Crucial stages of embryogenesis of <Emphasis Type="Italic">R. arvalis</Emphasis>: Part 1. Linear measurements of embryonic structures

Investigations of individual variability have allowed us to reveal the crucial (=nodal) stages in embryogenesis of the moor frog (Rana arvalis Nills.). These crucial stages are: the late gastrula stage (stages 18–20), the hatching stages (stages 32–33) and, apparently, early metamorphosis (stage 39). Moreover, we have found that each embryonic structure passes through its specific crucial stages. For example, stage 34 is crucial for the trait “tail width” but is internodal for all other embryonic traits. At this stage, larva passes from an attached to a free-swimming life style. We also found considerable differences between the different frog populations in the the level of developmental variability. These differences were associated with internodal developmental stages.     

Carry-over effects of ultraviolet-B radiation on larval fitness in Rana temporaria

A number of studies have failed to find evidence for negative effects of ultraviolet-B radiation (UVBR) on amphibian early-embryonic performance, leading to the conclusions, first, that the embryonic stages of many species are tolerant to UVBR, and second, that the increased amount of UVBR reaching the Earth's surface is not likely to have any direct negative effects on many amphibian populations. However, possible carry-over effects of exposure to UVBR in the embryonic stages to the larval stages have received less attention. We studied the effects of UVBR experienced during the embryonic stages (age less than 11 days) on the later performance (age 11-75 days) of common frog, Rana temporaria, larvae. In a factorial laboratory experiment, newly fertilized embryos were divided into three different UVBR treatments (no UVBR (control), 1.25 kJm(-2) (normal) and 1.58 kJm(-2) (26% enhanced)), after which the individual larvae were raised until metamorphosis in the absence of UVBR. No effects of UVBR on embryonic survival rates, frequency of developmental anomalies or hatchling size were found, corroborating the earlier results indicating that R. temporaria embryos are tolerant to UVBR. However, analyses of larval performance revealed that larvae exposed to enhanced levels of UVBR as embryos suffered from an increased frequency of developmental anomalies and metamorphosed later and at a smaller size than larvae that had been protected from UVBR as embryos. These results suggest, in contrast to the earlier studies, that UVBR has direct negative effects on R. temporaria embryos, but these effects are expressed mostly or only during the later life stages. To this end, our results support the contention that carry-over effects from one life stage to another may be an important source of phenotypic variation in fitness.     

Maturation of end-systolic stress-strain relations in chick embryonic myocardium

The embryonic myocardium increases functional performance geometrically during cardiac morphogenesis. We investigated developmental changes in the in vivo end-systolic stress-strain relations of embryonic chick myocardium in stage 17, 21, and 24 white Leghorn chick embryos (n = 10 for each stage). End-systolic stress-strain relations were linear in all developmental stages. End-systolic strain decreased from 0.50 +/- 0.02 to 0.31 +/- 0.01 (mean +/- SE, P < 0.05), while average end-systolic wall stress was similar at 3.29 +/- 0.34 to 4.19 +/- 0.43 mmHg (P = 0.14) from stage 17 to 24. Normalized end-systolic myocardial stiffness, a load-independent index of ventricular contractility, increased from 2.98 +/- 0.19 to 6.03 +/- 0.39 mmHg from stage 17 to 24 (P < 0.05). Zero-stress midwall volume increased from 0.024 +/- 0.002 to 0.124 +/- 0.004 microl from stage 17 to 24 (P < 0.05). These results suggest that the embryonic ventricle increases normalized ventricular "contractility" while maintaining average end-systolic wall stress over a relatively narrow range during cardiovascular morphogenesis.     

Clutch effects explain heart rate variation in embryonic frogs (Cave Coqui, Eleutherodactylus cooki)

Few physiological studies to date have focused on whether variation among sibling groups during development can account for often large, intraspecific physiological variation. In this study, we measured heart rate in the direct-developing frog Eleutherodactylus cooki throughout its embryonic development and examined heart rate variation among egg clutches comprising from 10 to 40 eggs. Clutches were collected in the wild in Yubucoa, Puerto Rico, and individual eggs were maintained under equivalent conditions in the lab. Heart rate showed large increases during development, rising from about 40 beats min(-1) in the earliest stages to about 110 beats min(-1) at hatching. The effect of stage (averaged across clutches) was highly significant (P<0.001). However, repeated-measures MANOVA also revealed that there were highly significant effects on heart rate associated with both clutch (variation among clutches averaged across development; P<0.001) and clutch-stage interactions (differences among clutches in the developmental change in heart rate; P<0.0001). These effects and interactions reveal that throughout development, heart rate in siblings is much more similar than in nonsiblings and that sib groups follow different heart rate trajectories during their development. Collectively, these data indicate that "clutch effects" caused by genetic and/or maternal influences can strongly affect patterns of heart function during development within cave coqui populations. This phenomenon also occurs in bird eggs and armadillo neonates, suggesting that physiological variation attributable to clutch effects might be a widespread phenomenon in vertebrates.     

Regulation of Raf-1-dependent signaling during early Xenopus development.

The Raf-1 gene product is activated in response to cellular stimulation by a variety of growth factors and hormones. Raf-1 activity has been implicated in both cellular differentiation and proliferation. We have examined the regulation of the Raf-1/MEK/MAP kinase (MAPK) pathway during embryonic development in the frog Xenopus laevis. We report that Raf-1, MEK, and MAPK activities are turned off following fertilization and remain undetectable up until blastula stages (stage 8), some 4 h later. Tight regulation of the Raf-1/MEK/MAPK pathway following fertilization is crucial for embryonic cell cycle progression. Inappropriate reactivation of MAPK activity by microinjection of oncogenic Raf-1 RNA results in metaphase cell cycle arrest and, consequently, embryonic lethality. Our findings demonstrate an absolute requirement, in vivo, for inactivation of the MAPK signaling pathway to allow normal cell cycle progression during the period of synchronous cell divisions which occur following fertilization. Further, we show that cytostatic factor effects are mediated through MEK and MAPK.     

Effects of transgenic overexpression of diapause hormone and diapause hormone receptor genes on non-diapause silkworm

Diapause is a state of developmental arrest that is most often observed in arthropods, especially insects. The domesticated silkworm, Bombyx mori, is a typical insect that enters diapause at an early embryonic stage. Previous studies have revealed that the diapause hormone (DH) signaling molecules, especially the core members DH and DH receptor 1 (DHR1), are crucial for the determination of embryonic diapause in diapause silkworm strains. However, whether they function in non-diapause silkworm strains remains largely unknown. Here, we generated two transgenic lines overexpressing DH or DHR1 genes in a non-diapause silkworm strain, Nistari. Our results showed that developmental expression patterns of DH and DHR1 are quite similar in transgenic silkworms: both genes are highly expressed in the mid to late stages of pupae and are most highly expressed in day-6 pupae but are expressed at very low levels in other developmental stages. Moreover, the overexpression of DH or DHR1 can affect the expression of diapause-related genes but is not sufficient to induce embryonic diapause in their offspring. This study provides new insights into the function of DH and DHR1 in a non-diapause silkworm strain.     

Orphan nuclear receptor LRH-1 is required to maintain Oct4 expression at the epiblast stage of embryonic development

Oct4 plays an essential role in maintaining the inner cell mass and pluripotence of embryonic stem (ES) cells. The expression of Oct4 is regulated by the proximal enhancer and promoter in the epiblast and by the distal enhancer and promoter at all other stages in the pluripotent cell lineage. Here we report that the orphan nuclear receptor LRH-1, which is expressed in undifferentiated ES cells, can bind to SF-1 response elements in the proximal promoter and proximal enhancer of the Oct4 gene and activate Oct4 reporter gene expression. LRH-1 is colocalized with Oct4 in the inner cell mass and the epiblast of embryos at early developmental stages. Disruption of the LRH-1 gene results in loss of Oct4 expression at the epiblast stage and early embryonic death. Using LRH-1(-/-) ES cells, we also show that LRH-1 is required to maintain Oct4 expression at early differentiation time points. In vitro and in vivo results show that LRH-1 plays an essential role in the maintenance of Oct4 expression in ES cells at the epiblast stage of embryonic development, thereby maintaining pluripotence at this crucial developmental stage prior to segregation of the primordial germ cell lineage at gastrulation.     

In search of the vertebrate phylotypic stage: a molecular examination of the developmental hourglass model and von Baer's third law

In 1828, Karl von Baer proposed a set of four evolutionary "laws" pertaining to embryological development. According to von Baer's third law, young embryos from different species are relatively undifferentiated and resemble one another but as development proceeds, distinguishing features of the species begin to appear and embryos of different species progressively diverge from one another. An expansion of this law, called "the hourglass model," has been proposed independently by Denis Duboule and Rudolf Raff in the 1990s. According to the hourglass model, ontogeny is characterized by a starting point at which different taxa differ markedly from one another, followed by a stage of reduced intertaxonomic variability (the phylotypic stage), and ending in a von-Baer-like progressive divergence among the taxa. A possible "translation" of the hourglass model into molecular terminology would suggest that orthologs expressed in stages described by the tapered part of the hourglass should resemble one another more than orthologs expressed in the expansive parts that precede or succeed the phylotypic stage. We tested this hypothesis using 1,585 mouse genes expressed during 26 embryonic stages, and their human orthologs. Evolutionary divergence was estimated at different embryonic stages by calculating pairwise distances between corresponding orthologous proteins from mouse and human. Two independent datasets were used. One dataset contained genes that are expressed solely in a single developmental stage; the second was made of genes expressed at different developmental stages. In the second dataset the genes were classified according to their earliest stage of expression. We fitted second order polynomials to the two datasets. The two polynomials displayed minima as expected from the hourglass model. The molecular results suggest, albeit weakly, that a phylotypic stage (or period) indeed exists. Its temporal location, sometimes between the first-somites stage and the formation of the posterior neuropore, was in approximate agreement with the morphologically defined phylotypic stage. The molecular evidence for the later parts of the hourglass model, i.e., for von Baer's third law, was stronger than that for the earlier parts.     

Alterations induced by E-cadherin and beta-catenin antibodies during the development of Bufo arenarum (Anura-Bufonidae)

E(epithelial)-cadherin is a member of a calcium-dependent family of cell surface glycoproteins involved in cell-cell adhesion and morphogenesis. Catenins are a large family of proteins that connect the cadherins to the cytoskeleton. They are important for cadherin function and for transducing signals involved in specification of cell fate during embryogenesis. The best characterized catenins include alpha-, beta-, gamma-, and p120-catenin. Using specific antibodies, we studied the expression and distribution of E-cadherin, and alpha- and beta-catenin in developmental stages of Bufo arenarum toad. The three proteins were found co-localized in stages 19 to 41 of development. Surprisingly, E-cadherin was the only of these three proteins found earlier than stage 19. To test whether E-cadherin and beta-catenin have a functional role in Bufo arenarum embryogenesis, stage 17 whole embryos were incubated with anti-E-cadherin and beta-catenin antibodies. Both anti-E-cadherin and anti-beta-catenin antibodies induced severe morphological alterations. However, while alterations produced by the anti-beta-catenin antibody, showed some variability from the most severe (neural tube and notochord duplication) to a simple delay in development, the alterations with anti-E-cadherin were homogeneous. These observations suggest a critical role for E-cadherin and beta-catenin in the early embryonic development of the Bufo arenarum toad. Our results are consistent with the developmental role of these proteins in other species. One of the most surprising findings was the blockage with the anti-beta-catenin antibodies on later embryo stages, and we hypothesize that the partial axes duplication could be mediated by the notochord induction.     

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。     

Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation

Laminins are components of all basement membranes and have well demonstrated roles in diverse developmental processes, from the peri-implantation period onwards. Laminin 1 (alpha1beta1gamma1) is a major laminin found at early stages of embryogenesis in both embryonic and extraembryonic basement membranes. The laminin gamma1 chain has been shown by targeted mutation to be required for endodermal differentiation and formation of basement membranes; Lamc1(-/-) embryos die within a day of implantation. We report the generation of mice lacking laminin alpha1 and laminin beta1, the remaining two laminin 1 chains. Mutagenic insertions in both Lama1 and Lamb1 were obtained in a secretory gene trap screen. Lamb1(-/-) embryos are similar to Lamc1(-/-) embryos in that they lack basement membranes and do not survive beyond embryonic day (E) 5.5. However, in Lama1(-/-) embryos, the embryonic basement membrane forms, the embryonic ectoderm cavitates and the parietal endoderm differentiates, apparently because laminin 10 (alpha5beta1gamma1) partially compensates for the absent laminin 1. However, such compensation did not occur for Reichert's membrane, which was absent, and the embryos died by E7. Overexpression of laminin alpha5 from a transgene improved the phenotype of Lama1(-/-) embryos to the point that they initiated gastrulation, but this overexpression did not rescue Reichert's membrane, and trophoblast cells did not form blood sinuses. These data suggest that both the molecular composition and the integrity of basement membranes are crucial for early developmental events.     

A potential role for differential contractility in early brain development and evolution

Differences in brain structure between species have long fascinated evolutionary biologists. Understanding how these differences arise requires knowing how they are generated in the embryo. Growing evidence in the field of evolutionary developmental biology (evo-devo) suggests that morphological differences between species result largely from changes in the spatiotemporal regulation of gene expression during development. Corresponding changes in functional cellular behaviors (morphogenetic mechanisms) are only beginning to be explored, however. Here we show that spatiotemporal patterns of tissue contractility are sufficient to explain differences in morphology of the early embryonic brain between disparate species. We found that enhancing cytoskeletal contraction in the embryonic chick brain with calyculin A alters the distribution of contractile proteins on the apical side of the neuroepithelium and changes relatively round cross-sections of the tubular brain into shapes resembling triangles, diamonds, and narrow slits. These perturbed shapes, as well as overall brain morphology, are remarkably similar to those of corresponding sections normally found in species such as zebrafish and Xenopus laevis (frog). Tissue staining revealed relatively strong concentration of F-actin at vertices of hyper-contracted cross-sections, and a finite element model shows that local contraction in these regions can convert circular sections into the observed shapes. Another model suggests that these variations in contractility depend on the initial geometry of the brain tube, as localized contraction may be needed to open the initially closed lumen in normal zebrafish and Xenopus brains, whereas this contractile machinery is not necessary in chick brains, which are already open when first created. We conclude that interspecies differences in cytoskeletal contraction may play a larger role in generating differences in morphology, and at much earlier developmental stages, in the brain than previously appreciated. This study is a step toward uncovering the underlying morphomechanical mechanisms that regulate how neural phenotypic differences arise between species.     

The evolution of genome size: what can be learned from anuran development?

Differences in nuclear DNA content in vertebrates have been shown to be correlated with cell size, cell division rate, and embryonic developmental rate. We compare seven species of anuran amphibians with a three-fold range of genome sizes. Parameters examined include the number and density of cells in a number of embryonic structures, and the change in cell number in the CNS during development. We show that genome size is correlated with cell proliferation rate and with developmental rate at different stages of embryonic development, but that the correlation between genome size and cell size is only evident at later stages. We discuss the evolution of genome size in amphibians. Our discussion takes into account data that reportedly support two conflicting hypotheses: the "skeletal DNA" hypothesis, which claims a selective role for differences in genome size, and the "junk DNA" hypothesis, which claims that differences in genome size are a random result of the accumulation of noncoding DNA sequences. We show that these supposedly conflicting hypotheses can be integrated into a more complex and inclusive model for the evolution of genome size.     

Characterization and development of rotational behavior in Helisoma embryos: role of endogenous serotonin.

Cilia-driven rotational behavior displayed by embryos of the pond snail Helisoma trivolvis was characterized in terms of its behavioral subcomponents, developmental changes, and response to exogenous serotonin. Rotation was found to be a complex behavior characterized by four parameters; rotational direction, rotation rate, rotational surges, and periods of inactivity. These parameters all exhibited characteristic developmental changes from embryonic stage E15 through stage E30. Notably, both rotation rate and frequency of rotational surges increased from stage E15 to E25 and declined to an intermediate level by stage E30. It appeared that the developmental increase in overall rotation rate was caused primarily by an increase in surge frequency, rather than an increase in the rate of nonsurge rotation. Immersion of embryos inserotonin-containing pond water resulted in a dose-dependent, reversible increase in rotation rate as well as a dose-dependent, reversible decrease in surge frequency. The serotonin antagonist, mianserin, abolished the excitatory effect of exogenous serotonin. Furthermore, application of mianserin alone reduced rotation rate and virtually abolished rotational surges. Taken together, these pharmacological results suggest that endogenous serotonin is responsible for generating rotational surges. Given that early embryos contain only a single pair of serotonergic neurons (Goldberg and Kater, 1989) during the stages when rotational surges are expressed, these results also prompt the hypothesis that these neurons, embryonic neurons C1, act as cilioexcitatory motor neurons during embryonic development.