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. 2023 Jan 28;242(6):1051–1066. doi: 10.1111/joa.13835

Embryonic and skeletal development of the albino African clawed frog (Xenopus laevis)

Zhixin Shan 1, Shanshan Li 1, Chenghua Yu 2, Shibin Bai 1, Junpeng Zhang 1, Yining Tang 1, Yutong Wang 1, David M Irwin 3, Jun Li 2,✉, Zhe Wang 1,✉
PMCID: PMC10184547  PMID: 36708289

Abstract

The normal stages of embryonic development for wild‐type Xenopus laevis were established by Nieuwkoop and Faber in 1956, a milestone in the history of understanding embryonic development. However, this work lacked photographic images and staining for skeleton structures from the corresponding stages. Here, we provide high‐quality images of embryonic morphology and skeleton development to facilitate studies on amphibian development. On the basis of the classical work, we selected the albino mutant of X. laevis as the observation material to restudy embryonic development in this species. The lower level of pigmentation makes it easier to interpret histochemical experiments. At 23°C, albino embryos develop at the same rate as wild‐type embryos, which can be divided into 66 stages as they develop into adults in about 58 days. We described the complete embryonic development system for X. laevis, supplemented with pictures of limb and skeleton development that are missing from previous studies, and summarized the characteristics and laws of limb and skeleton development. Our study should aid research into the development of X. laevis and the evolution of amphibians.

Keywords: embryonic development, skeletal development, skeleton staining, Xenopus laevis

The albino mutant of Xenopus laevis avoids the influence of pigmentation on the observation of development process; The albino mutant of X. laevis makes the results of histochemical experiments easier to be interpreted. The description of albino embryos and its skeleton development provides valuable information for the related research based on X. laevis model.

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1. INTRODUCTION

As a representative amphibian, Xenopus laevis is an important model organism for research, with advantages such as being easy to breed and laying eggs all year round (Liu et al., 2016). The diameter of X. laevis eggs and early embryos is about 1.1–1.3 mm, which aids in experiments such as microinjection, embryo cutting, and transplantation (Beck & Slack, 2001; Nakayama et al., 2020). Moreover, it is easy to observe and manipulate Xenopus embryos at all stages of development, thus they can be used in studies in basic and molecular biology, developmental biology, regenerative medicine, and toxicology (Bantle et al., 1999; Saide et al., 2019; Wagner et al., 2017). Xenopus are increasingly being used to model Human diseases (Kostiuk & Khokha, 2021). Extracts from Xenopus eggs have contributed to our underdoing of the maintenance of the genome as these extracts contain all factors required for extracellular DNA repair (Hoogenboom et al., 2017). The limbs and tail of X. laevis can regenerate, which has allowed this animal to become a valuable tool for studies in development and evolution (Phipps et al., 2020; Slack et al., 2004). X. laevis is an important model for studying spinal cord formation and development, brain development, and studies into the regeneration of the central nervous system (Borodinsky, 2017; Exner & Willsey, 2021). Xenopus embryos can also be used in chemical screens for new drugs (Schmitt et al., 2014). Eggs are also used in studies of mechanical stress and cell division (Stooke‐Vaughan et al., 2017).

The extracellular development of X. laevis embryos has made it an excellent model for understanding development (Borodinsky, 2017). The stages of normal embryonic development in wild‐type X. laevis were established by Nieuwkoop and Faber in 1956 and described in the book Normal Table of Xenopus laevis (Daudin) (hereafter referred to as Normal Table) (Nieuwkoop & Faber, 1994), one of the most widely cited works in vertebrate development. The most commonly used part of this book is a series of drawings of the changes seen in embryos as they develop from egg to adult, although studies on X. laevis development are ongoing. High‐definition color images of wild‐type X. laevis embryonic developmental stages 1–50 are provided in Xenbase (http://www.xenbase.org), which is a database of biological information for X. laevis and X. tropicalis (Fortriede et al., 2020; Karimi et al., 2018; Nenni et al., 2019). In addition, high‐quality schematic diagrams of the craniofacial development of tadpoles from stages 22 to 50 were drawn in 2017 (Zahn et al., 2017), with the authors hoping that additional views and stages would be described in the future. One hundred and thirty three new, high‐quality illustrations of X. laevis development, from fertilization to metamorphosis, were recently released (Zahn et al., 2022). With regard to research on skeletal development in X. laevis, the development of cartilaginous head morphology after stage 46 has been described in detail (Candioti, 2007; Trueb & Hanken, 1992). The developmental sequence and timing of stages 37–48 for the cartilaginous skull of X. laevis have also been described (Lukas & Olsson, 2018). Moreover, changes of Meckel's cartilage during metamorphosis in X. laevis were also summarized in a previous study (Thomson, 1986). Shoulder development, cartilage formation, ossification, and bone histological pattern of the chest band in X. laevis have also been reported (Shearman, 2008). The development of the pelvis and related structures (sacrum and iliosacral joint, etc.) of these animals, represented by seven types of tailless movement, has been described (Rocková & Rocek, 2005). These events mostly occur in stages 52–66 of Xenopus development.

Although there have been many studies on development in X. laevis, a complete description of the biological morphology and skeleton development processes for all stages of development has not yet been produced. Moreover, the albino Xenopus tadpole, which has less pigmentation, should be more helpful for these studies, as lower pigment levels should allow better observation of the development of tissues and organs with an anatomical microscope (Mogi et al., 2012). In addition, the lower levels of pigment allow more pronounced signals to be observed with in situ hybridization (Pera et al., 2015). Therefore, the goal of this study was to provide a complete characterization of embryonic development in the albino X. laevis. We include pictures for stages 51–66 that are not available in the Xenbase database, and also describe skeletal development after stage 40 in detail. Compared with the staging table in the Normal Table (Nieuwkoop & Faber, 1994), we add high‐definition images for each period and skeleton staining images after stage 40. Our work provides clear and intuitive images for embryonic and skeletal development that complement previous studies and provide a morphological basis for further studies on the development and evolution of amphibians.

2. MATERIALS AND METHODS

2.1. Samples

Experiments were carried out with albino X. laevis, which were obtained from an aquarium shop in Shenyang City. During mating, male and female frogs are padded with a support in a breeding box to prevent the parents from eating the eggs after laying (Figure 1).

FIGURE 1.

FIGURE 1

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Adult male and female albino Xenopus laevis. The photograph shows mating by male and female albino Xenopus laevis (with the smaller male above and the female below). White size bar = 2 cm.

2.2. Incubation and staining

Fertilized eggs were allowed to develop in a tadpole breeding house that was illuminated with biological lamps for 12 h followed by dark for 12 h each day. Air temperature was maintained at 24°C while the water temperature was kept at 23°C. The water tank was equipped with a heating thermostat. Tadpoles were raised to adults with feeding according to the description of Ishibashi and Amaya (2021). To reduce pain, operations were carried out under anesthesia. Male adults and embryos were anesthetized with 0.5% MS222. After anesthesia, embryos used for the collection of morphological images were fixed with 4% paraformaldehyde for 1 day, and embryos used for skeleton staining were fixed with 10% neutral formalin for 2 days. Staining of whole embryos followed the method of Wassersug with some modifications (Wassersug, 1976). Briefly, before dyeing the cartilage, samples were dehydrated through two changes with absolute ethanol. The concentration of Alcian blue 8GX used in our studies was reduced to 3 mg. For the stage 40–49 embryos, internal organs were not removed due to their small size. Embryos were stained with Alcian blue for about 12 h. If the blue was too dark, we washed them in 5% acetic acid for 2 h. For embryos after stage 50, the staining time was extended to 1 day, but not more than 2 days. Specimens were transferred to absolute ethanol for dehydration. Sample obtained in the previous step were then immersed in a solution containing 0.5% KOH and 0.005% Alizarin red for about 12 h. The staining of the samples can be observed during the dyeing process. When the bone has been sufficiently dyed, samples were removed from the staining solution to stop the dyeing processes and placed in a 25% glycerol solution containing 0.5% KOH. Glycerol gradients of 50% and 75% containing 0.5% KOH were then used for cleaning and to increase transparency. Detailed pictures of the limbs from stages 51 to 66 were collected on agarose. Due to the large degree of bending of the hind limbs of the tadpole during the metamorphosis stage, it is difficult to place the whole hind limb on a plane under the microscope, thus, only part of the hind limb is intercepted in the images (focusing on the soles of the hind limbs), while the complete hind limb can be seen in the lateral view. Pictures of embryo stages 1–50 and skeleton staining were obtained using a Leica stereomicroscope (M165FC; Leica).

3. RESULTS

Compared with the description of wild‐type X. laevis embryos, most of the characteristics of embryonic development of albino embryos are similar (Nieuwkoop & Faber, 1994), therefore, we only briefly describe the appearance of specific characteristics for each period here. However, full details of limb and skeleton development in these stages are presented. Since newly fertilized eggs have no pigment deposition, we cannot identify the dorsoventral side by color in stages 1–20. All of our partial pictures focus on the right limbs of the individual.

During stages 1 through 20, the embryo's shape basically does not change and remains round or oval (Figures 2 and 3). Stage 1 is just fertilized. From stages 2 to 6, the embryo undergoes cleavage. Stages 7–9 are the blastula, stages 10–12 are the gastrula and stages 13–20 are the neurula.

FIGURE 2.

FIGURE 2

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Embryonic development of albino Xenopus laevis at stages 1–10. Stage is indicated to the left of a pair of images for each stage: (A1–J1) and (A2–J2). Arrow above in (C1) identifies dorsal blastomeres. Arrow below in (C1) identifies ventral blastomeres. Arrows in (J1, J2) identify the visible blastopore lip. Bar in each image = 1 mm.

FIGURE 3.

FIGURE 3

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Embryonic development at stages 11–20. Stage is indicated to the left of a pair of images for each stage: (A1–J1) and (A2–J2). Arrows indicate: (C1) beginning of the neural plate; (D1) the blastopore is slit shaped; (D2) neural grooves that are elevated; (E2) visible neural folds; (F1) rectangular neural plate; (G1) long triangular anterior neural plate; (H2) parallel neural folds that have not yet touched; (J1) two eye anlagen that have appeared. Bar in each image = 1 mm.

Stage 1 (0 h): One cell stage, just after fertilization. The albino embryo has no pigmentation and the dorsal and ventral sides cannot be distinguished. The color of the fertilized egg is yellow to white and has a diameter of about 1.1–1.3 mm (Figure 2A1,A2).

Stage 2 (1.5 h): The 2‐cell stage. The first cleavage groove is clearly visible (Figure 2B1,B2).

Stage 3 (2 h): The 4‐cell stage. The second cleavage groove is obvious with the blastomeres on the dorsal side being smaller than those on the ventral side (Figure 2C1,C2).

Stage 4 (2.25 h): The 8‐cell stage. Blastomeres on the dorsal side being smaller than those on the ventral side (Figure 2D1,D2).

Stage 5 (2.75 h): The 16‐cell stage. Animal pole blastomeres are smaller than the vegetal pole (Figure 2E1,E2).

Stage 6 (3 h): The 32‐cell stage. The blastomeres are arranged in four rows, with eight in each row. Similarly, blastomeres on the dorsal side are smaller than those on the ventral side (Figure 2F1,F2).

Stage 7 (4 h): From this stage, the embryo becomes a blastula. Blastomeres are larger on the ventral side than on the dorsal side. About 10 small blastomere are distributed along the meridian from the animal's view (Figure 2G1,G2).

Stage 8 (5 h): Mid blastula stage. Cells become smaller and the surface of the embryo is not completely smooth (Figure 2H1,H2).

Stage 9 (7 h): Late blastula stage. There is no pigmentation in the embryo. Compared with wild‐type embryos, no pigmented lines show on the blastopore. Animal cells are smaller on the dorsal side than on the ventral side (Figure 2I1,I2).

Stage 10 (10 h): The initial gastrula stage. Blastopore groove forming (Figure 2J1,J2).

Stage 11 (11.75 h): With the development of the embryo, the yolk plug usually has a rounded rectangular shape that is slightly longer in the dorsum and ventral direction, with a diameter greater than about 2/5 of the egg diameter (Figure 3A1).

Stage 11.5 (12.5 h): The yolk plug became smaller, but is not very round. The diameter of the yolk plug is equal to or greater than 1/3 of the egg diameter (Figure 3A2).

Stage 12 (13.25 h): Medium yolk plug stage. No pigmented lines or pigmented area. The yolk plug is rounded and slightly less than 1/4 of the egg diameter (Figure 3B1,B2).

Stage 13 (14.75 h): From this stage, it became the neurula. The neural plate boundary is fuzzy. The anterior part is slightly raised and wide, and the posterior part is slightly flattened and narrow. The median groove formed on the caudal part. (Figure 3C1,C2).

Stage 14 (16.25 h): The median eminence is at the end of the median groove and the brain region of the neural plate is bent anteriorly. The initial neural folds are elevated, and the blastopore is slit shaped (Figure 3D1,D2).

Stage 15 (17.5 h): Neural folding stage. The anterior part of the neural plate is round, and the neural folds become obvious (Figure 3E1,E2).

Stage 16 (18.25 h): Middle stage of neural folding. The anterior part of the neural plate is rectangular, and the neural plate is contracted significantly in the middle. The elevation of the neural folds is becoming more obvious (Figure 3F1,F2).

Stage 17 (18.75 h): Anterior part of the neural plate is a long triangle. Neural folds are close to each other (Figure 3G1,G2).

Stage 18 (19.75 h): Anterior part of the neural plate narrows, usually toward the rostral end. Neural folds in the trunk area are parallel and very close to each other (Figure 3H1,H2).

Stage 19 (20.75 h): Neural folds are in contact with each other, with the brain becoming greatly extended. The outer contours of the embryo are still prominent (Figure 3I1,I2).

Stage 20 (21.75 h): Neural folds still have sutures. Two eye anlagen can begin to be seen. From the dorsal view, the embryo begins to extend (Figure 3J1,J2).

From stage 21, we mark the lateral, dorsal and ventral views to allow the description of more features. From stages 21 to 39, the embryo gradually grows, and the tail bud begins to appear as it gradually becomes a tadpole (Figures 4 and 5).

FIGURE 4.

FIGURE 4

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Embryonic development at stages 21–30. Stage (S) is indicated to the left of three images for each stage. (A1–I1) Lateral views of the entire embryo; (A2–I2) dorsal views of the entire embryo; (A3–I3) ventral views of the entire embryo. Arrows indicate: (A2) primary eye vesicles (arrow) and neural tube suture (triangular arrow); (B2) initial groove between gill area and jaw; (B3) cloaca opening; (C1) eyes protrude; (E2) gill area groove; (G1) the tail bud forms an inconspicuous contour on the side; (I1) inner translucent band (arrow) and outer transparent band (triangular arrow). Bar in each image = 1 mm.

FIGURE 5.

FIGURE 5

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Embryonic development at stages 31 to 39. Stage (S) is indicated to the left of three images for each stage. (A1–F1) Lateral views of the entire embryo; (A2–F2) dorsal views of the entire embryo; (A3–F3) ventral views of the entire embryo. (F1) Arrow indicated the 135° angle formed by the proctodeum and the tail. Bar in each image = 1 mm.

Stage 21 (22.5 h): From the dorsal view, the neural tube suture is almost closed. Eyes begin to bulge and the outer contour of the embryo is slightly concave. The outline of the abdomen is almost flat. Though hard to see, approximately 8–9 somites are segregated at this stage (Figure 4A1–A3).

Stage 22 (24 h): Eyes are prominent. Outline of the embryo's abdomen is more concave. The groove between the jaw and gill region can be seen on the lateral and dorsal sides. From the ventral view, the opening of the cloaca is displaced to the abdomen. About 9–10 somites (Figure 4B1–B3).

Stage 23 (1 day, 0.75 h): Eyes are a little more prominent. Jaw and gill regions are separated by grooves, and the ventral contour of the embryo is somewhat concave. About 12 somites (Figure 4C1–C3).

Stage 24 (1 day, 2.25 h): Eyes protrude less laterally than the gill region, which is more prominent than the jaw region. From this period, the embryo begins to respond to external stimulation. About 15 somites (Figure 4D1–D3).

Stage 25 (1 day, 3.5 h): Eyes protrude laterally the same or more than the gill regions. Fins are beginning to form. Gill regions grooved. About 16 somites (Figure 4E1–E3).

Stage 26 (1 day, 5.5 h): Otic vesicles are prominent. From the lateral view, fins on the back half of the body are slightly wider. Spontaneous movement begins in this stage. About 17 somites (Figure 4F1–F3).

Stage 27 (1 day, 7.25 h): From the dorsal view, a lateral thickening of eyes is observed. Fins are transparent, and the tail bud forms an inconspicuous contour in the lateral outline. About 19 somites (Figure 4G1–G3).

Stage 28 (1 day, 8.5 h): The fin is enlarged and clearly divided into external transparent and internal translucent bands from the lateral view. Fin extending to the cloaca. About 20–22 somites (Figure 4H1–H3).

Stage 29/30 (1 day, 11 h): The entire length of the fin from head to bottom is transparent. No pigmentation was found in the whole body of the embryo. The tail bud is distinct. About 24–25 somites (Figure 4I1–I3).

Stage 31 (1 day, 13.5 h): From this stage, the length of the tail bud is the main staging standard. Tail bud is equal in length and width. Embryo is formed from about 22 to 23 post‐otic somites (Figure 5A1–A3).

Stage 32 (1 day, 16 h): The length of the tail bud is about 1.5 times its width. Oval eye cups are obvious. About 26 post‐otic somites are formed (Figure 5B1–B3).

Stage 33/34 (1 day, 20.5 h): The length of the tail bud is about twice its width. From this period, a heartbeat begins. Eye cups are deeper and more obvious. About 32 post‐otic somites (Figure 5C1–C3).

Stage 35/36 (2 d, 2 h): Beginning to break through the jelly coat. The length of the tail bud is about three times its width. Posterior contour of the proctodeum is still curved. About 36 post‐otic somites (Figure 5D1–D3).

Stage 37/38 (2 days, 5.5 h): A few melanophores begin to appear on the eyes. From the ventral view, stomodeal invagination is round and deeper. The tail bud is longer. The posterior contour of the proctodeum is straight. About 40 post‐otic somites formed (Figure 5E1–E3).

Stage 39 (2 days, 8.5 h): The contour of the tail muscle and the proctodeum is at about 120°–135° angle. There are more melanocytes on the eyes. Duodenum bent to the left side. About 43 post‐otic somites (Figure 5F1–F3).

From stage 40, the digestive system of the embryo develops rapidly and becomes more transparent as a whole (Figures 6 and 8). Results for skeleton staining show that development of the cartilage begins at stage 40. As the individuals collected for the morphological images and the staining of the skeleton were different, some differences in the amount of melanin can be seen. Samples for skeletal stain imaging were taken on agarose, thus, the pigment deposition was more obvious (Figures 7 and 9). The skeletal system of the albino X. laevis consists of an axial and appendage skeleton. Axial skeletons include the skull and spine, while the accessory limb skeletons include the shoulder strap, sternum, front and rear limb skeletons, and the pelvic girdle.

FIGURE 6.

FIGURE 6

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Embryonic development at stages 40 to 46. Stage (S) is indicated to the left of three images for each stage. (A1–G1) Lateral views of the entire embryo; (A2–G2) dorsal views of the entire embryo; (A3–G3) ventral views of the entire embryo. (E3) Arrow points to the tentacle rudiment. Bar in each image = 1 mm.

FIGURE 8.

FIGURE 8

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Embryonic development at stages 47–50. Stage (S) is indicated to the left of three images for each stage. (A1–D1) Lateral views of the entire embryo; (A2–D2) dorsal views of the entire embryo; (A3–D3) ventral views of the entire embryo. Arrows indicate: (A1) hindlimb bud is visible; (A2) forelimb bud anlagen is visible; (A3) edge of gill cap forms quarter circle. Bar in each image = 2 mm.

FIGURE 7.

FIGURE 7

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Skeleton staining at stages 40–47. Stage (S) is indicated to the left of a pair of images for each stage. (A1–H1) Dorsal views of the embryo; (A2–H2) ventral views of the embryo. Bar in each image = 1 mm. ba, branchial arches; bhb, basihyobranchial; ch, ceratohyal; cm, Meckel's cartilage; oc, otic capsule; pmpq, processus muscularis palatoquadrati; pq, palatoquadrate; tp, trabecular plate.

FIGURE 9.

FIGURE 9

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Skeleton staining at stages 48–51. Stage (S) is indicated to the left of a pair of images for each stage. (A1–D1) Dorsal views of the entire embryo; (A2–D2) ventral views of the entire embryo. Bar in each image = 1 mm. ch, ceratohyal; ctm, commissura terminalis; exo, external occipital; io, infrarostral; o, occipital; oc, otic capsule; se, sphenethmoid; tec, tentacular cartilage; v, vertebrae.

Stage 40 (2 days, 18 h): Contour of the tail muscle and the proctodeum form a right angle. Mouth breaks open. About 45 post‐otic somites formed (Figure 6A1–A3). Before stage 40, the stroma forming the future skull increasingly aggregates. At stage 40, two presumptive trabeculae plates are approaching each other under the forebrain (Figure 7A1,A2).

Stage 41 (3 days, 4 h): The yolk mass form left and right caudal furrows and the middle part is twisted at about 45°. The conical proctodeum is at an angle of about 60° to the tail myotomes (Figure 6B1–B3). The trabeculae plate and palatoquadrate have become cartilaginous (Figure 7B1,B2).

Stage 42 (3 days, 8 h): Angle of the intestinal volvulus is larger, at about 90°. Opercular folds begin to form. The proctodeum is connected to the yolk mass through a little intestinal tube (Figure 6C1–C3). Cartilage around the viscera and palate began to form (Figure 7C1,C2).

Stage 43 (3 days, 15 h): There are more melanophores on the head and the abdomen. Intestinal volvulus is about 180°. The proctodeum is narrowed, showing a S‐shape (Figure 6D1–D3). The skull cartilage begins to form (Figure 7D1,D2).

Stage 44 (3 days, 20 h): The curly part of the intestine is in a S‐shaped ring. The fold of the opercular protrudes further, and the gills become smaller. Emergence of the tentacle rudiment (Figure 6E1–E3). Visceral skeletons extend further to the skull, most of the jawbone is cartilaginized and the Michael's cartilage is obvious (Figure 7E1,E2).

Stage 45 (4 days, 2 h): Intestine is spiral, displaying one and a half circles. Feeding occurs from this stage, and the gill cap partially covers the gills. The ectoderm is thickened in the bud area of the hindlimb. Heart‐beat (~120 pulses per minute) was readily seen at this stage (Figure 6F1–F3). The otic capsule is in a state of anterior cartilage. Most of the three anterior branchial arches are cartilaginized (Figure 7F1,F2).

Stage 46 (4 days, 10 h): The intestine is composed of two to two and a half circles. Xanthophores appear on the abdomen, and the edge of the gill cap protrudes (Figure 6G1–G3). The otic capsule is cartilaginized and the visceral skeletons have been completely cartilaginized. The fourth branchial arch is cartilage (Figure 7G1,G2).

Stage 47 (5 days, 12 h): Tentacles became longer and more obvious. The forelimb bud anlagen can be identified for the first time. Hindlimb buds become larger. The intestinal tract is two‐and‐a‐half to three‐and‐a‐half circles, and the edge of the gill cap has a quarter‐round shape (Figure 8A1–A3). The anterior occipital arch is cartilaginized and the trabecular has become larger. The branchial arch expands continuously in the later growth process. Anterior cartilaginization of the atlas is manifested by the contraction of the mesenchymal cells behind the occipital arch (Figure 7H1,H2).

Stage 48 (7 days, 12 h): Tissue above the abdomen forelimb bud can be distinguished from the dorsal aspect. The hindlimb bud is semicircular in the side view, and the abdomen becomes golden in color. There are more melanophores on the head (Figure 8B1–B3). The boundary between the occipital base and the atlas is not obvious (Figure 9A1,A2).

Stage 49 (ca. 12 days): Forelimb bud becomes more obvious. The hindlimb bud is longer, its end is round and its base does not shrink (Figure 8C1–C3). The bottom of the atlas begins to form cartilage (Figure 9B1,B2).

Stage 50 (ca. 15 days): Forelimb buds are clearly visible from the dorsal view. The shape of the bud is similar to an ellipse. The base of the hindlimb shrank and the front end becomes pointed, with the length almost equal to its width (Figure 8D1–D3). The development of the axial skeleton is along the craniocaudal direction. Cartilage is beginning to form in 4–5 pairs of vertebrae and the occipital arch (Figure 9C1,C2).

From stage 51, pictures of the limbs are included to facilitate a more accurate staging through the shape of the limbs (Figures 10, 12 and 14). Images of limb skeleton development are also added to describe progress in their development (Figures 11, 13 and 15). At stage 51, embryos did not form ossific, thus, we still did Alcian blue staining. Therefore, the picture for skeleton staining for stage 51 was placed in Figure 9. From stage 52, we suspected that osteogenesis was occurring and performed Alcian blue and Alizarin red staining.

FIGURE 10.

FIGURE 10

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Embryonic development at stages 51–57. Stage (S) is indicated to the left of five images for each stage. F: Forelimb. H: Hindlimb. (A1–G1) Lateral views of the entire embryo; (A2–G2) dorsal views of the entire embryo; (A3–G3) ventral views of the entire embryo. Bar in each whole embryo image = 1 cm. (A4–G4) Partial view of the forelimb; (A5–G5) partial view of the hindlimb. Bar in each limb image = 1 mm.

FIGURE 12.

FIGURE 12

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Embryonic development at stages 58–60. Stage (S) is indicated to the left of five images for each stage. F: Forelimb. H: Hindlimb. (A1–C1) Lateral views of the entire embryo; (A2–C2) dorsal views; (A3–C3) ventral views. Bar in each whole‐embryo image = 1 cm. (A4–C4) Partial views of the forelimb. (A5–C5) Partial views of the hindlimb. Bar in each limb image = 1 mm.

FIGURE 14.

FIGURE 14

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Embryonic development at stages 61–66. Stage (S) is indicated to the left of five images for each stage. F: Forelimb. H: Hindlimb. (A1–F1) Lateral views of the entire embryo; (A2–F2) dorsal views; (A3–F3) ventral views. Bar in each whole‐embryo image = 1 cm. (A4–F4) Partial views of the forelimb. (A5–F4) Partial views of the hindlimb. Bar in each limb image = 1 mm.

FIGURE 11.

FIGURE 11

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Skeleton staining at stages 52–57. Stage (S) is indicated to the left of five images for each stage. Stage (S) is indicated to the left of three images for each stage. (A1–G1) Dorsal views; (A2–G2) partial views of the forelimb. (A3–G3) Partial views of the hindlimb. Arrow indicates ossification. Bar in each image = 1 mm. as, astragalus; ca, calcaneum; cp, carpals; f, femur; fb, fibula; hm, humerus; il, ilium; mc, metacarpals; mt, metatarsals; ph, phalanges; rd, radius; ru, radioulna; tb, tibia; tf, tibiofibula; ts, tarsals; ul, ulna; Arrowheads indicate the location of the initial ossification.

FIGURE 13.

FIGURE 13

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Skeleton staining at stages 58–60. Stage (S) is indicated to the left of four images for each stage. F: Forelimb. H: Hindlimb. (A1–C1) Dorsal views of the entire embryo; (A2–C2) ventral views. Bar in each whole‐embryo image = 1 cm. (A3–C3) Partial views of the forelimb. (A4–C4) Partial views of the hindlimb. Bar in each limb image = 1 mm. cl, clavicle; il, ilium; n, nasal; o, occipital; oc, otic capsule; ss, suprascapula.

FIGURE 15.

FIGURE 15

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Skeleton staining at stages 61–66. Stage (S) is indicated to the left of four images for each stage. F: Forelimb. H: Hindlimb. (A1–F1) Dorsal views; (A2–F2) ventral views. Bar in each whole embryo image = 1 cm. (A3–F3) Partial views of the forelimb. (A4–F4) Partial views of the hindlimb. Bar in each limb image = 1 mm. an, angulosplenial; cl, clavicle; co, coracoid; d, dentary; fp, frontoparietal; ic, ischium; il, ilium; n, nasal; pmx, premaxilla; ps, parasphenoid; pt, pterygoid; pu, pubis; q, quadrate; r, rib; sc, scapula; se, sphenethmoid; su, fused sacrourostyle.

Stage 51 (ca. 17 days): Forelimb buds become oval. Hindlimb buds are long and conical and the length is longer than their width. From the dorsal view, tentacles become longer (Figure 10A1–A5). The accessory sphenethmoid is located below the skull, and the posterior edge is connected to the external occipital bone. Cartilaginization of the base of the atlas was complete. The pelvic zone begins to develop and is initially represented by a stroma (cell mass) at the base of the two limb buds (Figure 9D1,D2).

Stage 52 (ca. 21 days): Buds for the forelimbs are long and conical. The ankle part of the hindlimb buds begins to contract. The muscle tissue of the hindlimbs begins to thicken (Figure 10B1–B5). The pedicle is cartilaginized. The spinous processes appear. During this stage, limb skeletons have not appeared (Figure 11A1–A3).

Stage 53 (ca. 24 days): Forelimb bud appears to have the shape of a palm. The length of the hindlimbs, except the foot, is slightly longer than wide, with the fourth and fifth toes slightly protruding. Muscle fibrogenesis begins in the thigh and leg (Figure 10C1–C5). Cartilaginization of the occipital arch is complete. The back of the first and second neural arches is still connected by connective tissue. The pelvic zone begins to cartilaginize, and the pedicle canal was cartilaginized to the lower and anterior part of the pedicle. Cartilages of the forelimbs and hindlimbs first appeared in this stage. A little humerus appears. The tibia and fibula are separated, and there is skeletal progress in the femur and fibular tarsus and the fourth toe. This is the state of anterior cartilage (Figure 11B1–B3).

Stage 54 (ca. 26 days): Edges of the fingers become clear. The position where the third finger forms is the most obvious. Five toes become clear. The length of hindlimbs, except the foot, is about twice as long as the wide. The fourth and fifth toes are the most prominent (Figure 10D1–D5). The humerus of the forelimbs is lengthened. The radius and ulna can also be clearly separated. Cartilages appear first in the position of the third finger. In addition to the lengthening of the femur and tibiofibula, there are tarsals and metacarpals of the third and fourth toes. The metatarsal of the second toe is faintly visible. At this time, it is in the state of complete cartilage. The approximate position of the tibiotarsal (astragalus) and peroneal tarsals (calcaneum), which are joined with the tibiofibula, is visible at stage 53, when they are largely cartilaginized. The position of the ilium is vaguely identifiable, and the ischium is separated (Figure 11C1–C3).

Stage 55 (ca. 32 days): Palms buckle inward. The length and width of the first finger is almost equal, while all other fingers are about twice as long. The length of the fourth and fifth toes is about four times their width (Figure 10E1–E5). The humerus has been completely cartilaginized. The radius and ulna are close to fusion and six carpals form. All four metacarpals are visible. Progress has also been made in the remaining phalanges. Hindlimbs are the same as the forelimbs with all five metatarsals cartilaginized, and three tarsals appear. The vertebrae of this stage are all cartilage. The ilium is developing rapidly. The spine extends to the entire length of the torso (Figure 11D1–D3).

Stage 56 (ca. 38 days): The length of the fingers is about 3–4 times their width, and the length of the fourth and fifth toes is about six times their width. Wrists are clearly visible. The first and fourth fingers are almost the same length. Webbing of forelimbs has almost completely disappeared. The tips of the first and second toes are more prominent (Figure 10F1–F5). The radius and ulna are fused, and the fused bone and humerus were slightly ossified near the middle. Slight ossification of the femur, tibiofibular, and the third to fifth metatarsals. The four fingers and five toes become clear and recognizable. From then on, the perichondrium begins to ossify. Ossification of the frontoparietal bone progresses rapidly. The proximal part of the coracoid process is slightly ossified, and the middle part of the ilium begins to ossify (Figure 11E1–E3).

Stage 57 (ca. 41 days): Elbow bent at more than 90°. The fingers become longer, about seven times as long as their width, and point to the position of the heart. The first finger is longer than the fourth finger (Figure 10G1–G5). At stage 57− (ca. 39.5 days), the humerus and fusion skeleton were further ossified and become seven wrist joints. All knuckles are clearly displayed. All five metatarsals were ossified in the middle area. The third to fifth proximal phalanges are ossified. The first and second pairs of ribs are cartilaginized. The second pair of ribs is longer than the first pair and extend outward to the shoulder blades (Figure 11F1–F3). At stage 57, increase to eight wrist joints, with slight ossification of the fingertips. Ossification occurs in the proximal phalanges. The phalangeal formula and phalangeal formula are 3, 3, 2, 2 and 2, 2, 3, 4, 3. Cornification of the first to third toes. Ossification occurs between the five toes. A synovial cavity forms between the humerus and radius and ulna. Ossification occurs in almost all skeletons except the carpals and tarsals. There is a small hook like spur on the inside of the first metatarsal. The ilium extended toward the head (Figure 11G1–G3).

From stage 58, the tadpole's head becomes smaller and its tail shrinks. From the dorsal view, it looks increasingly like an adult (Figures 12 and 14). Ossification is progressing rapidly (Figures 13 and 15).

Stage 58 (ca. 44 days): Forelimbs brake through the membrane. At this stage, body length is the longest. Three black claws appear on hindlimbs for the first time (Figure 12A1–A5). The tibia and fibula are fused and are wrapped in the same bone sheath. It is very different from stage 57. The third pair of the distal ribs is cartilaginized. The 10th and 11th pairs of neural arches are fused. The ilium is further ossified, the sciatic bones are close to each other, and the nasal bone is partially ossified (Figure 13A1–A4).

Stage 59 (ca. 45 days): Tentacles begin to shorten. Hindlimbs extend toward the tail. The forelimbs reach the bottom of hindlimbs when extended. The tail begins to shrink (Figure 12B1–B5). All four fingertips are ossified, and cornification between the three toes is more obvious. The occipital bone of the anterior ear is partially ossified, the suprascapula is obviously enlarged, but the size of the scapula is almost unchanged (Figure 13B1–B4).

Stage 60 (ca. 46 days): The extended forelimbs extend beyond the base of the hindlimbs. From the ventral view, the leading edge of the skin area becomes narrow. Forelimbs are behind the heart level. The trunk has significantly narrowed (Figure 12C1–C5). The degree of ossification of all skeletons is further increased. The occipital joint appears. Clavicle cartilaginization is more obvious. The 12th pair of the medullary arch is fused. The ilium progresses rapidly. From the ventral side, there are two future anterior structures of the upper pubic cartilage (Figure 13C1–C4).

Stage 61 (ca. 48 days): Tentacles became shorter, and most of them are bent backward. The head becomes narrower. Fins became narrower. The position of the forelimbs is in the back half of the heart. The skin of the abdomen begins to cover the posterior half of the heart. Openings of the gill cavities are significantly narrowed (Figure 14A1–A5). The four fingers became more slender, and the metatarsals and phalanges are almost completely ossified. The nasal bone is largely ossified, and the clavicle begins to ossify. The iliac, sciatic, and pubic bones form a V‐shaped acetabulum. The left and right pubis are connected to form the abdominal wall of the acetabulum. The ischium forms the posterior wall of the acetabulum (Figure 15A1–A4).

Stage 62 (ca. 49 days): Tentacles become shorter and straight. Pelvic fins are no longer visible in the abdomen. The opening of the operculum is narrowed. The head is a little wider than the trunk and the corners of the mouth are in front of the eyes (Figure 14B1–B5). Ossification of the metacarpals and phalanges increases and the hind feet become larger. The pair of pterygoid bones are still cartilage, and their main branches connect to the end of the quadrate. All gill commissures disappear. Ossification of the middle of the three pairs of ribs progresses rapidly, and the proximal and distal ends are still cartilage. The two sternal primordia are cartilage, the ilium is further ossified and close to the midline of the abdomen, and the sacral caudal bone fusion bone begins to form cartilage (Figure 15B1–B4).

Stage 63 (ca. 51 days): The tentacles disappear completely and the operculum is closed. The tail is slightly longer than the body length. The trunk is slightly wider than the head. The position of the forelimbs is horizontal with the front half of the heart. Three black claws became more obvious (Figure 14C1–C5). From stage 63 to 66, the bones of the front and rear limbs developed more completely. The quadrate is small and short and is located on both sides of the outer edge of the rear end of the maxillary. The accessory sphenoid bone extends below the nasal septum. From the ventral side, the two coracoid processes are close to each other, and the front end of the ileum is close to the torso (Figure 15C1–C4).

Stage 64 (ca. 53 days): The tail is about one‐third the length of the body. In the lateral view, the corners of the mouth are behind the eyes. The boundary lines between the skin areas are clear (Figure 14D1–D5). The two sternal primordia are fused into a sternum, which is composed of a middle sternal body and two wide and flat lateral wings. The front part of the sternal body forms the sternal pouches (Figure 15D1–D4).

Stage 65 (ca. 54 days): The tail is turned into a slightly longer triangle of about 1/10 the length of the body, and the boundary lines between the skin areas disappear in this part (Figure 14E1–E5). The tarsals are partially ossified at stages 65 and 66, but the carpals are still cartilage. The suprascapula has been completely ossified, the sacral caudal bone has been fused, and ossification is completed (Figure 15E1–E4).

Stage 66 (ca. 58 days): The tail degenerates into a very small yellow‐red triangle, and not visible in the abdomen. The boundary lines between the skin areas disappear (Figure 14F1–F5). The premaxilla is located at the front of the maxilla. The front end of the angulosplenial is connected with the dentary. The transverse process of the vertebral body in sections 5–8 will prolong in subsequent growth. The ileum is almost completely ossified and the whole pelvic zone is almost fully developed. Most of the ribs are completely ossified (Figure 15F1–F4).

4. DISCUSSION

Development from a fertilized albino X. laevis egg to complete degeneration of the tail takes about 58 days. Compared with the description in the Normal Table (Nieuwkoop & Faber, 1994), development of the albino Xenopus embryos is basically the same as seen for wild‐type embryos, with skeleton development also being similar. However, since albino embryos have little or no pigmentation in their early stages, this facilitated the staging of the embryos as they could be stained with Nile blue after being dejellied. Albino individuals can also be used for histochemical reaction experiments without the need to bleach them. Compared with wild‐type embryos, the staining signal is more obvious (Pera et al., 2015).

The speed of embryo development can differ under different conditions (Godfrey & Sanders, 2004), with the biggest impact on speed being temperature. Studies have shown that X. laevis develops well at 16–25°C, and can tolerate long times even at 12°C. In the early stages of cleavage, the rate of cell division increases with temperature. On the whole, the growth rate at 25°C is significantly faster than that at 22°C (Khokha et al., 2002). In our experiments, to keep embryo development speed as consistent as possible, we cultured the fertilized eggs at a temperature of 23°C. With the development of embryos, dead eggs and impurities are continuously removed. Although our external conditions were as consistent as possible, some differences in the speed of development were seen in some individuals, which becomes more obvious in the later stages of embryonic development. Thus, our work is most useful for specifying the exact stage for an albino individual. Compared with staging only according to time, the appearance of characteristics of an embryo should become the standard for staging (Onimaru et al., 2018).

Skin pigmentation differs between embryos. Periodic albinism is a recessive mutation (Hoperskaya, 1975). However, the specific reason for the formation of periodic albino mutants in X. laevis has not been determined (Fukuzawa, 2021). It has been reported that the melanin content of the retinal pigment epithelium and melanophores decreases in later stages of development and that they gradually decolorize during metamorphosis (Hoperskaya, 1975, 1981). In a recent study, it was determined that the HPS4 gene is the cause of the albino mutation in X. laevis (Fukuzawa, 2021), and that mutation of HPS will also affect pigmentation in X. tropicalis (Nakayama et al., 2017). Additional studies on the causes of the albino phenotype in Xenopus are needed.

There are few studies on the limb skeleton of X. laevis, whether wild‐type or albino. In this study, compared with the normal table (Nieuwkoop & Faber, 1994), we provide a more detailed description of the developmental processes of the limb skeleton, which should help studies on the developmental biology of limbs. Before stage 57, the hind limbs of X. laevis have the ability to regenerate (Dent, 1962; Overton, 1963), and X. laevis is an indispensable and important regeneration model (Keenan & Beck, 2016; Simon & Tanaka, 2013). Our skeleton staining images show that there is a claw‐like protrusion on the inner side of the first metatarsal of the hindlimb at stage 57, in both males and females, which is consistent with a previous investigation (Hayashi et al., 2015). Unlike X. tropicalis, the fingertip of this claw process is not ossified in stage 59. Accordingly, the sixth finger of the albino X. laevis should be the same as in the X. tropicalis, which is a degenerate digit.

Our work is based on existing research with wild‐type X. laevis, with the purpose of providing high‐definition pictures of the biological morphology of albino X. laevis embryos in all developmental stages complemented with skeleton staining pictures. We hope these efforts can provide help for future experimental research in amphibians.

AUTHOR CONTRIBUTIONS

Zhixin Shan performed the experiments and wrote the manuscript. Shanshan Li participated in the experimental design and revised the manuscript. Chenghua Yu and Shibin Bai revised the manuscript., Yining Tang and Yutong Wang participated in the experiments. David M. Irwin revised the manuscript and improved the language. Junpeng Zhang, and Zhe Wang reviewed the findings. Jun Li and Zhe Wang supervised the whole research process. All authors read and approved the final version of the manuscript.

FUNDING INFORMATION

This work was supported by a grant from the Organization Department of Liaoning Provincial Committee of China (LiaoNing Revitalization Talents Program, no. XLYC1907018).

5. Ethics statement

All procedures were approved by the Animal Ethics Committee of Shenyang Agricultural University (approval ID: 2022030401).

ACKNOWLEDGMENTS

The authors thank Xinyu Wang, Junnan Chen, Xiaoping Li, Wei Teng, Shiyu Zhu and Kefan Hong for experimental assistance.

Shan, Z. , Li, S. , Yu, C. , Bai, S. , Zhang, J. , Tang, Y. et al. (2023) Embryonic and skeletal development of the albino African clawed frog (Xenopus laevis). Journal of Anatomy, 242, 1051–1066. Available from: 10.1111/joa.13835

Zhixin Shan, Shanshan Li, and Chenghua Yu contributed equally to this work.

Contributor Information

Jun Li, Email: 15640216688@163.com.

Zhe Wang, Email: zwang@syau.edu.cn.

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

REFERENCES

  1. Bantle, J.A. , Finch, R.A. , Fort, D.J. , Stover, E.L. , Hull, M. & Kumsher‐King, M. (1999) Phase III interlaboratory study of FETAX. Part 3. FETAX validation using 12 compounds with and without an exogenous metabolic activation system. Journal of Applied Toxicology, 19(6), 447–472. [DOI] [PubMed] [Google Scholar]
  2. Beck, C.W. & Slack, J.M. (2001) An amphibian with ambition: a new role for Xenopus in the 21st century. Genome Biology, 2(10), REVIEWS 1029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borodinsky, L.N. (2017) Xenopus laevis as a model organism for the study of spinal cord formation, development, function and regeneration. Frontiers in Neural Circuits, 11, 90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Candioti, M. (2007) Anatomy of Anuran tadpoles from lentic water bodies: systematic relevance and correlation with feeding habits. Zootaxa, 1600, 1–175. [Google Scholar]
  5. Dent, J.N. (1962) Limb regeneration in larvae and metamorphosing individuals of the South African clawed toad. Journal of Morphology, 110, 61–78. [DOI] [PubMed] [Google Scholar]
  6. Exner, C.R.T. & Willsey, H.R. (2021) Xenopus leads the way: frogs as a pioneering model to understand the human brain. Genesis (New York, N.Y.: 2000), 59(1–2), e23405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fortriede, J.D. , Pells, T.J. , Chu, S. , Chaturvedi, P. , Wang, D. , Fisher, M.E. et al. (2020) Xenbase: deep integration of GEO & SRA RNA‐seq and ChIP‐seq data in a model organism database. Nucleic Acids Research, 48(D1), D776–D782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fukuzawa, T. (2021) Periodic albinism of a widely used albino mutant of Xenopus laevis caused by deletion of two exons in the Hermansky‐Pudlak syndrome type 4 gene. Genes to Cells, 26(1), 31–39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Godfrey, E.W. & Sanders, G.E. (2004) Effect of water hardness on oocyte quality and embryo development in the African clawed frog (Xenopus laevis). Comparative Medicine, 54(2), 170–175. [PubMed] [Google Scholar]
  10. Hayashi, S. , Kobayashi, T. , Yano, T. , Kamiyama, N. , Egawa, S. , Seki, R. et al. (2015) Evidence for an amphibian sixth digit. Zoological Letters, 1, 17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hoogenboom, W.S. , Klein Douwel, D. & Knipscheer, P. (2017) Xenopus egg extract: a powerful tool to study genome maintenance mechanisms. Developmental Biology, 428(2), 300–309. [DOI] [PubMed] [Google Scholar]
  12. Hoperskaya, O.A. (1975) The development of animals homozygous for a mutation causing periodic albinism (ap) in Xenopus laevis . Journal of Embryology and Experimental Morphology, 34, 253–264. [PubMed] [Google Scholar]
  13. Hoperskaya, O.A. (1981) Induction‐the main principle of melanogenesis in early development. Differentiation, 20, 104–116. [DOI] [PubMed] [Google Scholar]
  14. Ishibashi, S. & Amaya, E. (2021) How to grow Xenopus laevis tadpole stages to adult. Cold Spring Harbor Protocols, 2021(3), pdb.prot106245. Available from: 10.1101/pdb.prot106245 [DOI] [PubMed] [Google Scholar]
  15. Karimi, K. , Fortriede, J.D. , Lotay, V.S. , Burns, K.A. , Wang, D.Z. , Fisher, M.E. et al. (2018) Xenbase: a genomic, epigenomic and transcriptomic model organism database. Nucleic Acids Research, 46(D1), D861–D868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Keenan, S.R. & Beck, C.W. (2016) Xenopus limb bud morphogenesis. Developmental Dynamics, 245(3), 233–243. [DOI] [PubMed] [Google Scholar]
  17. Khokha, M.K. , Chung, C. , Bustamante, E.L. , Gaw, L.W. , Trott, K.A. , Yeh, J. et al. (2002) Techniques and probes for the study of Xenopus tropicalis development. Developmental Dynamics: An Official Publication of the American Association of Anatomists, 225(4), 499–510. [DOI] [PubMed] [Google Scholar]
  18. Kostiuk, V. & Khokha, M.K. (2021) Xenopus as a platform for discovery of genes relevant to human disease. Current Topics in Developmental Biology, 145, 277–312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Liu, L.S. , Zhao, L.Y. , Wang, S.H. & Jiang, J.P. (2016) Research proceedings on amphibian model organisms. Zoological research, 37(4), 237–245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lukas, P. & Olsson, L. (2018) Sequence and timing of early cranial skeletal development in Xenopus laevis . Journal of Morphology, 279(1), 62–74. [DOI] [PubMed] [Google Scholar]
  21. Mogi, K. , Adachi, T. , Izumi, S. & Toyoizumi, R. (2012) Visualisation of cerebrospinal fluid flow patterns in albino Xenopus larvae in vivo. Fluids and Barriers of the CNS, 9, 9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nakayama, T. , Grainger, R.M. & Cha, S.W. (2020) Simple embryo injection of long single‐stranded donor templates with the CRISPR/Cas9 system leads to homology‐directed repair in Xenopus tropicalis and Xenopus laevis . Genesis, 58(6), e23366. [DOI] [PubMed] [Google Scholar]
  23. Nakayama, T. , Nakajima, K. , Cox, A. , Fisher, M. , Howell, M. , Fish, M.B. et al. (2017) No privacy, a Xenopus tropicalis mutant, is a model of human Hermansky‐Pudlak syndrome and allows visualization of internal organogenesis during tadpole development. Developmental Biology, 426, 472–486. [DOI] [PubMed] [Google Scholar]
  24. Nenni, M.J. , Fisher, M.E. , James‐Zorn, C. , Pells, T.J. , Ponferrada, V. , Chu, S. et al. (2019) Xenbase: facilitating the use of Xenopus to model human disease. Frontiers in Physiology, 10, 154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nieuwkoop, P.D. & Faber, J. (1994) Normal table of Xenopus laevis (Daudin). NewYork: Routledge. [Google Scholar]
  26. Onimaru, K. , Motone, F. , Kiyatake, I. , Nishida, K. & Kuraku, S. (2018) A staging table for the embryonic development of the brownbanded bamboo shark (Chiloscyllium punctatum). Developmental Dynamics: An Official Publication of the American Association of Anatomists, 247(5), 712–723. Available from: 10.1002/dvdy.24623 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Overton, J. (1963) Patterns of limb regeneration in Xenopus laevis . Journal of Experimental Zoology, 154, 153–161. [DOI] [PubMed] [Google Scholar]
  28. Pera, E.M. , Acosta, H. , Gouignard, N. & Climent, M. (2015) Whole‐mount In situ hybridization and immunohistochemistry in Xenopus embryos. NeuroMethods, 99, 151–167. [Google Scholar]
  29. Phipps, L.S. , Marshall, L. , Dorey, K. & Amaya, E. (2020) Model systems for regeneration: Xenopus . Development (Cambridge, England), 147(6), dev180844. [DOI] [PubMed] [Google Scholar]
  30. Rocková, H. & Rocek, Z. (2005) Development of the pelvis and posterior part of the vertebral column in the Anura. Journal of Anatomy, 206(1), 17–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Saide, K. , Sherwood, V. & Wheeler, G.N. (2019) Paracetamol‐induced liver injury modelled in Xenopus laevis embryos. Toxicology Letters, 302, 83–91. [DOI] [PubMed] [Google Scholar]
  32. Schmitt, S.M. , Gull, M. & Brändli, A.W. (2014) Engineering Xenopus embryos for phenotypic drug discovery screening. Advanced Drug Delivery Reviews, 69‐70, 225–246. [DOI] [PubMed] [Google Scholar]
  33. Shearman, R.M. (2008) Chondrogenesis and ossification of the lissamphibian pectoral girdle. Journal of Morphology, 269(4), 479–495. [DOI] [PubMed] [Google Scholar]
  34. Simon, A. & Tanaka, E.M. (2013) Limb regeneration. Wiley Interdisciplinary Reviews in Developmental Biology, 2(2), 291–300. [DOI] [PubMed] [Google Scholar]
  35. Slack, J.M. , Beck, C.W. , Gargioli, C. & Christen, B. (2004) Cellular and molecular mechanisms of regeneration in Xenopus . Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 359(1445), 745–751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stooke‐Vaughan, G.A. , Davidson, L.A. & Woolner, S. (2017) Xenopus as a model for studies in mechanical stress and cell division. Genesis (New York, N.Y.: 2000), 55, 1–2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Thomson, D.A. (1986) Meckel's cartilage in Xenopus laevis during metamorphosis: a light and electron microscope study. Journal of Chemical Neuroanatomy, 149, 77–87. [PMC free article] [PubMed] [Google Scholar]
  38. Trueb, L. & Hanken, J. (1992) Skeletal development in Xenopus laevis (Anura: Pipidae). Journal of Morphology, 214(1), 1–41. [DOI] [PubMed] [Google Scholar]
  39. Wagner, N. , Müller, H. & Viertel, B. (2017) Effects of a commonly used glyphosate‐based herbicide formulation on early developmental stages of two Anuran species. Environmental Science and Pollution Research International, 24(2), 1495–1508. [DOI] [PubMed] [Google Scholar]
  40. Wassersug, R.J. (1976) A procedure for differential staining of cartilage and bone in whole formalin‐fixed vertebrates. Stain Technology, 51(2), 131–134. [DOI] [PubMed] [Google Scholar]
  41. Zahn, N. , James‐Zorn, C. , Ponferrada, V.G. , Adams, D.S. , Grzymkowski, J. , Buchholz, D.R. et al. (2022) Normal table of Xenopus development: a new graphical resource. Development, 149(14), dev200356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zahn, N. , Levin, M. & Adams, D.S. (2017) The Zahn drawings: new illustrations of Xenopus embryo and tadpole stages for studies of craniofacial development. Development (Cambridge, England), 144(15), 2708–2713. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

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