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. 2015 Sep 15;405(2):291-303.
doi: 10.1016/j.ydbio.2015.06.007. Epub 2015 Jul 11.

Budgett's frog (Lepidobatrachus laevis): A new amphibian embryo for developmental biology

Nirav M Amin  1 , Mandy Womble  1 , Cristina Ledon-Rettig  2 , Margaret Hull  1 , Amanda Dickinson  3 , Nanette Nascone-Yoder  4
Affiliations

Budgett's frog (Lepidobatrachus laevis): A new amphibian embryo for developmental biology

Nirav M Amin et al. Dev Biol. .

Abstract

The large size and rapid development of amphibian embryos has facilitated ground-breaking discoveries in developmental biology. Here, we describe the embryogenesis of the Budgett's frog (Lepidobatrachus laevis), an unusual species with eggs that are over twice the diameter of laboratory Xenopus, and embryos that can tolerate higher temperatures to develop into a tadpole four times more rapidly. In addition to detailing their early development, we demonstrate that, like Xenopus, these embryos are amenable to explant culture assays and can express exogenous transcripts in a tissue-specific manner. Moreover, the steep developmental trajectory and large scale of Lepidobatrachus make it exceptionally well-suited for morphogenesis research. For example, the developing organs of the Budgett's frog are massive compared to those of most model species, and are composed of larger individual cells, thereby affording increased subcellular resolution of early vertebrate organogenesis. Furthermore, we found that complete limb regeneration, which typically requires months to achieve in most vertebrate models, occurs in a matter of days in the Budgett's tadpole, which substantially accelerates the pace of experimentation. Thus, the unusual combination of the greater size and speed of the Budgett's frog model provides inimitable advantages for developmental studies-and a novel inroad to address the mechanisms of spatiotemporal scaling during evolution.

Keywords: Amphibian; Embryo; Lepidobatrachus; Scaling; Stages; Xenopus.

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Figures

Figure 1
Figure 1
The schedule of Lepidobatrachus laevis embryonic development (versus Xenopus laevis). Developmental trajectories from fertilization (Gosner stage 1; GS1) to early tadpole (GS22) were plotted for L. laevis (blue diamonds, 28°C) and X. laevis (black circles, 23°C). Data points for L. laevis were calculated from the average time to achieve the developmental milestones characteristic of each stage in at least three separate clutches of embryos from at least two different adult breeding pairs. Developmental timing of Xenopus laevis was plotted from published reports (Nieuwkoop and Faber, 1994). For clarity, stages are clustered into cleavage/blastula (pink), gastrula (green), neurula (yellow) and organogenesis (blue) phases. The dashed line at 30 hpf indicates a break in the x-axis, to accommodate the substantial difference (~50 hours) in the developmental schedule of the two species (at their respective optimal temperatures—note that both organisms develop at the same rate when raised at mutually permissive temperatures; see Figure 5). Gosner (GS) and Nieuwkoop and Faber (NF) stages are indicated.
Figure 2
Figure 2
Early cleavage and gastrulation patterns in L. laevis are similar to Xenopus. Representative images of L. laevis fertilization (A; GS1); 2-cell (B; GS3); 4-cell (C; GS4); 8-cell (D; GS5); 16-cell (E; GS6); mid-blastula (F; GS8); late blastula (G; GS9); and early gastrula (H; GS10). “D” and “V” indicate the prospective dorsal and ventral sides of the embryo, respectively. (I) A side-by-side comparison of a L. laevis 2-cell stage embryo with a X. laevis gastrula demonstrates the substantial size difference between the two species. J) The dorsal lip (DL) is obvious as a pigmented depression (arrow) in the early gastrula (GS10). K) The dorsal lip (arrow) expands circumferentially (arrowheads). L) The yolk plug is still slightly protuberant in the midgastrula (GS 11). A–I are animal views; J and K are latero-vegetal views; L is a vegetal view. Scale bar = 1 mm.
Figure 3
Figure 3
Neurulation and organogenesis in L. laevis. The neural plate (A; GS12.5), neural folds (yellow arrowheads, B; GS14) and migrating streams of neural crest (arrows, B; GS14) are highly prominent in dorsal views of early neurulae. C) Neural tube closure is complete and individual somites (S) are evident by GS16. D-F) The cement gland (CG) and pronephros (P) are visible at the earliest tailbud stages (GS17-19). G–H) Later tailbud stages (GS20-22) show rapid tail elongation, the development of gills and craniofacial features (see also Figure 4), and early gut morphogenesis. NP= neural plate; H=heart; L=liver; G=gills; FG=foregut; MG=midgut; HG=hindgut. Scale bar = 1 mm.
Figure 4
Figure 4
Craniofacial morphogenesis in L. laevis. The embryonic head is shown in lateral (A,D,G,J; anterior to the left) and frontal views (B,E,H,K). Arrows indicate the location of the stomodeum (stom), or mouth. Higher magnification views (C,F,I,L) detail the gradual maturation of the mouth, including the perforating buccopharyngeal membrane (bm). The face and mouth widen dramatically as development proceeds, and the cement gland (cg), which initially forms a horseshoe shape (B), becomes bifurcated as the face widens (K). Gosner Stages (GS) are as indicated. Scale bars in A–B, D–E, G–H and J–K = 500μm; scale bars in C,F,I,L = 250μm. Other abbreviations: gb=gill buds; n=nasal pits; op=optic cup; ot=otic vesicle, pa=pharyngeal arches; so= somites.
Figure 5
Figure 5
Thermal tolerance of L. laevis and X. laevis embryos. L. laevis (solid lines, n=10) and X. laevis (dotted lines, n=20) were assayed for growth from 2-cell stage (GS3) to tadpole (GS22) at 37°C (purple), 32°C (green), 28°C (black), 23°C (orange), 16°C (blue). Asterisks (*) indicate time points at which at least 50% of embryos fail to gastrulate, indicating decreased viability. Gosner (GS) and Nieuwkoop and Faber (NF) stages are indicated.
Figure 6
Figure 6
L. laevis embryos are amenable to microinjection of exogenous reagents for fate-mapping and expression of synthetic mRNA. Embryos were injected at the 2-cell stage (A–C), 8-cell stage (D–F) or 128-cell stage (G–I) with red fluorescent dextran and/or capped eGFP mRNA. By 48 hours post fertilization (hpf), injection into a single blastomere at the 2-cell stage results in predominantly unilateral labeling (B) and expression of eGFP (C, green), while injection into a single blastomere at the 8-cell stage results in fluorescence limited to the developing gut (E). Injection of dextran into a single blastomere at the 128-cell stage labels a limited region of only 4 cells by the mid-blastula stage (H). Merge of brightfield and fluorescent images in (F, I). Scale bars = 1 mm.
Figure 7
Figure 7
L. laevis explants are amenable to animal cap explant culture. Untreated animal caps from X. laevis (A) or L. laevis (B) heal into balls of ciliated epidermis. In contrast, treatment of freshly microdissected caps with activin results in significant elongation of dissected tissue in both X. laevis (C) and L. laevis (D). The smaller rightmost cap in B and D was trimmed to a reduced size. Scale bars = 1 mm.
Figure 8
Figure 8
Heart morphogenesis occurs at a larger scale in L. laevis. The scale of L. laevis cardiogenesis is illustrated by comparing a dissected L. laevis heart tube (GS18) with a whole X. laevis neurula embryo (A), and by comparing mature hearts isolated at an equivalent stage from each species (B; GS22). Immunofluorescence analysis of tissue architecture was performed on transverse sections through the heart of GS21 (C–F) and GS20 (G–J) embryos. C–F) Localization of beta-catenin (βcat; green) in X. laevis and actin (actin; green) in L. laevis reveals the cellular architecture of the heart. G–H) Localization of the microtubule marker alpha-tubulin (αtub; green) and beta-catenin (βcat; red) in the L. laevis heart tube. I–J) Localization of the centrosome marker gamma-tubulin (γtub; green), and filamentous actin (F-actin; red), in a neighboring section through the L. laevis heart tube. Nuclei are counterstained with TO-PRO-3. White boxes in C, D, G, I indicate regions of the heart magnified in E, F, H, J, respectively. c, conus; v, ventricle; endo, endocardial layer; myo, myocardium. Scale bars = 100 μm.
Figure 9
Figure 9
Limb regeneration occurs at a rapid pace in L. laevis. Amputation was conducted on 8-day tadpoles at the indicated level (red dotted line; A). A blastema was identified within 24 hours of amputation (arrow; B). Full regeneration was observed in the operated limb by 10 days post amputation (C).
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