Expression levels of ina.S and nif.S proteins in developing tiger-skinned frog tissues
To certify generated polyclonal anti-ina.S and anti-nif.S antibodies could be expressed in developing tiger-skinned frog tissues, western blotting were performed for protein identification. Brain, retina and liver were taken and extracted. The brain tissues were specified in diencephalon including pineal gland. St.46, st.54, st.58 and adult were selected to perform the stages from developing to mature (Fig. 6). Immunoblotting used anti-ina.S antibody predicted 55-60 kDa and anti-nif.S predicted 53-54 kDa. The result revealed that both ina.S and nif.S could be observed in frog brain and retina.
Immunohistochemical patterns of photoreceptor marker, recoverin and XAP1 in developing tiger-skinned frog retina
Previous study has shown that the antibody against recoverin labels only cone cells in mouse retina (Biswas et al., 2014). Also, XAP1 reactivity was primarily expressed on the surfaces of the outer segments of both rods and cones in Xenopus (Harris et al., 1992). In the study, these two antibodies, recoverin and XAP1, were used in immunohistochemistry staining to recognize tiger-skinned frog retina. The
immunofluorescence staining of recoverin and XAP1 were performed at retina of tadpoles at st.46, st.54, st.58, young adult and adult. The immunoreactivity of recoverin could be detected in the cell body of photoreceptors in outer nuclear layer in retina (Fig.
7, A-E) in every stages of frogs that were collected. Same as the patterns of recoverin in developing retina, the immunoreactivity of XAP1 could be observed in the outer
segment of photoreceptor (Fig. 7, A’-E’). The result demonstrated that the photoreceptor cells in tiger-skinned frog retina could be identified by both recoverin and anti-XAP1 antibodies.
Immunohistochemical patterns of ina.S in developing tiger-skinned frog retina
In previous studies, both zebrafish inaa (Liao et al., 2016) and chicken α-internexin (chkINA) (Liu et al., 2013; Hao, 2018) could be identified in the photoreceptor-like cells in both retina and pineal gland. To test whether the antibody of ina.S is able to identify photoreceptor-like cells, we used epitope-specific polyclonal anti-ina.S antibody to exam the patterns of tiger-skinned frog retina via immunohistochemistry.
The retina of tadpoles at st.46, st.54, st.58, young adult and adult were performed by the immunofluorescence staining. The immunoreactivity of ina.S could be found only in the
retinal neurons of ganglion cell layer and inner nuclear layer, but not in photoreceptors (Fig. 8, A-E). The distribution pattern of ina.S is similar to that of zebrafish inab (Liao et al., 2016).
Immunohistochemical patterns of nif.S in developing tiger-skinned frog retina
Since anti-ina.S failed to be identified in photoreceptors of frog retina, another α-internexin-like protein, frog nif.S, were tried to examine the immunoreaction in frog retina by way of immunohistochemistry. The stages of immunofluorescence staining were performed at tadpoles at st.46, st.54, st.58, young adult and adult. The
immunopositive staining of nif.S in the retina could be detected not only in ganglion cell layer and inner nuclear layer, but also in the outer segment of photoreceptors (Fig.
9, A-E). From a single optic section of the confocal image (Fig. 9, F”), nif.S could be colocalized with XAP1 in the outer segment of photoreceptors of adult retina. Based on this observation, this study suggests that the distribution pattern of nif.S is similar to that of zebrafish inaa (Liao et al., 2016) and chkINA (Liu et al., 2013; Hao, 2018).
Immunohistochemical patterns of photoreceptor marker, recoverin and XAP1 in developing tiger-skinned frog pineal gland
Pineal tissues from five stages of frog brains were collected and sectioned:
tadpoles at st.46, st.54, st.58, young adult and adult. The immunostaining of anti-recoverin could be detected within pineal tissues at st.46 (Fig.10a, A), and decreased at the following developmental stages (Fig. 10a, B-E). The expression of XAP1 could be detected in the photoreceptor-like cells of pineal gland at all stages (Fig. 10a, B’-E’).
The immunopositive staining of recoverin could be found in some fields of adult pineal gland (Fig. 10b, G), but failed to be colocalized with XAP1 (Fig. 10b, J, white arrow).
Furthermore, according to statistical analysis, the distribution patterns of XAP1 in the developmental pineal gland did not show any significant changes (P>0.05) (Fig. 10b;
11b; 12b, F). From the results, it is suggested that some photoreceptor cells might exist in frog pineal gland during development.
Immunohistochemical patterns of ina.S in developing tiger-skinned frog pineal gland
From the previous studies, the distributions of inaa and inab, both the ortholog of mammalian α-internexin, could be discovered in developing zebrafish pineal gland
(Liao et al., 2016; Liao et al.,2018), and chkINA could be identified in the developing chicken retina as well as pineal gland (Liu et al., 2013; Hao, 2018). Therefore, we further examined the distribution patterns of internexin neuronal intermediate filament protein alpha S homeolog, ina.S in frog pineal gland via immunohistochemistry. The immunofluorescence staining of ina.S was performed at pineal gland of tadpoles at st.46, st.54, st.58, young adult and adult. The immunopositive staining of anti-ina.S could be rarely detected in the development of pineal gland (Fig. 11a, A’-E’). The immunopositive staining of ina.S could be found in some observation fields of adult pineal glands (Fig. 11b, G). However, ina.S could not be colocalized with XAP1 within pineal tissues from this study’s observations (Fig. 11 b, J, white arrows).
Immunohistochemical patterns of nif.S in developing tiger-skinned frog pineal gland
To examine the expression pattern of nif.S in the developing pineal gland of frogs, this study performed immunostaining of anti-nif.S antibody at pineal gland of tadpoles at st.46, st.54, st.58, young adult and adult. The expression of nif.S could be found in some cells of the early developing pineal gland (Fig. 12a, A-B), but decreased in the following stages (Fig. 12a, C-E). From the observation, the immunopositive staining of
nif.S could be only found in some areas in the pineal tissues from adults (Fig. 12b, G).
Yet, it failed to be colocalized with XAP1 (Fig. 12b, J, white arrow). Based on this study observations, it can be suggested that anti-nif.S might not be as good as a photoreceptor marker for identifying photoreceptor-like cells in pineal gland of tiger-skinned frogs.