Embryogenesis and Laboratory Maintenance of the Foam-Nesting Tu´ Ngara Frogs, Genus (Physalaemus ؍) Engystomops

DEVELOPMENTAL DYNAMICS 238:1444–1454, 2009

SPECIAL ISSUE TECHNIQUES

Embryogenesis and Laboratory Maintenance of the Foam-Nesting Tu´ ngara Frogs, Genus (Physalaemus ؍) Engystomops

Andre´s Romero-Carvajal,1 Natalia Sa´ enz-Ponce,1 Michael Venegas-Ferrı´n,1 Diego Almeida-Reinoso,2 Chanjae Lee,3 Jennifer Bond,4 Michael J. Ryan,4 John B. Wallingford,3 and Eugenia M. del Pino1*

The vast majority of embryological research on amphibians focuses on just a single genus of frogs, Xenopus. To attain a more comprehensive understanding of amphibian development, experimentation on non-model frogs will be essential. Here, we report on the early development, rearing, and embryological analysis of tu´ ngara frogs (genus Engystomops, also called Physalaemus). The frogs Engystomops pustulosus, Engystomops coloradorum, and Engystomops randi construct floating foam-nests with small eggs. We define a table of 23 stages for the developmental period in the foam-nest. Embryos were immunostained against Lim1, neural, and somite-specific proteins and the expression pattern of RetinoBlastoma Binding Protein 6 (RBBP6) was analyzed by in situ hybridization. Due to their brief life-cycle, frogs belonging to the genus Engystomops are attractive for comparative and genetic studies of development. Developmental Dynamics 238:1444–1454, 2009. © 2009 Wiley-Liss, Inc.

Key words: gastrulation modes; somitogenesis; neural development; Colostethus machalilla; Engystomops coloradorum; Engystomops randi; Engystomops pustulosus; Gastrotheca riobambae

Accepted 10 March 2009

INTRODUCTION ing the rainy season; the male attracts ing tadpoles of other frogs (Ryan, the female with a characteristic call, 1985). Moreover, the hardened sur- Many frogs deposit their eggs in the and amplexus takes place. During am- face of the nest may reflect solar radi- water; other frogs, however, have evolved adaptations that diminish or plexus, the male beats the egg-jelly ation, decreasing heat absorption, and eliminate the aquatic requirement for with its legs and feet. This action pro- preventing internal desiccation. At reproduction (Duellman and Trueb, duces a floating mass of white foam, in hatching, the tadpoles fall into the wa- 1986). A deviation of the aquatic re- which numerous eggs are embedded ter (Davidson and Hough, 1969; Ryan, productive mode occurs in the tu´ ngara (Davidson and Hough, 1969; Ryan and 1985). Engystomops pustulosus is ac- frogs, genus Engystomops (ϭ Physa- Rand, 2003). In this way, E. pustulo- tive at night and displays a complex laemus), Leiuperidae, and consists of sus eggs are placed over, and develop reproductive behavior. The ecology, the formation of foam-nests that float above, water. The strategy of placing sexual selection, and communication over temporal pools. In Engystomops foam-nests over temporal pools pro- strategies of this frog have been exten- pustulosus, reproduction occurs dur- tects embryos from predators, includ- sively studied (reviewed in Ryan,

Additional Supporting Information may be found in the online version of this article. 1Laboratorio de Biologıá del Desarrollo, Escuela de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica del Ecuador, Quito, Ecuador 2Museo de Zoologıá, Seccioń de Herpetologıá, Escuela de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica del Ecuador, Quito, Ecuador 3Section of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology University of Texas, Austin, Texas 4Section of Integrative Biology, University of Texas, Austin, Texas Grant sponsor: Pontificia Universidad Cato´lica del Ecuador; Grant sponsor: The Academy for the Developing World (TWAS); Grant number: 07-017 LDC/BIO/LA-UNESCO FR 3240144821; Grant sponsor: NIH/NIGMS. *Correspondence to: Eugenia M. del Pino, Pontificia Universidad Cato´ lica del Ecuador, Escuela de Ciencias Biolo´ gicas, Apartado 17-01-2184, Avenida 12 de Octubre 1076 y Roca, Quito, Ecuador. E-mail: [email protected] DOI 10.1002/dvdy.21952 Published online 20 April 2009 in Wiley InterScience (www.interscience.wiley.com).

1985; Ryan and Rand, 2003; Ron et Pino et al., 2007). Furthermore, as in RESULTS AND DISCUSSION al., 2006). X. laevis, embryonic development oc- Frogs of the genus Engystomops in- curs rapidly in comparison with the Engystomops Developmental habit the forest floor in lowland re- slow-developing embryos of the den- Stages gions from Central Mexico to the Am- drobatid frog Colostethus (ϭ Epipe- Development of E. pustulosus, E. colo- azon basin, and the lowlands of W dobates) machalilla (Grant et al., radorum, and E. randi embryos in the Ecuador and NW Peru (reviewed by 2006), and of the marsupial frog Gas- Ron et al., 2006). The widely studied, foam-nest was divided into 23 stages trotheca riobambae (del Pino et al., (st). Development in the foam-nest Engystomops pustulosus, occurs at the 2007). Tail bud stage embryos of E. Smithsonian Tropical Research Insti- lasted 3 days in all of these frogs. At coloradorum and E. randi differ from hatching, the foam-nest became some- tute in Panama´, among other loca- X. laevis embryos in their general tions (Davidson and Hough, 1969; what liquid and the tadpoles moved shape, in the mode of somitogenesis, into the water. The criteria used to Ryan, 1985; Ron et al., 2006). Other and size of cranial neural crest cell congenerics that have been analyzed define the developmental stages are streams (del Pino et al., 2007). are Engystomops coloradorum, and E. given in Table 1. Developmental Analysis of Lim1 (Lhx1) gene ex- randi, which inhabit the western low- stages from fertilization to the early pression at the protein level in gastru- lands of Ecuador (Ron et al., 2006). neurula were defined according to the lae of E. coloradorum and E. randi Mitochondrial DNA analysis of these X. laevis table of development (st allowed detection of the organizer, three species revealed a close relation- 1–14; Nieuwkoop and Faber, 1994). ship. Accordingly, these frogs have and of the dorsal mesoderm (del Pino Thereafter, the generalized table of been placed in the Engystomops pus- et al., 2007; Venegas et al., 2009). In frog development (Gosner, 1960) was tulosus species group (Ron et al., embryos of these frogs, elongation of used to stage embryos, as the embryonic morphology of Engystomops devi- 2006). In this study, we describe the the notochord overlaps with involu- ated from that of X. laevis embryos early development of these Engysto- tion at the blastopore lip, as in X. lae- (Table 1) The external views of em- mops species, methods for the han- vis embryos (del Pino et al., 2007). In bryos are provided to complement the dling of eggs and embryos, and frog addition, in the mid gastrula, the pre- description of developmental stages laboratory maintenance. chordal plate and notochord were si- (Figs. 1A–P, 2A–G). The free-living In the search for an anuran amphib- multaneously recognized by the ex- tadpoles were not studied. ian with synchronous oogenesis, Da- pression of the protein Lim1, a The foam-nests of E. pustulosus, E. vidson and Hough (1969) determined pattern equivalent with that of Xlim1 coloradorum, and E. randi measured that E. pustulosus was appropriate for mRNA expression in X. laevis em- about 5 cm in diameter and were 4–5 developmental work. The environ- bryos (Taira et al., 1992; 1994a,b; cm high. The white eggs and embryos mental changes of wet and dry sea- Venegas et al., 2009). In contrast, in of these frogs were effectively camou- sons were reproduced in the labora- embryos of the dendrobatid frog C. flaged in the white foam of the nest. tory to analyze the modifications of machalilla, only the prechordal plate Each nest contained 130 Ϯ 60 eggs of oogenesis. Oogenesis changes from was recognized during gastrulation. 1.3-mm diameter in E. coloradorum synchronous during the dry season to Elongation of the notochord occurred (21 foam-nests with a total of 2,072 asynchronous in the wet season. Dur- after blastopore closure in this frog eggs); and 110 Ϯ 69 eggs of 1.1-mm ing the dry season, many oocytes (Venegas et al., 2009). This uncou- diameter in E. randi (17 foam-nests reach their full growth (stage 6) and pling of developmental events associ- with a total of 1,952 eggs). The foam- remain in the ovary until reproduction ates with the modular nature of frog nests of E. pustulosus contain an av- is triggered by the first rains. Besides gastrulation (Ewald et al., 2004; Moya erage of 238 Ϯ 97 eggs, based on 68 these large oocytes, only previtello- et al., 2007; del Pino et al., 2007; Vene- foam-nests collected in the field at the genic oocytes were detected in the gas et al., 2009). Barro Colorado Island, Panama´ ovary by the end of the dry season. In In this study, we immunostained (Ryan, 1985). The means and stan- contrast, during the wet season, the Engystomops embryos with several dard deviations are given. full range of oocyte sizes occurs in the antibodies, to detect the neural tube, ovary. After the first mating of the cranial neural crest cells, somites, rainy season, ovarian oocytes grow, Cleavage and the Blastula pronephros, and performed in situ hy- producing oocytes of all sizes. There- (st 1–9) after, in preparation for the impend- bridization to analyze the expression ing mating, the largest oocytes com- of the RetinoBlastoma Binding Pro- External views of cleavage stage em- plete their growth. This adaptation tein 6 (RBBP6) during development. A bryos are shown in Figure 1A–E, and facilitates repeated mating during the particular advantage for developmen- of the blastula in Figure 1F,G. In the wet season. Maternal transcripts are tal studies is the brief life-cycle of three species analyzed, eggs had a retained in E. pustulosus embryos to these frogs, which can be as short as 4 uniform white appearance. Therefore, the tadpole stage (Davidson and months in E. pustulosus (Davidson the animal hemisphere could not be- Hough, 1969; Hough et al., 1973). and Hough, 1969). Our aim is to pro- detected at the one-cell stage (Fig. Eggs of E. coloradorum and E. vide a methodological base-line for 1A). Similarly, a gray crescent could randi are small and white, and resem- further comparative studies of Engys- not be detected in these pale-colored ble Xenopus laevis albino embryos (del tomops development. embryos. Cleavage was holoblastic 1446 ROMERO-CARVAJAL ET AL.

TABLE 1. Stages of Engystomops Development

Stagea Age in:b EXGCharacteristics of embryos Hours Days 1 1 1 Fertilization (Fig. 1A). 0.0 2 2 3 Two-cell stage. 0,3 3 3 4 Four-cell stage (Fig. 1B). 0,6 4 4 5 Eight-cell stage (Fig. 1C). 1 5 5 6 Sixteen-cell stage (Fig. 1D, 3A). 1,3 6 6 7 Thirty-two-cell stage (Fig. 1E). 1,7 7 7 8 Large-cell blastula (Fig. 1F,3B). 3 8 8 - Medium-cell blastula (Fig. 1G,3C–D). 5 9 9 9 Advanced blastula. 7.2 10 10 10 Early gastrula. Embryos develop the dorsal blastopore lip (Figs. 1H, 3E–G). 11,5 11 11 11 Mid gastrula with a large yolk plug that measures about one-half of the 16,2 embryo’s diameter (Fig. 1I). 12 12 12 Late gastrula. The yolk plug measures one-third of the embryo’s diameter or 19,7 less (Figs. 1J–K, 3J–K, M). 13 13 13 Slit blastopore stage. Some embryos have a small blastopore (Figs. 1L, 3L). 1 The neural plate becomes visible in the late stage 13 (Fig. 3N). 14 14 14 Early neural fold stage (Figs. 1M, 3O, 4A) 1.1 15 15 Mid neural fold stage (Figs. 1N–O, 4B–H). 1.2 16 16 Closure of the neural tube (Figs. 1P, 4I–L). 1.3 17 17 Tail bud stage. The tail bud and the head region protrude beyond the yolky 1.5 endoderm (Figs. 2A, 5A–F). 18 18 Muscular activity. The branchial arches protrude on the sides of the head. The 2 cement gland is visible. The tail becomes elongated (Figs. 2B–C, 5G–H). 19 19 The heart beats, and the gill buds develop (not shown). 2.3 20 20 Circulation to the external gills. The first gill pair has 3 branches at this stage. 2.5 Buds of the second gill pair are visible (Fig. 2D). 21 21 The first gill pair has 4 branches, and the second gill pair has 3 branches (not 2.8 shown). 22 22 Blood circulation in the tail fin is detected. Five branches occur in the first pair 3.1 of external gills, and 4 branches in the second pair. Embryos have a tadpole appearance (Fig. 2E). 23 23 The external gills have reached their full size. There are 6 gill branches in the 3.4 first pair and 5 branches in the second pair of external gills. The opercular fold is forming. Embryos have a white appearance, although they contain some dark pigment (Fig. 2F–G).

aThe stages of development for Engystomops (E) are compared to the table of stages of X. laevis (X) (Nieuwkoop and Faber, 1994), and to the general staging table for frogs (G) (Gosner, 1960). bDevelopmental times for E. coloradorum embryos maintained at room temperature (18–23°C) in a humid chamber. and synchronous, and new cleavage these embryos is white, dark pig- were detected in animal cells of blas- furrows were detected every 20 min ment was detected around nuclei of tula stage embryos (Fig. 3D). during the early cleavage cycles (Ta- surface cells (Fig. 1F). ble 1). The first cleavage furrow di- Development of the blastocoel is vided the egg into two blastomeres shown in Figure 3A–C. The blastocoel Gastrulation (st 10–13) (st. 2, not shown). At the next three was detected as a small cavity in the cleavage cycles, new cleavage fur- animal region of 8-cell-stage embryos External views of the gastrula are rows developed, and divided the em- (st 4, not shown). The blastocoel en- given in Figures 1H–L, 3J. The mor- bryo into 4, 8 (st 3–4, Fig. 1B,C), 16, larged from cleavage to the onset of phology of the gastrula and the ex- and 32 blastomeres (st 5–6; Fig. gastrulation (st 5–10.5; Fig. 3A–C,F). pression of the protein Lim1 are 1D,E). The planes of these cleavage Dark pigment was detected around in- shown in Figure 3E–G, J–N. A prom- furrows were equivalent with those ternal nuclei, as observed in the blas- inent dorsal blastopore lip developed of X. laevis embryos (Nieuwkoop and tocoel roof (Fig. 3B), in the vegetal in the subequatorial region at the Faber, 1994). Animal blastomeres region (not shown), and in surface onset of gastrulation (st 10.5; Figs. were smaller than the vegetal blas- cells of the equatorial region (st 7; Fig. 1H, 3E), as in embryos of X. laevis. tomeres, as shown for blastula stage 1F). In spite of this light pigmenta- Time-lapse movies of gastrulation embryos (st 7–9; Fig. 1F,G). Al- tion, embryos had a white appear- revealed that the blastopore closes though the external appearance of ance. Transcripts of the gene RBBP6 asymmetrically, moving ventrally EMBRYOS OF FOAM-NESTING FROGS 1447 over the yolk plug as gastrulation (Fig. 1M–O). Neural folds were far and streams of cranial neural crest cells proceeded (see Supp. Movie, which is more pronounced than those in X. lae- is comparable with the previously de- available online), as has been ob- vis (Fig. 4G,H). Similarly, the gastro- tected expression in G. riobambae em- served in X. laevis. The archenteron coel roof plate was larger than in X. bryos (del Pino and Medina, 1998). The was small at 10.5 (Fig. 3F,G), and laevis (Figs. 3N, 4H). Transcripts of closed neural tube and optic vesicles bottle cells were found at the ante- the gene RBBP6 were restricted to the were detected by immunostaining rior tip of the archenteron (Fig. 3G), presumptive eye region (st 14, Fig. against NCAM (st 17; Fig. 5D). Em- as in X. laevis embryos. The thick- 4A). Somites were detected in st-15 bryos of st 17 had about 12 somites ness of the blastocoel roof decreased embryos (Fig. 4B,C). The neural folds pairs, as detected by immunostaining from 5–6 cell-layers in a large-cell were positive for antigen 2G9 in st-15 against sarcomeric meromyosin (st 17; blastula (st 7; Fig. 3B) to 3 layers in embryos (Fig. 4D). The protein Lim1 Fig. 5E). Pronephros, immunostained the mid gastrula (st 11; Fig. 3H), and was used as marker of the notochord, against Lim1, were larger than in st-15 finally to 2 cell layers in the late specific cells of the cns, and prone- embryos (Fig. 5F). gastrula (st 12; Fig. 3I). During gas- phros (st 15; Fig. 4E,F). The notochord Embryos of st 18 were characterized trulation, the blastopore decreased was covered by endoderm in the ros- by muscular activity (Table 1). Em- in size until it was just a slit at st 13 tral region (Fig. 4G), and it was ex- bryos of this stage acquired an elon- (Fig. 1I–L). Some embryos of st 13, posed in the caudal region of st-15 em- gated shape, somites were numerous, however, had a small yolk plug (Figs. bryos (Fig. 4H), as detected previously and the tail was longer (Fig. 5G). The 1L, 3L). The dorsal blastopore lip of in st 13–14 (Fig. 3N,O). The neural heart was positive for sarcomeric mero- st-10.5 embryos of E. coloradorum folds gradually closed in the midline myosin (Fig. 5G). Pronephros and pro- gave a positive signal for the protein during st 16 (Figs. 1P, 4I). The nephric ducts were fully developed in Lim1 (Fig. 3E), as previously de- streams of cranial neural crest cells embryos of this stage (Fig. 5H). Spe- tected in E. randi embryos (Venegas were detected by the expression of an- cific cns cells were Lim1-positive (not et al., 2009). In st-12 embryos (Fig. tigen 2G9 in st-16 embryos (Fig. 4J). shown). Embryos developed the ce- 3J), the archenteron elongated and Somites were detected by immuno- ment gland, which was clearly detect- became inflated (Fig. 3K), and the staining against sarcomeric meromy- able at st 18 (not shown). blastocoel was still large (Fig. 3K). osin in st-15 embryos (not shown) and Buds of the external gills developed In slightly more advanced embryos, became more clearly detectable in in st-19 embryos (not shown). Further the archenteron was fully elongated st-16 embryos (Fig. 4K). Somites con- development of the external gills oc- and inflated (st 13, Fig. 3L). Small tain numerous cells of round shape curred from st 20–23 (Fig. 2D–G). At remnants of the blastocoel were de- (Fig. 4L), a feature that greatly differs hatching, external gills were small tected (Fig. 3L). Elongation of the from the X. laevis mode of somitogen- and contained 6 gill branches in the notochord started in the mid gas- esis, which involves fewer cells and first pair and 5 branches in the second trula, as detected by immunostain- cell rotation (Radice et al., 1989). pair (St 23; Fig. 2G). The tail became ing against the protein Lim1 (not gradually elongated from st 18–23, shown). The notochord of a st-12.5 The Tail Bud and More and embryos acquired the shape of a embryo is shown in Figurer 3M. The tadpole (Fig. 2C,D,F). The opercular notochord was covered by endoderm Advanced Embryos (st 17–23) fold was detected in st-22–23 embryos in the rostral region of st-13, and -14 External views of tail bud and more ad- (not shown). A remarkable feature of embryos (Fig. 3O). In the caudal re- vanced embryos are given in Figure Engystomops development is the al- gion, however, the notochord was not 2A–G. Expression of neural and somite most complete absence of dark pig- covered by endoderm, and was ex- markers are shown in Figure 5. Em- ment during development in the foam posed in the gastrocoel roof plate bryos were curved around a large yolky nest. The body and the head developed (Fig. 3N). These findings are consis- endoderm, and differed in shape from some dark pigment in st-20–23 em- tent with the previous observation the elongated X. laevis tailbud (st bryos (Fig. 2D–G). In spite of the lim- that the mode of gastrulation in En- 17–18; Figs. 2A–C, 5A). Prominent ited pigmentation, hatching embryos gystomops resembles that in X. lae- branchial arches were visible in living had a white appearance (Fig. 2F). Em- vis (del Pino et al., 2007). and fixed embryos (st 17; Fig. 2A–C). bryos of the three species of Engysto- Transcripts of the gene RBBP6 were mops hatched at st 23 (Table 1). Neurulation and Somite detected in the neural region (Fig. 5A). Formation (st 15–16) After immunostaining against antigen, Comparison of Engystomops 2G9 rhombomeres and streams of cra- Development With Other Frogs The morphology of the neurula, and nial neural crest cells were visible (st. the expression of neural and somite 17; Fig. 5B,C). Rhombomeres 3 and 5 Early development in E. pustulosus, markers are shown in Figure 4. Exter- were negative for antigen 2G9 (Fig. E. coloradorum, and E. randi greatly nal views of the neurula are given in 5B,C). The branchial anterior stream resembles X. laevis development. Figures 1M–P, 4I. The neural plate was weakly stained in comparison with Eggs and early embryos are white, de- was detected in st-13–14 embryos the mandibular, hyoid, and branchial velop rapidly, and resemble X. laevis (Fig. 3N,O). The open neural folds posterior streams of cranial neural crest albino embryos in size and appear- were detected in embryos of st 14 and cells (st 17; Fig. 5C). The expression ance. It should be noted, however, were clearly visible in st-15 embryos pattern of antigen 2G9 in rhombomeres that blastomeres contain dark pig- Fig. 1. ment in the Engystomops species analyzed. Pigment surrounds the nuclei, instead of being localized to the surface of animal blastomeres (Figs. 1F, 3B). The patterns of cleavage, blastula formation, and gastrulation resemble closely the developmental patterns observed in X. laevis embryos. Elonga- tion of the notochord overlaps with other processes of gastrulation in these rapidly developing frogs, as in X. laevis. In contrast, in the slow developing embryos of the dendrobatid frogs, C. machalilla and G. riobambae, the process of notochord elongation occurs after blastopore closure (del Pino,1996; Benı´tez and del Pino, 2002; Venegas et al., 2009). The limitations imposed by the aquatic reproductive mode of X. laevis and Engystomops Fig. 2. EMBRYOS OF FOAM-NESTING FROGS 1449

Fig. 3. Morphology of early embryos. A: Blastocoel of a 16-cell embryo. B: Blastocoel of a large-cell blastula. The arrow signals a darkly pigmented nucleus. C: Blastocoel of a medium-cell blastula. D: RBBP6 in situ hybridization signal is in the animal hemisphere. E: Lim1-positive nuclei occurred in the dorsal blastopore lip. F: Sagittal section of an early gastrula. G: Higher magniﬁcation from the embryo in F. The arrow signals a bottle cell. H: Three cell layers in the blastocoel roof of a mid gastrula. I: Two cell layers in the blastocoel roof of a late gastrula. J: Blastopore of a late gastrula. K: Sagittal section of a late gastrula. L: Sagittal section of a stage-13 embryo. M: Lim1-positive notochord of a late gastrula. N: Transverse section through the caudal region of a late gastrula. Arrows indicate the lateral endodermal crests at the limits of the gastrocoel roof plate. O: Transversal section through the rostral region of an early neurula. The endoderm covered the archenteron roof. a, archenteron; bl, blastocoel; dl, dorsal lip; n, notochord; np, neural plate; yp, yolk plug. Scale bars ϭ 50 ␮m in G,N,O; 100 ␮m in H,I; 200 ␮m in A–D,E,M; 250 ␮m in F,J,K,L.

Fig. 1. External views of embryos from cleavage to the neurula. A: Eggs within the foam-nest. B: Four-cell stage. C: Eight-cell stage. D: Sixteen-cell stage. E: Thirty-two cell stage. F: Large-cell blastula. Nuclei were darkly pigmented (arrow). G: Medium-cell blastula. H: Early gastrula. I: Mid gastrula. J: Late gastrula. K: Late gastrula with a small yolk plug. L: Stage-13 embryo that shows formation of the neural groove. This embryo has a yolk plug. Other embryos had a slit blastopore. M: Early neural fold stage. N: Mid neural fold stage. O: Caudal view of the embryo in N. P: Closure of the neural tube. The pink color of embryos is an artifact of fixation. In this and the following figures, numbers in the top right-hand corner give the developmental stage, and letters indicate the species: Ec, E. coloradorum; Ep, E. pustulosus; Er, E. randi. b, blastopore; c, cleavage furrow; dl, dorsal lip; f, jelly-foam; ng, neural groove; np, neural plate; nt, formation of the neural tube; yp, yolk plug. Scale bars ϭ 300 ␮m in B–P; 500 ␮minA. Fig. 2. External views of embryos from tail bud to hatching. A: Tail bud stage. B: Embryo at the muscular response stage. C: Dorsal view of the embryo in B. D: Embryo at the circulation to the external gills stage. The trunk acquired some dark pigment. E: Head region of a stage-22 embryo. Dark pigment was detected in the head region. F: Embryo at hatching. G: Higher magnification of the head region from the embryo in F. The external gills reached their maximum length. br, branchial arch; g, gills; tf, tail fin. Scale bars ϭ 300 ␮minA;400␮minE;500␮minG;600␮mB–D;1mminF. 1450 ROMERO-CARVAJAL ET AL.

Fig. 4. may have favored the acceleration of early development and, consequently, the overlap of developmental process during gastrulation. Although early development of tu´ ngara frogs closely resembled the developmental pattern of X. laevis, more advanced embryos greatly differed from X. laevis. The external morphology of advanced embryos of Engystomops resembled the morphology of Rana embryos (Pollis- ter and Moore, 1937), with a major difference in pigmentation, as foam- nest embryos of tu´ ngara frogs lack dark surface pigment. Tu´ ngara frog embryos were staged according to the X. laevis table of stages only until st 14 (Nieuwkoop and Faber, 1994). Thereafter, the embryonic morphology differed from X. laevis. Differences detected included the general shape of tail bud embryos (st 17), pattern of somitogenesis, and size of the cranial neural crest cell streams. Somitogenesis involves numerous small cells and cell intercala- tion in E. coloradorum and E. randi, whereas in X. laevis, each somite consists of cells, which are already elon- Fig. 5. EMBRYOS OF FOAM-NESTING FROGS 1451 gated, and the mode of somitogenesis human chorionic gonadotropin (hCG, gradually removed for analysis. The involves cell rotation (Radice et al., Davidson and Hough, 1969). Addition- jelly layers were somewhat liquid and 1989; del Pino et al., 2007). Moreover, ally, phonotaxis is induced in adult E. were successfully removed with for- the neural plate and neural folds of pustulosus females with hCG, which ceps. In some instances, embryos were tu´ ngara frog embryos are larger than will eventually cause the female to ovu- chemically dejellied according to the in X. laevis. The cranial neural crest late (Lynch et al., 2006). Ovulation was X. laevis method (Sive et al., 2000). cell streams of E. randi embryos are similarly triggered in three females of also more conspicuous than in X. lae- E. randi by the subcutaneal adminis- Handling of embryos, vis embryos and are comparable to the tration of three doses of 100, 200, and immunostaining, and time-lapse pattern observed in the marsupial 300 IU of hCG, respectively. The hor- recordings. frog G. riobambae (del Pino and Me- mone was administered at 3-day inter- dina, 1998; del Pino et al., 2007). vals. Embryos of the tu´ ngara frogs were Therefore, although frogs of the genus handled according to X. laevis meth- Engystomops and X. laevis develop Removal of the egg jelly. ods (Sive et al., 2000), including time- rapidly, and have a similar morphol- We have found that embryos of E. pus- lapse recordings. To maintain em- ogy of early development, there are tulosus can be easily released from bryos in the foam-nest, we placed the important differences in their ad- foam nests with an adaptation of the foam-nests of E. coloradorum and E. vanced morphogenetic patterns. X. laevis de-jellying method (Sive et randi in deep plastic dishes of about al., 2000). Foam nests were removed 15 cm in diameter that were half-filled from terraria and placed onto the sur- with chlorine-free water. Dishes were Methods for the Study of face of de-jellying solution (3% cys- covered with plastic film to prevent Engystomops Development teine in 33% MMR, pH 7.2) in a wide desiccation and were maintained at room temperature (18–22°C). Some Methods used to study X. laevis devel- beaker. The beaker was gently swirled embryos were moved to a Petri dish opment (Sive et al., 2000) were suc- for several minutes. At intervals, em- filled with 15% Steinberg’s solution cessfully adapted to analyze the embryos dropped from the nest into the (Rugh, 1962) to study embryogenesis. bryogenesis of tu´ ngara frogs. solution, and were immediately removed to a dish containing 33% MMR Procedures for fixation, vibratome sec- Induction of ovulation. (MMR solution was prepared accord- tioning of embryos, and immunostain- Ovulation in tu´ ngara frogs can be in- ing to Sive et al., 2000). In some cases, ing were previously described (del duced by adding water to terraria and agitation of the foam with forceps has- Pino et al., 2004; Venegas et al., 2009). by water spraying to simulate rain, as tened release of embryos. Embryos explained in the section on mainte- were then reared as per X. laevis. Injection of mRNA, gene nance and reproduction. As in X. laevis, In contrast, embryos of E. colo- isolation, and in situ it is known that E. pustulosus responds radorum and E. randi were main- hybridization. with ovulation to the administration of tained within the foam nest, and were Injections of mRNA into early cleavage stage embryos were straightfor- ward using the same method em- ployed for X. laevis (not shown) (Sive Fig. 4. Morphology of the neurula. A: Neurula stage, anterior view, dorsal to the top. In situ et al., 2000). We have also adapted the hybridization signal of RBBP6 was detected in the presumptive eye region. B: Para-sagittal section Xenopus in situ hybridization method. through the somites. C: Higher magnification, of the embryo in B. D: The antigen 2G9–positive We cloned a partial cDNA for the neural folds. E: The Lim1-positive notochord, cns cells, and pronephros anlage in a stage-15 RBBP6 gene out of E. pustulosus, and embryo. F: Higher magnification of the embryo in E. The arrows in E,F signal a cns Lim 1-positive nucleus. G: Transversal section through the rostral region. The archenteron roof is covered by by in situ hybridization we found it to endoderm. H: Transversal section through the caudal region. The notochord is exposed in the be expressed in the animal region of gastrocoel roof plate. Arrows signal the lateral endodermal crests. I: A late neurula in dorsal view. the blastula (Fig. 3D) and neural tis- J: The antigen 2G9–positive neural tube and cranial neural crest cell streams. K: Embryo immu- sues, especially the presumptive eye nostained against sarcomeric meromyosin (indicated as Myosin in the image). L: Horizontal section through the somites. a, archenteron; cnc, cranial neural crest; n, notochord; nt, neural tube; p, (Figs. 4A, 5A). We have also per- pronephros anlage; s, somite. Scale bars ϭ 100 ␮m in C,F–H,J,L; 200 ␮m in D,E; 250 ␮m in A,B,K; formed in situ hybridization to the 400 ␮minI. Shroom2 gene (Lee et al., unpublished data). Fig. 5. Morphology of tailbud embryos. A: RBBP6 in situ hybridization signal was detected in the neural tube. B: Dorsal view of an embryo immunostained against antigen 2G9. Rhombomere 5 is 2G9-negative. C: Lateral view of the embryo in B that shows the streams of cranial neural crest. D: Dorsal view of an embryo immunostained against NCAM. The neural tube and optic vesicles are Maintenance and NCAM-positive. E: Somites are sarcomeric meromyosin–positive. F: The Lim 1–positive prone- Reproduction in Captivity phros. G: Stage-18 embryo. The somites and heart were positive for sarcomeric meromyosin (sarcomeric meromyosin is indicated as Myosin in the images E,G). H: Stage-18.5 embryo. The Lim Tadpoles, juveniles, and adults of E. 1–positive pronephros and pronephric ducts are Lim 1 positive. The Lim 1–positive cns cells are not pustulosusand E. randi were kept in shown. ba, branchial anterior stream of cranial neural crest cells; bp, branchial posterior stream of terraria, with successful reproduction. cranial neural crest cells; br, branchial arch; h, heart; hy, hyoid stream of cranial neural crest cells; m, mandibular stream of cranial neural crest cells; nt, neural tube; o, optic vesicle; p, pronephros; Maintenance in captivity of E. pustu- pd, pronephric duct; r5, rhombomere 5; s, somite. Scale bars ϭ 200 ␮m in B,C,F,H; 300 ␮minA; losus at the University of Texas, Aus- 400 ␮m in D,E; 500 ␮minG. tin, differed from the method em- 1452 ROMERO-CARVAJAL ET AL. ployed with E. randi at the Pontificia ported by legs. Small-sized gravel par- inhibited reproduction. Accordingly, Universidad Cato´lica del Ecuador. tially covered the plastic mesh. Ter- we changed the water and flooded the Both methods are described. raria were slightly tilted, resulting in terraria to induce reproduction. The E. pustulosus rearing labora- the formation of a shallow water pool The 32 E. randi adults produced a tory at The University of Texas, Aus- of about 1 cm in depth in the gravel- total of 80 foam-nests in 2 years (Feb- tin is an adaptation of the enclosures free area. Water was changed every 2 ruary 2006 to March 2008). The used at the National Aquarium in Bal- months. Coconut shells and a plant, a monthly production ranged from timore (MD) and the San Antonio Zoo Phylodendrum sp., provided hiding 0–16, with an average of 3 Ϯ 4 foam- (TX). The laboratory has a daily light places. Each terrarium was covered nests per month (the mean and stan- cycle of 12 hr and is maintained at with plastic mesh and two glass plates dard deviation are given). Foam-nests 27–28°C with humidity at 30–50% that were placed slightly apart from were removed and cultured in aquaria year round. Terraria are placed on each other. This cover arrangement al- until metamorphosis. Development in wooden frames with a downward an- lowed aeration, prevented frogs from the foam-nest took 3 days until hatch- gle of 30° and include 5.5-, 10-, and breaking away, and insured conditions ing (observation of 9 foam-nests). 15-gallon glass aquaria that have a of high humidity. Terraria with a lower Based on the analysis of 121 tadpoles, hole with a connector and plastic tub- water level did not contain water pools, the aquatic tadpole stages lasted ing to allow for drainage. A 2” layer of and were used to keep young frogs. about one month until metamorphosis autoclaved and rehydrated sphagnum Foam-nests, embryos, and tadpoles (34 Ϯ 6 days; mean and standard de- moss covers the bottom of each terrar- were maintained under equivalent con- viation are given). A maximum of 20 ium. Moss is hydrated using room tem- ditions of light in 50 ϫ 30 ϫ 30 cm juveniles were maintained per terrar- perature chlorine-free water. Plastic aquaria. The aquaria were half-filled ium. Six months after metamorphosis, huts made of plant pots and artificial with chlorine-free water, and the water young frogs of the first and second plants are placed in terraria to act as temperature fluctuated between 24– generations produced foam-nests, an refugia. Terrarium covers are made of 26°C. Pumps provided aeration. Water indication of sexual maturity (193 and plastic frames with mesh screens and was changed at weekly intervals. 201 days, respectively). lined with Velcro and weather-strip- Tadpoles were fed with a mixture of Engystomops randi was success- ping to prevent escapes. dried and finely ground plant leaves fully maintained in small terraria and Reproduction is induced by adding a and invertebrates, as follows: the wa- reached adulthood in 7–8 months af- water pond (small container with rocks ter fern Azolla sp., Taraxacum, Spir- ter fertilization. In contrast, Davidson and chlorine-free water), increasing hu- ulina, Cyclops, Daphnia, and Tubifex. and Hough (1969) maintained from 50 midity, and sometimes simulating rain- Newly metamorphosed frogs were fed to 400 individuals of E. pustulosus in fall by spraying the terraria with chlo- daily for about 10 days with collem- beds of sterilized earth placed in the rine-free water. Pairs produce a foam bola (Folsomia candida). Somewhat floor of a room of 8 ϫ 12 feet with nest that is then transferred to a 5-gal- larger frogs received newly hatched controlled conditions of light, temper- lon aquarium filled with chlorine-free crickets, Gryllus sp., as prey. Adults ature, and humidity. Tadpoles of E. water. were fed every other day with 15-day- pustulosus required 3–4 weeks to Tadpoles hatch after 2 days. hatched crickets and Drosophila. reach metamorphosis, and young Tadpoles are housed in aquaria filled The presence of a water reservoir at frogs reproduced 2–3 months after with chlorine-free water and each day the bottom of each terrarium was ad- metamorphosis (Davidson and Hough, are fed JurassiDiet, a pellet food for vantageous for the maintenance of sub- 1969). aquatic reptiles. Within 6 weeks, there strate humidity. Additionally, when the Tu´ ngara frogs are attractive for de- are newly metamorphosed frogs. These water level of the reservoir was in- velopmental work due to the ease of frogs are fed wingless Drosophila only, creased, a water pool was formed, and their maintenance in captivity and while juvenile and adult frogs are fed a the frogs used it for reproduction. In rapid development. In addition, repro- combination of Drosophila and 7-day contrast, reduction of the water level in duction can be induced by increasing hatched crickets three times per week. the reservoir diminished or eliminated the environmental humidity and by Engystomops randi was maintained the water pool. Young adults of E. randi flooding. The synchronous oogenesis, at the Herpetology Section of the Mu- reproduced often, whereas one-year-old rapid development, and short life cy- seum of Zoology of Pontificia Univer- and older frogs reproduced less fre- cle of these frogs are attractive for sidad Cato´lica del Ecuador. Frogs quently or not at all. studies that involve the induction of were kept in terraria, which were One terrarium contained 12 frogs (6 developmental mutations and genetic stacked in shelves inside a room with males and 6 females) collected from studies of development. controlled environmental conditions. nature. In two additional terraria, we The daily light-cycle was 12 hr, and maintained 5 males and 5 female lab- room-temperature fluctuated between oratory-raised frogs per terrarium. EXPERIMENTAL 24–28°C. Terraria measured 60 ϫ These frogs belonged to the first and 35 ϫ 35 cm and were arranged as fol- second generations. We observed that PROCEDURES lows: Chlorine-free water filled the reproduction occurred when the level Immunostaining Methods floor to a height of about 5 cm. The of the water in the reservoir was in- water was covered with a sheet of creased until the pool was flooded. In Embryos were immunostained in sturdy plastic mesh that was sup- contrast, reduction of the water level whole mount against Lim1, neural, EMBRYOS OF FOAM-NESTING FROGS 1453 and somite-specific proteins. We used Frog Collection Sites heavy chain during avian myogenesis in the monoclonal antibody MF-20 vivo and in vitro. J Cell Sci 95:763–770. Foam nests of E. pustulosus were ob- (Bader et al., 1982) against adult Benı´tez MS, del Pino EM. 2002. The ex- tained from frogs maintained at The pression of Brachyury during develop- chicken pectoralis myosin to detect University of Texas at Austin. E. pus- ment of the dendrobatid frog Colostethus muscle development. The monoclonal tulosus were collected at various times machalilla. Dev Dyn 225:592–596. Davidson EH, Hough BR. 1969. Synchro- antibody (4d) against NCAM from from the Panama´ Canal area in Pan- chick brain membranes (Watanabe et nous oogenesis in Engystomops pustulo- ama´ with permission of the Smithso- sus, a neotropic anuran suitable for lab- al., 1986) was used to detect the neu- nian Tropical Research Institute and oratory studies: localization in the ral tube. These two antibodies were with scientific collecting permits from embryo of RNA synthesized at the lamp- obtained from the Developmental Autoridad Nacional del Ambiente, brush stage. J Exp Zool 172:25–48. Studies Hybridoma Bank (Iowa City, Panama´. del Pino EM. 1996. The expression of IA). The monoclonal antibody 2G9 Brachyury (T) during gastrulation in the Six couples of E. randi were collected marsupial frog Gastrotheca riobambae. against a neural antigen from X. lae- from 8 to 23 February 2005 in Ecuador, Dev Biol 177:64–72. vis (Jones and Woodland, 1989) was Province of “El Oro,” road to the Puy- del Pino EM, Medina A. 1998. Neural De- used to detect the neural tube and the ango Forest. The altitude is 288 m velopment in the marsupial frog Gas- neural crest (del Pino and Medina, above sea level. The geographic coordi- trotheca riobambae. Int J Dev Biol 42: 723–731. 1998). This antibody was donated by nates of this site are W 80.083319, S del Pino EM, A´ vila ME, Pe´rez OD, Benı´tez E. A. Jones (University of Warwick, 3.88184. Three couples of E. colo- MS, Alarcoń I, Noboa V, Moya IM. 2004. Warwick, UK). Some embryos were radorum were collected on January 30, Development of the dendrobatid frog Co- immunostained with a polyclonal an- 2005 from Ecuador, Province of Pichin- lostethus machalilla. Int J Dev Biol 48: tibody against Lim1, donated by M. cha, secondary road near Tinalandia 663–670. north bank of the Toachi river. The al- del Pino EM, Venegas-Ferrıń M, Romero- Taira (University of Tokyo, Japan). Carvajal A, Montenegro-Larrea P, Secondary antibodies were sheep anti- titude of this site is 938 m above sea Saénz-Ponce N, Moya IM, Alarcoń I, Su- rabbit IgG and sheep anti-mouse IgG. level, and the geographic coordinates dou N, Yamamoto S, Taira MA. 2007. Both antibodies were conjugated to al- are W 79.05028, S 0.29546. Frogs were Comparative analysis of frog early development. Proc Natl Acad Sci USA 104: kaline phosphatase (Roche Molecular collected by S. R. Ron, G. Romero, and E. Tapia. Authorization 016-IC-FAU- 11882–11888. Biochemicals, Mannheim, Germany). Duellman WE, Trueb L. 1986. Biology of Immunostaining was done as previ- DNBAP-MA from the Ministry of the amphibians. New York: McGraw-Hill. ously described (del Pino et al., 2004; Environment allowed the collection of 670 p. Venegas et al., 2009). Some sections frogs from Ecuador. Ewald AJ, Peyrot SM, Tyszka JM, Fraser SE, Wallingford JB. 2004. Regional re- were cleared, stained with Hoechst ACKNOWLEDGMENTS quirements for Dishevelled signaling 33258 (Sigma-Aldrich, St. Louis, MO), during Xenopus gastrulation: separable mounted in glycerol, and examined We thank L. A. Coloma, S. Ron, and effects on blastopore closure, mesendo- with fluorescent optics. other members of the Herpetology Sec- derm internalization and archenteron tion, Museum of Zoology of the Pontifi- formation. Development 131:6195–6209. cia Universidad Cato´lica del Ecuador, Gosner KL. 1960. A simplified table for for the donation of several E. colo- staging anuran embryos and larvae with In Situ Hybridization notes on identification. Herpetologica 16: radorum adults and for their help with 183–190. E. pustulosus embryos fertilized natu- the reproduction of E. randi (its repro- ϫ Grant, T, Frost DR, Caldwell JP, Gagliardo rally were de-jellied in 1/3 modified duction received financial support from R, Haddad CFB, Kok PJR, Means DB, Marc’s Ringer (MMR) with 3% (w/v) the research program “Balsa de los Sa- Noonan BP, Schargel WE, Wheeler WC. cysteine at pH 7.8. They were fixed at pos”). We thank E. A. Jones (University 2006. Phylogenetic systematics of dart- poison frogs and their relatives (Am- the neurula and tailbud stage with 1ϫ of Warwick, UK) and M. Taira (Univer- phibia: Athesphatanura: Dendrobati- MEMFA and were dehydrated with sity of Tokyo, Japan) for the donation of dae). Bull Am Mus Nat Hist 299:1–262. 100% methanol (according to Sive et antibodies. We acknowledge the help of Hough BR, Yancey PH, Davidson EH. al., 2000). O. D. Pe´rez, and P. Montenegro-Larrea 1973. Persistence of maternal RNA in To synthesize a probe for RBBP6 with frog care and discussions, and the Engystomops embryos. J Exp Zool 185: transcripts (RetinoBlastoma Binding assistance of J. Davis and D. Davis with 357–368. Jones EA, Woodland HR. 1989. Spatial as- Protein 6), partial RBBP6 cDNA was language revision of a previous version. pects of neural induction in Xenopus lae- cloned into a PCRII vector (Invitro- E.M.P. was supported by grants from vis. Development 107:785–791. gen) by using FirstChoice RLM-RACE Pontificia Universidad Cato´lica del Ec- Lynch KS, Crews DC, Ryan MJ, Wilczyn- Kit (Ambion) and the probe was syn- uador: 2006, 2007, 2008, and The Acad- ski W. 2006. Hormonal state influences thesized with T7 polymerase (Pro- emy for the Developing World (TWAS), aspects of female mate choice in the tuń- Grant 07-017 LDC/BIO/LA-UNESCO gara frog (Physalaemus pustulosus). mega). In situ hybridization for E. Horm Behav 49:450–457. pustulosus was performed with X. lae- FR 3240144821. J.B.W. and C.J.L. were Moya IM, Alarcoń I, del Pino EM. 2007. vis in situ hybridization protocol (Sive supported by NIH/NIGMS. Gastrulation of Gastrotheca riobambae et al., 2000). RBBP6-expressing re- in comparison with other frogs. Dev Biol 304:467–478. gions were colorized with alkaline REFERENCES Nieuwkoop PD, Faber J. 1994. Normal ta- phosphatase (AP) and BM purple AP Bader D, Masaki T, Fischman DA. 1982. ble of Xenopus laevis (Daudin). New substrate (Roche). Immunochemical analysis of myosin York: Garland Publishing. 252 p. 1454 ROMERO-CARVAJAL ET AL.

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