Mesoderm formation in Eleutherodactylus coqui: body patterning in a frog with a large egg.

The direct developing frog, Eleutherodactylus coqui, develops from a large egg (diameter 3.5 mm). To investigate the effect of egg size on germ-layer formation, we studied mesoderm formation in E. coqui and compared it to that of Xenopus laevis (diameter 1.3 mm). First, we identified the position of prospective mesoderm in the 16-cell E. coqui embryo by cell-lineage tracing. Although the animal blastomeres are small, they form most of the blastocoel roof and make extensive contributions to some mesodermal tissues. Second, we performed recombinant analysis with X. laevis animal caps to define the distribution of mesoderm-inducing activity. Mesoderm-inducing activity in E. coqui was restricted around the marginal zone with strong activity in the superficial cells. Neither the vegetal pole nor the blastocoel floor had activity, although these same regions from X. laevis induced mesoderm. Third, we cloned Ecbra, a homologue of Xbra, an early mesoderm marker in X. laevis. Ecbra was expressed in the marginal ring close to the surface, similar to X. laevis, but E. coqui had weaker expression on the dorsal side. Our results suggest that mesoderm formation is shifted more animally and superficially in E. coqui compared to X. laevis.     

Status of RNAs, localized in Xenopus laevis oocytes, in the frogs Rana pipiens and Eleutherodactylus coqui

Early development in the frog model, Xenopus laevis, is governed by RNAs, localized to the vegetal cortex of the oocyte. These RNAs include Xdazl RNA, which is involved in primordial germ cell formation, and VegT RNA, which specifies the mesoderm and endoderm. In order to determine whether orthologues of these RNAs are localized and have similar functions in other frogs, we cloned RpDazl and RpVegT from Rana pipiens, a frog that is phylogenetically distant from X. laevis. RNAs from both genes are localized to the vegetal cortex of the R. pipiens oocyte, indicating that the vegetal localization is likely the basal state. The animal location of EcVegT RNA in Eleutherodactylus coqui that we found previously (Beckham et al., 2003) is then a derived state, probably due to the great increase in egg size required for direct development of this species. To answer the question of function, we injected RpVegT or EcVegT RNAs into X. laevis embryos, and assayed animal caps for gene expression. Both of these RNAs induced the expression of endodermal, mesodermal, and organizer genes, showing that the function of RpVegT and EcVegT as meso-endodermal determinants is conserved in frogs. The RNA localizations and the function of VegT orthologues in germ layer specification may be synapomorphies for anuran amphibians.     

Development in frogs with large eggs and the origin of amniotes

The origin of the amniote egg is one of the most significant events in the evolution of terrestrial vertebrates. This innovation was probably driven by increased egg size, and to find potential parallels, we can examine the derived development of extant amphibians with large eggs. The embryo of the Puerto Rican tree frog, Eleutherodactylus coqui, exhibits an alteration of its fate map and a secondary coverage of its yolky cells, reflecting the large 3.5 mm egg. Comparable changes may have occurred with the derivation of an amniote pattern of development. Future investigations should focus on the molecular organization of the egg. In the model amphibian for development, Xenopus laevis, information for embryonic germ layers, the dorsal axis, and germ cells is stored mainly as localized RNAs at the vegetal pole of the egg. These localizations would likely be changed with increased egg size. A review of the orthologues of the key X. laevis genes raises the possibility that their activities are not conserved in other vertebrates.     

Use of Hybrids between Xenopus laevis and Xenopus borealis in Chimera Formation: Dorsalization of Ventral Cells

The combination of Xenopus borealis and X. laevis provides an excellent cell marking system. The potential availability of this system for chimera formation has also been suggested. However, eggs and early embryos of these species differ in size and the fusion of blastomeres of different sizez results in some disturbance in arrangement of blastomeres of a chimera. This disturbance was avoided by use of embryos from X. laevis eggs fertilized with X. borealis sperm, instead of X. borealis embryos. The cells of these hybrids could also be distinguished from the cells of X. laevis.
The fate of animal ventral cells placed in the dorsal region was followed by making a chimera by fusing a right lateral half of an 8-cell X. laevis embryo with that of an 8-cell hybrid embryo. The animal ventral cells in the "dorsal" region were found to become "dorsalized", giving rise to a lateral half of dorsal axial structures. This observation explains a previous finding that the replacement of dorsal cells by ventral ones had no effect on embryogenesis in a composite embryo.     

Novel regulation of yolk utilization by thyroid hormone in embryos of the direct developing frog Eleutherodactylus coqui

Thyroid hormone (TH) is required for metamorphosis of the long, coiled tadpole gut into the short frog gut. Eleutherodactylus coqui, a direct developing frog, lacks a tadpole. Its embryonic gut is a miniature adult form with a mass of yolky cells, called nutritional endoderm, attached to the small intestine. We tested the TH requirement for gut development in E. coqui. Inhibition of TH synthesis with methimazole arrested gut development in its embryonic form. Embryos treated with methimazole failed to utilize the yolk in their nutritional endoderm, and survived for weeks without further development. Conversely, methimazole and 3,3',5-tri-iodo-l-thyronine, the active form of TH, stimulated gut development and utilization and disappearance of the nutritional endoderm. In Xenopus laevis, the receptor for TH, TRβ, is upregulated in response to TH. Similarly, EcTRβ, the E. coqui ortholog, was upregulated by TH in the gut. EcTRβ expression was high in the nutritional endoderm, suggesting a direct role for TH in yolk utilization by these cells. An initial step in the breakdown of yolk in X. laevis is acidification of the yolk platelet. E. coqui embryos in methimazole failed to acidify their yolk platelets, but acidification was stimulated by TH indicating its role in an early step of yolk utilization. In addition to a conserved TH role in gut development, a novel regulatory role for TH in yolk utilization has evolved in these direct developers.     

Blastomeres show differential fate changes in 8-cell Xenopus laevis embryos that are rotated 90° before first cleavage

To study the mechanisms of dorsal axis specification, the alteration in dorsal cell fate of cleavage stage blastomeres in axis-respecified Xenopus laevis embryos was investigated. Fertilized eggs were rotated 90° with the sperm entry point up or down with respect to the gravitational field. At the 8-cell stage, blastomeres were injected with the lineage tracers, Texas Red- or FITC-Dextran Amines. The distribution of the labeled progeny was mapped at the tail-bud stages (stages 35–38) and compared with the fate map of an 8-cell embryo raised in a normal orientation. As in the normal embryos, each blastomere in the rotated embryos has a characteristic and predictable cell fate. After 90° rotation the blastomeres in the 8-cell stage embryo roughly switched their position by 90°, but the fate of the blastomeres did not simply show a 90° switch appropriate for their new location. Four types of fate change were observed: (i) the normal fate of the blastomere is conserved with little change; (ii) the normal fate is completely changed and a new fate is adopted according to the blastomere's new position; (iii) the normal fate is completely changed, but the new fate is not appropriate for its new position; and (4) the blastomere partially changed its fate and the new fate is a combination of its original fate and a fate appropriate to its new location. According to the changed fates, the blastomeres that adopt dorsal fates were identified in rotated embryos. This identification of dorsal blastomeres provides basic important information for further study of dorsal signaling in Xenopus embryos.     

Independence of two microtubule systems in fertilized frog eggs: the sperm aster and the vegetal parallel array

Two microtubule-containing structures are implicated in dorsoventral polarization of the frog egg, and we examined the relationship between them. The sperm aster provides a directional cue for a cortical rotation specifying polarity, and a vegetal cortical array of parallel microtubules is likely part of the rotational machinery. The growing aster has an accumulation of microtubules marking the path of the sperm pronucleus, and its microtubules extend into the egg cortex as well as the cytoplasm. To test whether the vegetal parallel array was an extension of astral cortical growth, fertilized or activated eggs were bisected into animal and vegetal fragments. The vegetal fragments formed parallel arrays, even when isolated within a few minutes of egg activation. Neither the sperm centrosome nor another microtubule organizing center in the animal half of the egg is required for formation of the parallel array, but some animal half activity is involved in its disappearance. Correspondence to: R.P. Elinson     

The Location of Dorsal Information in Frog Early Development

Dorsal information is necessary for the development of the dorsal axial structures which characterize the vertebrates. The nature of dorsal information in early embryos is not known, but its presence is required for the formation of dorsal mesoderm with Spermann organizer activity. In frogs, the dorsoventral axis is specified by a cortical/cytoplasmic rotation in the egg shortly after fertilization, and this dorsal information is limited to a few cells in the equatorial and vegetal region of early cleavage embryos. At the 8-cell stage, 2–4 cells can promote dorsal development, and at the 32-cell stage, 4–6 cells have dorsal information. Recent experiments have shown that growth factors can induce cells to form dorsal mesoderm and that lithium can act in those cells to enhance the induction. It will be important to determine the relationship between the location of dorsal information defined embryologically and factors involved in the development of dorsal mesoderm.     

High-frequency twinning of Xenopus laevis embryos from eggs centrifuged before first cleavage

In embryos of the frog Xenopus laevis, the dorsal structures normally develop from regions of the egg opposite the side of sperm entry. Gravity and centrifugal force, applied at an angle of 90 degrees to the animal-vegetal axis of the egg, can override this topographic relationship and can cause the dorsal structures to be positioned according to the force vector (S. Black and J. Gerhart, 1985, Dev. Biol. 108, 310-324). We report here that at time 0.40 (40% of the first cleavage interval), an average of 60% of eggs centrifuged at 30g for 4 min in this orientation form conjoined twins with one body axis arising from the centripetal side of the egg and one arising from the centrifugal side of the egg. This positioning is observed regardless of the orientation of the side of sperm entry in the centrifugal field. If, after the 0.40 centrifugation, the eggs are inclined with the centripetal side up, they do not make twins; instead, they make only a single axis at the centripetal side. This indicates that the second axis in twins is caused to form by postcentrifugation gravity-driven internal rearrangements of materials that were displaced by the centrifugation. Twins also form at high frequency in eggs centrifuged twice, first at an inclination of 90 degrees, and then at an inclination of 0 degrees. The second centrifugation yields secondary axes even when it is begun midway in the second cell cycle, well after the time of grey crescent formation. Double centrifugation also causes twinning ("double rescue") of uv-irradiated eggs which otherwise would not develop axial structures. This suggests that the internal displacements caused by the centrifugations can substitute for a step in the normal axis specification process that is impaired in irradiated eggs.     

凹耳臭蛙胚胎发育及其分类学意义(英文)

通过人工受精的方法获得的凹耳臭蛙(Odorrana tormota)的早期胚胎及胚后幼体的发育过程,根据胚胎发育过程中的形态及生理特征变化规律进行分期。把凹耳臭蛙的发育过程分成两个阶段:1)早期胚胎发育阶段,即从蛙卵受精到鳃盖完成期,在18～23℃水温下,凹耳臭蛙早期胚胎发育阶段历时324h;2)蝌蚪发育阶段,即从鳃盖完成期结束到尾部被完全吸收,本阶段在20～24℃水温条件下历时1207h。凹耳臭蛙蝌蚪未发现腹吸盘特征,从形态特征上支持了分子系统分类学将之从湍蛙属划出的观点。实验中发现,多数胚胎在8细胞期为纬裂,16细胞期为经裂,同时有小部分胚胎(1.5%)在8细胞期为经裂,16细胞期为纬裂。该文进一步讨论了影响卵裂率、孵化率、发育速度,以及生态适应的因素。     

A transient array of parallel microtubules in frog eggs: potential tracks for a cytoplasmic rotation that specifies the dorso-ventral axis

The dorsoventral axis of the frog embryo is specified by a rotation of the egg cytoplasm relative to the cortex. When eggs undergoing the cortical/cytoplasmic rotation were examined by immunocytochemistry and electron microscopy, an extensive array of parallel microtubules was found covering the vegetal hemisphere of the egg. The microtubules were 1-3 microns deep from the plasma membrane and were aligned parallel to the direction of rotation. They formed at the start of rotation and disappeared at its completion. Colchicine and uv irradiation, inhibitors of the rotation, prevented the formation of the parallel microtubules. Based on these properties, we suggest that the parallel microtubules serve as tracks for the cortical/cytoplasmic rotation which specifies the dorsoventral axis of the embryo.     

Parasite loss and introduced species: a comparison of the parasites of the Puerto Rican tree frog, (<Emphasis Type="Italic">Eleutherodactylus coqui</Emphasis>), in its native and introduced ranges

The Puerto Rican frog, Eleutherodactylus coqui has invaded Hawaii and reached densities far exceeding those in their native range. One possible explanation for the success of E. coqui in its introduced range is that it lost its co-evolved parasites in the process of the invasion. We compared the parasites of E. coqui in its native versus introduced range. We collected parasite data on 160 individual coqui frogs collected during January-April 2006 from eight populations in Puerto Rico and Hawaii. Puerto Rican coqui frogs had higher species richness of parasites than Hawaiian coqui frogs. Parasite prevalence and intensity were significantly higher in Hawaii, however this was likely a product of the life history of the dominant parasite and its minimal harm to the host. This suggests that the scarcity of parasites may be a factor contributing to the success of Eleutherodactylus coqui in Hawaii.     

Two UV-sensitive targets in dorsoanterior specification of frog embryos

Previous work has shown that ultraviolet (UV) irradiation of fertilized frog eggs yields embryos that lack dorsal and anterior structures. The eggs fail to undergo the cortical/cytoplasmic rotation that specifies dorsoventral polarity, and they lack an array of parallel microtubules associated with the rotation. These eggs can be rescued by tilting with respect to gravity, and normal dorsoanterior development occurs. We find here that UV irradiation of Xenopus prophase I oocytes or Rana metaphase I oocytes also causes the dorsoanterior deficient syndrome, but the UV target is different from that in fertilized eggs. Tilting eggs, irradiated as oocytes, with respect to gravity, does not rescue dorsoanterior development, although lithium treatment does. The UV dose required to produce dorsoanterior deficiency for Rana metaphase I oocytes is much less than that for fertilized eggs, and the oocytes can form the array of parallel microtubules and undergo the cortical/cytoplasmic rotation after fertilization. Despite these features of normal development, no dorsoanterior structures form. While the UV target in fertilized eggs is thought to be the parallel microtubules (Elinson & Rowning, 1988; Devl Biol. 128, 185-197), the UV target in the oocytes may be a dorsal determinant.     

The gastrocoel roof plate in embryos of different frogs

The morphology of the gastrocoel roof plate and the presence of cilia in this structure were examined in embryos of four species of frogs. Embryos of Ceratophrys stolzmanni (Ceratophryidae) and Engystomops randi (Leiuperidae) develop rapidly, provide comparison for the analysis of gastrocoel roof plate development in the slow-developing embryos of Epipedobates machalilla (Dendrobatidae) and Gastrotheca riobambae (Hemiphractidae). Embryos of the analyzed frogs develop from eggs of different sizes, and display different reproductive and developmental strategies. In particular, dorsal convergence and extension and archenteron elongation begin during gastrulation in embryos of rapidly developing frogs, as in Xenopus laevis. In contrast, cells that involute during gastrulation are stored in the large circumblastoporal collar that develops around the closed blastopore in embryos of slow-developing frogs. Dorsal convergence and extension only start after blastopore closure in slow-developing frog embryos. However, in the neurulae, a gastrocoel roof plate develops, despite the accumulation of superficial mesodermal cells in the circumblastoporal collar. Embryos of all four species develop a ciliated gastrocoel roof plate at the beginning of neurulation. Accordingly, fluid-flow across the gastrocoel roof plate is likely the mechanism of left-right asymmetry patterning in these frogs, as in X. laevis and other vertebrates. A ciliated gastrocoel roof plate, with a likely origin as superficial mesoderm, is conserved in frogs belonging to four different families and with different modes of gastrulation.     

Differential Protein Distribution Related to Dorsoventral Polarity in Pleurodeles waltl Cleaving Egg

The molecular basis for the initial specification of dorsoventral polarity in the Amphibian egg prior to the mid-blastula transition still remains an open question. Regional differences in the protein pattern of Pleurodeles egg were investigated during early cleavage (8- and 512-cell stages, prior to the mid-blastula transition). Animal-dorsal, animal-ventral, vegetal-dorsal and vegetal-ventral quarters were separated and proteins were analyzed by 2D-electrophoresis. The comparison of acidic protein patterns from dorsal and ventral quarters revealed differences between vegetal cells but no difference was detected between animal cells. One protein (p11, 30 kDa) was characterized in the dorsal side as early as the 8-cell st. and two dorsal spots were detected at the 512-cell st. (p11 and p5, 65 kDa). Similarly one protein (p7b, 46 kDa) appears to be ventral-specific from the 8-cell st. The p11 spot was shown to appear in ventral cells as a consequence of a dorsalizing LiCl-treatment at the 32-cell stage. Conversely, p11 disappeared from dorsal cells and p5 did not appear at 512-cell stage after UV-irradiation of the uncleaved egg, which results in the expression of the ventral-specific protein p7b in the dorsal part of the egg. Therefore differential protein expression is already present at very early cleavage stages. Its significance needs further investigation.     

Protamines in the internally fertilizing neobatrachian frog Eleutherodactylus coqui

The internally fertilizing primitive frog Ascaphus truei (family Ascaphidae) from the Pacific Northwest is the only frog with an intromittent organ. The more advanced neobatrachian frog Eleutherodactylus coqui (family Leptodactylidae) from Puerto Rico has secondarily acquired internal fertilization but mates by cloacal apposition. Nonetheless, both frogs have introsperm with an elongated head containing highly condensed chromatin. Characterization of sperm nuclear basic proteins (SNBPs) in E. coqui by acid-urea polyacrylamide gel electrophoresis indicates that, as in A. truei, testes from a single animal contain several protamines. Amino acid analysis indicates a composition for the most rapidly moving protamine of each species as follows: in E. coqui, ARG (35.6 mol %) + LYS (3.8 mol %) + HIS (7.6 mol %) = 47 mol % total basic residues and in A. truei, ARG (42.1 mol %) + LYS (11.1 mol %) = 53.2 mol % total basic residues. Transmission electron microscopy shows that E. coqui introsperm, like those in A. truei, are elongate with highly condensed chromatin. However, E. coqui introsperm lacks an axial perforatorium that extends into an endonuclear canal. These morphological features are plesiomorphic (primitive) and shared by A. truei with urodeles and basal amniotes (Jamieson et al. (1993) Herpetologica 49:52-65). In E. coqui introsperm, the nucleoprotein complex has a cross-sectional axis of 420 + 20 angstroms and shows a knobby chromatin structural organization in TEM. The presence of arginine-enriched protamines in both a basal anuran like the ascaphid A. truei and a more advanced neobatrachian like the leptodactylid E. coqui supports the hypothesis that internal fertilization acts as a constraint on the range of SNBP diversity in animals.     

A comparative study of the membrane potential from before fertilization through early cleavage in two frogs, Rana pipiens and Xenopus laevis

The membrane potential of Rana pipiens eggs (-55.0 mV +/- 11.2(16)) was more likely to recover from impalement and was always more negative than that of eggs of Xenopus laevis (-19.3 mV +/- 4.2(68)). It was also much more negative than previously reported. Essentially similar membrane resistance changes were measured in the two frog species through fertilization and cleavage. Small transient depolarizations only associated with the onset of the fertilization potential in Xenopus could be prevented by hyperpolarizing the egg membrane prior to fertilization. Repolarization was variable and longer in Rana and often accompanied by large transient spontaneous depolarizations. Insemination time, the time between fertilization and cleavage and the first cleavage division cycle, were all about twice as long in Rana. Xenopus egg cleavage was invariably accompanied by pronounced transient hyperpolarizations that were essentially absent in Rana.     

Gavity and Microtubules in Dorsoventral Polarization of The Xenopus Egg

In Xenopus laevis , the dorsoventral axis of the embryo is specified by a 30° relative rotation between the cortex and the cytoplasm of the fertilized egg, and a cortical array of parallel microtubules may be part of the rotation machinery (7). The parallel microtubules are aligned with the sperm entry point in most of the eggs as expected, since the dorsoventral axis is usually defined by the sperm entry point. We show that gravity can play two roles in the formation of the dorsoventral axis. First, a simple 90° tilt off-axis before the start of the rotation overcomes the influence of the sperm and determines the orientation of the parallel microtubules. Second, a 90° tilt off-axis can specify the dorsoventral axis even in the absence of the parallel microtubules. Therefore, gravity can affect dorsoventral polarity by orienting the parallel microtubules or by moving cytoplasm directly without microtubules.