The animal model: Xenopus laevis and Hoplobatrachus rugulosus
The African clawed frog Xenopus laevis is an amphibian species and frequently used in laboratory research for developmental biology and cell biology (Feehan et al., 2017). There are several proper properties for Xenopus to be used. The first reason is that standard protocols for Xenopus laboratory breeding and husbandry are available, which is comparably simpler and with lower cost than those required by rodents.
Secondly, it is easy to gain eggs and embryos because females lay large clutches of thousands of eggs and egg laying could be inducible by hormone priming (Browne et al., 2006).
On the other side, unlike X. laevisis who are native to sub-Saharan Africa,
Hoplobatrachus rugulosus’ natural habitats are in East Asian freshwater marshes, arable
land, pasture land, ponds, irrigated land, seasonally flooded agricultural land, and so forth. They are more familiar to us rather than Xenopus. This species is commonly referred to as being “field chicken” or “tiger-skinned frog” in Taiwan. Moreover, this thesis will refer to H. rugulosus as “tiger-skinned frog”. H. rugulosus is a kind of large, robust frog. In snout-vent length, males are about 6 to 8 cm, and females are 6 to 12 cm.
They can grow up to 15 cm. Observed from the appearance, the head length is slightly
larger than the head width, and the snout is sharp and long. Females are usually larger than males. This species of frogs’ breeding season is from spring to early summer. They are primarily carnivore. Their color is diversity, from yellow-green to dark gray. Also, they have dark speckles (Diesmos et al., 2004; Lin and Ji, 2005; Lue, 2012). When they are tadpoles, they are larger than other frogs. Their total length is about 5 cm and the tail length can be twice as long as the body length. Their back is green-brown with some small black spots, and on the eyes and mouth, some golden yellow spots can be observed. The tadpoles are demersal and likes to live in still waters, so they could usually be seen in ponds (Chou and Lin, 1997). In addition, their developmental processes are similar to X. laevisis.
Retina
Adult frogs are essentially dependent on vision. They feed on worms, flies, and other insects which they catch them by striking with the tongue or directly with the mouth. For this functional reflex, they use visual clues. Furthermore, frogs prey only on moving insects, and their attention is never attracted by stationary creatures or objects (Maturana et al., 1960). Moreover, when tiger-skinned frog are tadpoles, they could exercise all day, not limited by day-night cycle. Besides the eye vision, tadpoles
could also use the electrotactile stimulation of the skin to transduce visual data
(Blackiston and Levin, 2013). The function of the eyes is important for the daily life of adult frogs.
Looking into the eye, the amphibian retina shows the typical five-layered structure of vertebrates (Fig. 1). The outer nuclear layer (ONL), the inner nuclear layer (INL), and the ganglion cell layer (GCL) are separated by two fiber layers: the outer plexiform layer (OPL), which is thin, and the much thicker inner plexiform layer (INL). The two plexiform layers are the main site of synaptic contacts between five major types of retinal cells: photoreceptors, bipolar cells, ganglion cells, horizontal cells, and amacrine cells. (Heatwole et al., 1998; Purves et al., 2001). In the previous study, the ONL was formed by two layers of cell bodies in most frogs and salamanders. The rod cells nuclei are aligned at the distal side of the ONL, and the cone cells nuclei are more proximal (Gordon and Hood, 1976). In most other vertebrates, it is the opposite situation. Another special point is that the GCL consists of more than one row of ganglion cells in the amphibian (Heatwole et al., 1998).
Unlike primates or birds, there is no fovea in the retina of the amphibian. Only a few species even have a concentrated area sophisticated enough to be considered an area centralis, an analogue of the macula (Schwab, 2004). In some frogs, Hyla raniceps
(Bousfield and Pessoa, 1980), Heleioporus eyrie (Dunlop and Beazley, 1981), Bufo marinus (Nguyen and Straznicky, 1989), a streak of high cell density exists in the GCL.
The same situation expresses in the ONL (Zhang and Straznicky, 1991) and the INL (Zhu et al., 1990).
Photoreceptors
The eye is a specialized organ whose job is to convert or transduce light energy into neural impulses. A photoreceptor cell is a specialized type of neuroepithelial cell found in the retina that is capable of visual phototransduction. Photoreceptor cells of the vertebrate retina are highly specialized cells which respond to the stimulus of light, and transmit this response to adjoining neurons for ultimate relay to the visual centers of the brain (Young, 1967). Photoreceptor cells of amphibians, like those of other vertebrates, exhibit two main cells, rod cells and cone cells.
(i) Rod cells
In vertebrates, rod cells can functionalize in less intense light than the other types of visual photoreceptor. So, rods are responsible for dusk or even night vision.
Also, the rod pigments have a maximum absorption at 496 nm of visual spectrum and the image provided is one composed of gray tones, meaning that for seeing in black
and white (Pawlina et al., 2018). As for location, in the human eye, rods can be found everywhere in the retina, except in and near the fovea, and are used in peripheral vision. The number of rod cells is much higher than that of cone cells. There are approximately 90 million rod cells in the human retina (Curcio et al., 1990).
In the amphibians, rod cells are the largest photoreceptors and the most frequent receptors (Heatwole et al., 1998; Yovanovich et al., 2017). Rods are named after the cylindrical shape of their outer segments. The outer segment is the photosensitive region of the photoreceptor, and it is densely filled with free-floating disks formed by a double-layered membrane. As the reaction to light and darkness, photoreceptors can change their position. The myoid, the area between ellipsoid and nucleus, can be shortened or lengthened (Heatwole et al., 1998). The rod cells are narrower than the cones and distributed differently across the retina (Fig. 2).
Different from typical mammals which have a brightness-sensitive system and a three-color cone system, rods and cones of frog retina are able to both distinguish color (Denton and Wyllie, 1955; Heatwole et al., 1998; Yovanovich et al., 2017).
There are two types of rod cells, red rods and green rods. The most frequent
photoreceptors are red rods. The pigment of red rods in frogs is rhodopsin, which has its maximum absorbance in the green spectrum at about 502 nm. The green rods
contain the pigment with an absorbance at 432 nm, which is similar to the blue-sensitive cones in mammals, and have much shorter outer segments (Denton and Wyllie, 1955; Kelber et al., 2003; Yovanovich et al., 2017).
(ii) Cone cells
As opposed to rod cells, cones perform best in relatively bright light and are more sensitive to red, green, and blue regions of the visual spectrum in mammals.
Therefore, cones exist in three classes: L (588 nm), M (531 nm), and S (420 nm) (long-, middle-, and short-wavelength sensitive, respectively), but cannot be distinguished morphologically (Pawlina et al., 2006). In earlier studies, there were found to be about 4 to 7 million cones in a human eye and most are concentrated towards the macula (Osterberg et al., 1935; Curcio et al., 1990). Cone cells are densely packed in the fovea centralis, a nearly 0.3 mm diameter rod-free area with thin, densely packed cones which reduce in number towards the periphery of the retina. From shape, the outer segment of cone cells is much shorter and cone-shaped (Fig. 2).
Cone cells could be differentiated into two types, single and double in frog retina (Bowmaker, 1990; Heatwole et al., 1998; Pignatelli et al., 2010). The cone pigment is sensitive to yellow light at about 575 to 580 nm. The component of a double cone cell
carries a typical single cone and an accessory cone which pigment is the green-sensitive rhodopsin of the red rod (Heatwole et al., 1998). While the outer segments of the double cone cells are separated, the inner segments are fused.
The organization and function of the pineal gland
The pineal gland, also called the median third eye, develops as an evagination of the neuroepithelium of the dorsal diencephalon and serves as a photoreceptive
neuroendocrine organ (Sapede and Cau, 2013). The shapes and organizations of pineal glands depend on different species. In cold-blooded animals, pineal gland is located beneath the skull and is connected to the diencephalon by a stalk (Collin, 1971;
McNulty, 1984). The pineal of fish and frogs are a vesicle connected to the roof of the diencephalon by a slender stalk, and the lumen is opened to the third ventricle and is thus filled with cerebrospinal fluid (CSF) (Falcón et al., 1992; Omura and Oguri, 1969).
In lizards and avian, the pineal gland becomes follicular. In mammals, the pineal gland becomes glandular and more compact (Collin, 1971; Falcon, 1999; Vollrath, 1981).
In some of the lower vertebrates, pineal gland has the sensory function of light.
Since, this kind of pineal gland has the existence of photoreceptors, and is superficially situated in the brain, it is so-called the parietal eye (Kappers, 1979). Although the pineal
glands of postnatal mammalians no longer have photoreceptors, there are other nerve conduction pathways that indirectly affect the pineal gland for photosensory responses (Klein, 1985; Relkin, 1966). The mammals’ pineal gland is only endocrine. The function of the pineal gland is to act as the endocrine gland that secretes melatonin (N-acetyl-5-methoxytryptamine). Melatonin can regulate the circadian rhythm of the organism due to the difference in light duration, which includes changes in the daily cycle and seasonal changes (Falcón, 1999).
Intermediate filaments
Intermediate filaments (IFs) (~10 nm diameter) are expressed in cell-, tissue- and differentiation-specific function and play important roles in maintaining the mechanical stability through interconnection with other filamentous systems and provide
specialized functions via decorated by a variety of proteins (Oshima, 2007; Toivola et al., 2005). The IF proteins exhibit a common tripartite domain structure, with non-helical amino (head) and carboxyl-terminal (tail) domains flanking a central coil-coil α-helical core region (310-352 amino acids) (Kim and Coulombe, 2007; Sihag et al., 2007). IFs have been classified into six types based on the gene structure and amino acid sequence. IFs of neurons, or neuronal intermediate filaments (nIFs), are made of
five subunit proteins: the low, middle and high molecular mass neurofilament (NF) triplet proteins (designated as NF-L, NF-M and NF-H), α-internexin and peripherin (Thyagarajan et al., 2007). These proteins provide support and scaffold in outgrowth and stabilization of axon and dendrite (Lariviere and Julien, 2004). The developing nervous system undergoes progressive changes in the molecular composition of nIFs. α-Internexin, a 66 kDa nIF protein, is wildly expressed from early development through adulthood. Its expression in developing central nervous system (CNS) of mammals precedes the NF triplet proteins, arrives at its highest level before birth and declines postnatally (Fliegner et al., 1994; Kaplan et al., 1990). In adult mammals, chickens, and zebrafish, α-internexin has been found in retina, olfactory bulb, telencephalon, optic tectum and cerebellum (Fliegner et al., 1994; Chien and Liem, 1995; Chien et al., 1996;
Chien et al., 1998; Liu and Chien, 2013; Liu et al., 2013; Liao et al., 2016; Liao et al.,2018; Hao, 2018). These studies suggest that α-internexin might play a key role in neuronal cytoskeleton network during development.
The neuronal intermediate filaments (nIFs) in frog
According to the GenBank of NCBI (http://www.ncbi.nlm.nih.gov/genbank/), there are three α-internexin-like (ina.S, nif.L and nif.S), two NF-M, one NF-L and two
peripherin (plasticin) genes in the X. laevis. These nIF proteins could be classified into the same nIF categories, except nif.L (Charnas et al., 1992; Klein et al., 2002;
Strausberg et al., 2002) and nif.S (Klein et al., 2002; Strausberg et al., 2002). Some of the general features of expression of these nIF proteins have been documented in X.
laevis, including ina.S (internexin neuronal intermediate filament protein alpha S
homolog, known as xefiltin, an ortholog of mammalian α-internexin), nif.S (low molecular weight neuronal intermediate filament S homolog, also known as xefiltin), NF-M and NF-L (Sharpe et al., 1989; Gervasi and Szaro, 1997; Zhao and Szaro, 1997a;
b; Gervasi et al., 2000; Klein et al., 2002; Strausberg et al., 2002). However, to our best knowledge, there are very few studies to investigate what the role the nIFs play in the development of photoreceptors of frogs, so based on the phylogenetic analysis of Xenopus nIFs both ina.S and nif.S are candidates for studying the nIFs in the development of retina and pineal gland in tiger-skinned frogs.
Specific Aims
In previous studies of our laboratory, the distribution of α-internexin, inaa and inab, both the ortholog of mammalian α-internexin, could be discovered in the developing zebrafish pineal glands. In addition, inaa was found in cone photoreceptors of zebrafish
retina and also distinctively expressed in the photoreceptor-like cells of pineal gland, where inab was sparsely detected (Liao et al., 2016; Liao et al., 2018). Also, the expression of chicken α-internexin (chkINA) could be detected in the early stage of developing brains and found as the major IF protein in the parallel processes of
cerebellar granule neurons (Liu and Chien, 2013). Moreover, chkINA was expressed in all neuronal lineages of the developing chicken retina and pineal gland (Liu et al., 2013;
Hao, 2018). In the mouse model, α-internexin was reported to be detected postnatally in some pinealocytes and nerve processes (Ko et al., 2005). Moreover, some of
pinealocytes are shown to have the similar function to the photoreceptor of retina in many non-mammal vertebrates (Klein, 2004). However, from an evolutionary point of view, there is still lack of evidence to illustrate the temporal and spatial distribution of frog α-internexin protein in the developing pineal gland. The aims of this study are to characterize the expression patterns of α-internexin-like proteins, ina.S and nif.S, and to examine which are expressed in the pineal photoreceptor-like cells in the developing pineal gland of frog H. rugulosa. The photoreceptor markers, recoverin and XAP1, are also applied for the study of the developing retina and pineal gland in tiger-skinned frogs.
Chapter 2: Materials and Methods