the central region of the T. guttata retina reached a peak at E8-9, whereas the highest density values were found by E7.5-8 in C. coturnix (present study) and in G. gallus (Boije et al., 2009). Therefore, the onset of appearance of non-apical mitoses occurs at later stages in the zebra finch than in the quail.

Non-apical mitoses have been linked to horizontal cell differentiation in fish (Godinho et al., 2007; Weber et al., 2014; Engerer et al., 2017) and birds (Edqvist and Hallböök, 2004; Boije et al., 2009, 2016; Shirazi Fard et al., 2013, Shirazi Fard et al., 2014a,b). We used specific markers of horizontal cells to establish a possible relationship between non-apical mitosis and horizontal cell differentiation. Prox1 is considered a pan-marker of horizontal cells, whereas the transcription factor Isl1 is expressed by the axon-less horizontal cell subtype (Edqvist et al., 2008; Boije et al., 2009). A similar staining to that described by those authors in the chicken retina has been detected in the T. guttata (Álvarez-Hernán et al., 2020) and in the C. coturnix retinal tissue (present study). Although we never detected co-labeling of these horizontal cell markers with non-apical pHisH3 mitoses, there was a close spatiotemporal relationship between the onset of the appearance of non-apical pHisH3-immunoreactive mitoses and the onset of Prox1-expression in the different retinal sectors, in concordance with the results obtained in the chicken (Edqvist et al., 2008; Boije et al., 2009). These findings suggest a possible relationship between the appearance of non-apical mitoses and horizontal cell differentiation in the retina of both bird species. Again, these results may suggest that the onset of horizontal cell differentiation is delayed in the altricial bird retina.

In conclusion, the present study provides comprehensive data on distinct patterns of retinal development between the altricial zebra finch and the precocial Japanese quail, which may serve as empirical reference in future studies. While the order of different events involved in retinal maturation is the same during development, their specific timing differs between the altricial and precocial bird species. The retina of the precocial birds exhibits higher degree of maturation at hatching, thus providing evidence for the notions that precocial species might have acquired the morphological machinery required to attain their higher functional state at hatching. Together, our results expand our current understanding of the timing and cellular differences that regulate patterns of avian retinal growth and maturation, and provides a better understanding of the evolution of avian altriciality and precociality.