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.

Variation in anuran embryogenesis: differences in sequence and timing of early developmental events.

Comparative embryology of closely related species can shed light on the evolution of developmental processes. An important mechanism in the evolution of developmental processes, which can lead to significant changes in larval or adult form, is variation in the sequence and timing of developmental events. We compared the development of 12 species of anurans, including a wide taxonomic range as well as a number of congeneric species. The comparison consisted of monitoring a series of external morphological markers and histological markers. For each species we noted the timing of each of the markers, using a uniform parameter of normalized time. We compared the normalized time of each of these events among the species, as well as the sequence of the events. Our analysis revealed many differences in sequence and in timing of developmental events. We mapped these differences on a cladogram of the studied species, using sequence units as discrete characters. The differences do not seem to be connected to the phylogenetic relations between the species or to any obvious ecological factors. We suggest a hypothetical ancestral sequence of developmental events, and discuss the possible factors that could have caused the observed variations from the ancestral sequence.

Variations in anuran embryogenesis: yolk-rich embryos of Hyperolius puncticulatus (Hyperoliidae).

Anuran development is usually described using model species, most notably Xenopus laevis and Rana pipiens. We describe the development of the East African Reed Frog, Hyperolius puncticulatus, a species displaying development that is highly divergent from the "classic" anuran developmental pattern. Although having small eggs, the eggs of H. puncticulatus are characterized by a large amount of yolk, and embryonic development is reminiscent of species with large eggs. The eggs are teleolecithal and the cleavage is holoblastic, with a "pseudo-meroblastic" pattern. Gastrulation proceeds primarily at the dorsal lip and is characterized by reduced embryonic cavities. Gastrulation ends with a thickened "embryonic mantle" that sits upon a large yolk mass and forms most of the tissues of the embryo. The embryonic axis curves across the yolk mass, instead of the typical lengthening of anuran embryos. The tadpole hatches with a large ventral yolk mass which is gradually absorbed. We hypothesize about the developmental mechanisms that underlie this unusual development, based on comparisons with other anuran and fish species. We suggest that this type of development is not unique to this species, but can be found in many species of different anuran taxonomic groups. Comparing the development of H. puncticulatus and similar species to what is known about the development of model species, such as X. laevis, shows us the variation in early anuran embryogenesis. Knowing the existing diversity is a prerequisite to understanding the evolution of early anuran development and the changes in patterning mechanisms in different lineages.

Limited left-right cell migration across the midline of the gastrulating avian embryo.

During avian development the earliest phase in which the avian embryo expresses axial features of a left-right axis is at the primitive streak stage. Until the stage of definitive primitive streak (streak 4 H&H), the axis seems to possess morphological bilateral symmetry. Morphological asymmetry begins only during the next few hours of incubation, with development of overt morphological and molecular asymmetry within Hensen's node (stage 5 H&H). In this report, we present an experimental study aimed at following the pattern of cell movements during primitive streak formation and gastrulation of specific left-right regions from earlier stages of the avian embryo. To determine the origin of cells contributing to each side of the primitive streak, we applied the dye Lysinated-Rodamine-Dextran (LRD) to one half, either left or right, of the pre-streak blastoderm (stages X-XIII, EG&K). We tried to estimate the relative cell contribution to primitive streak formation, and to the three germ layers evolving during gastrulation in the context of the left-right axis. Moreover, we asked whether the midline serves as a border, that is, as a physiological barrier preventing cell passing during gastrulation. Our results demonstrate that on each side of the axis, either the right or the left, most of the cells originate from the same half of a pre-streak blastoderm, populate the same half of the PS and contribute to tissues largely confined to that particular side. However, along the primitive streak, a few cells were detected on the opposite side of the midline. Moreover, variation in the number of cells crossing the midline at specific regions along the primitive streak was found. Most crossing cells were located near the mid rostrocaudal extent of the primitive streak, from 25-85% of its length. At the posterior end of the primitive streak, fewer crossing cells were detected. At the anterior region of the PS, that is, within Hensen's node, cells do not cross the midline. These results suggest that differences occur in the process of ingression along the rostrocaudal extent of the PS.

The ability to initiate an axis in the avian blastula is concentrated mainly at a posterior site.

Cell interactions during early vertebrate development are crucial for embryonic mesoderm induction and axis initiation. In the avian embryo two unique layers of cells, the epiblast and the hypoblast, constitute the blastoderm before the primitive streak develops (stage XIII). It was suggested that cells of the hypoblast have the ability to induce competent cells in the epiblast to form the mesoderm and to initiate the embryonic axis. Recent results suggest, however, that at stage XIII the hypoblast does not act by inducing the epiblast to form a primitive streak. Since the hypoblast at stage XIII does not induce the epiblast, experiments were designed in this work to examine whether other subregions of the avian blastula have the ability to initiate the embryonic axis. To distinguish the contribution of a particular fragment to the formation of the embryonic axis, cell-marking examinations with lysinated rhodamine dextran (LRD) were designed. The results of the experimental series discussed in this report suggest that at stage XIII it is mainly the posterior side of the marginal zone and/or of the posterior region of the epiblast layer which has the abilities to initiate the embryonic axis. However, the posteriolateral part of the marginal zone region also has such abilities, which are inhibited during normal development. LRD examinations have demonstrated that a graft of a particular posterior blastoderm region, or posteriolateral marginal zone, can initiate an ectopic streak, and is able to recruit other neighboring cells to the developing ectopic streak. No evidence was found that Koller's sickle itself can initiate an ectopic axis in the epiblast at stage XIII. It is proposed that the cells which are important to initiate the avian embryonic axis are concentrated mainly at the region of the posterior marginal zone including Koller's sickle and in the posterior region of the epiblast layer. The cells in this region, which also express the goosecoid and cVg1 genes, may have organizer properties which induce the mesoderm and determine the initiation site of gastrulation in the chick embryo.

High proliferation rate characterizes the site of axis formation in the avian blastula-stage embryo.

Localized zones of high cell proliferation have been thought to be important in determining several phases of axis formation at early stages of chick development. It was suggested that a developmental center, a center of cellular activity such as proliferation and movement, is located in the posterior half of the area pellucida in the unincubated chick blastoderm. In the work reported here, we have used the bromodeoxyuridine (BrdU) incorporation procedure followed by immunoperoxidase detection to assess the rate of cell proliferation at particular sub-regions of pregastrulating chick blastoderms (stages X-XIII). Examination of whole-mount and histological sections of stages X through XII blastoderms, pulsed with BrdU, showed no distinguishable difference in labeled cells between particular regions of these blastoderms and also that there are no specific zones of high cellular proliferation in either the hypoblast or the epiblast layers of the area pellucida. However, our observations have shown a striking difference in the stage XIII blastoderm, in which a relatively high amount of labeled cells were detected all around the posterior region of the area opaca, the marginal zone, Koller's sickle and the epiblast. The relatively high proportion of cell divisions observed at the posterior end of a stage XIII blastoderm, the blastula stage of the avian embryo, may be associated with the major developmental ability of this region to initiate an embryonic axis. Directional axis formation, therefore, may be attributed to a region of proliferation in the posterior side of a stage XIII blastoderm.

Axis formation in half blastoderms of the chick: Stage at separation and the relative positions of fused halves influence axis development.

The blastoderm of the avian embryo acts during the early stages of development as an integrative system programmed to form a single embryonic axis. Isolated parts of the blastoderm are known to each form an axis, owing to the system's properties. In the work reported here, the regulative capability of the right and left halves of chick blastoderms to form an embryonic axis was examined systematically at different stages. This revealed a progressive change in the developing blastoderm. After early separation, the axis in each half will form at some distance from the blastoderm's original midline, while with late separation the axis will form next to the original midline and may even lack one row of somites at the medial rim. Since development stops in culture after about 2 days, axis development after early separation ceases before somites are formed, whereas after late separation somites and brain vesicles can develop. In addition, an attempt was made to learn whether the two halves of blastoderm, when shifted along the midline and then reunited in staggered fashion, act as a single or two separate embryonic fields. When reunion of the right and left halves was achieved so that the posterior end of one half was adjoining the posterior area pellucida region of the other half, a single embryonic axis developed. When, on the other hand, the shift was larger so that the posterior end was fused to the central area pellucida of the other half, two separated embryonic axes developed.

The rotated hypoblast of the chicken embryo does not initiate an ectopic axis in the epiblast.

In the amniotes, two unique layers of cells, the epiblast and the hypoblast, constitute the embryo at the blastula stage. All the tissues of the adult will derive from the epiblast, whereas hypoblast cells will form extraembryonic yolk sac endoderm. During gastrulation, the endoderm and the mesoderm of the embryo arise from the primitive streak, which is an epiblast structure through which cells enter the interior. Previous investigations by others have led to the conclusion that the avian hypoblast, when rotated with regard to the epiblast, has inductive properties that can change the fate of competent cells in the epiblast to form an ectopic embryonic axis. Thus, it has been suggested that the hypoblast normally induces the epiblast to form a primitive streak at a specific locus. In the work reported here, an attempt was made to reexamine the issue of induction. In contrast to previous reports, it was found that the rotated hypoblast of the chicken embryo does not initiate formation of an ectopic axis in the epiblast. The embryonic axis always initiates and develops according to the basic polarity of the epiblast layer. These results provoke a reinterpretation of the issues of mesoderm induction and primitive streak initiation in the avian embryo.

Genomic organization of a gene encoding the spicule matrix protein SM30 in the sea urchin Strongylocentrotus purpuratus.

We report the characterization of a genomic clone containing portions of two tandemly arranged genes that encode a spicule matrix protein, SM30, of the sea urchin Strongylocentrotus purpuratus. The isolated 18.4-kilo-base genomic clone contains the complete genomic sequence of one SM30 gene, designated SM30-alpha, and a portion of another SM30 gene, designated SM30-beta. Southern blot analysis shows that SM30 protein is encoded by a small gene family of two to four members. RNase protection assays indicate that the SM30-alpha gene is expressed at the time of spicule formation in the sea urchin embryo. In addition, mapping of SM30-alpha shows that a large single intron interrupts the coding sequence. Comparison of the nucleic acid and amino acid sequences of the SM30-alpha genomic sequence and the previously isolated SM30 cDNA reveals them to be very similar, but not identical. We also demonstrate that 2.6 kilobases of upstream sequence of SM30-alpha are sufficient to direct primary mesenchyme cell-specific expression of a reporter gene construct.

The potency of the first two cleavage cells in echinoderm development: the experiments of Driesch revisited.

A hundred years have passed since Driesch performed the classical experiment of separating sea urchin blastomeres from a two-cell-stage embryo, finding that each developed into a complete though smaller larva. The earlier studies of Roux using frogs showed that inactivating one of the two blastomeres by a heated needle resulted, during the early stages of development, in the formation of a half embryo. In this type of experiment, in which the two blastomeres are not separated, the live blastomere continues its development while it is still attached to an inactivated neighbour. In the work reported here, Roux's experimental design was used on two-cell-stage embryos of sea urchins. In contrast to the findings of Roux using amphibians, it was found (as claimed by Driesch) that the living blastomere developed as in the case of separated blastomeres.

Interactions of different vegetal cells with mesomeres during early stages of sea urchin development.

It has been known from results obtained in the classical experiments on sea urchin embryos that cell isolation and transplantation showed extensive interactions between the early blastomeres and/or their descendants. In the experiments reported here a systematic reexamination of recombination of mesomeres and their progeny (which come from the animal hemisphere) with various vegetal cells derived from blastomeres of the 32- and 64-cell stage was carried out. Cells were marked with lineage tracers to follow which cell gave rise to what structures, and newly available molecular markers have been used to analyze different structures characteristic of regional differentiation. Large micromeres form spicules and induce gut and pigment cells in mesomeres, conforming to previous results. Small micromeres, a cell type not heretofore examined, gave rise to no recognizable structure and had very limited ability to evoke poorly differentiated gut tissue in mesomeres. Macromeres and their descendants, Veg 1 and Veg 2, form primarily what their normal fate dictated, though both did have some capacity to form spicules, presumably by formation from secondary mesenchyme. Macromeres and their descendants were not potent inducers of vegetal structures in animal cells, but they suppress the latent ability of mesomeres to form vegetal structures. The results lead us to propose that the significant interactions during normal development may be principally suppressive effects of mesomeres on one another and of adjacent vegetal cells on mesomeres.

The influence of cell interactions and tissue mass on differentiation of sea urchin mesomeres.

The developmental potential of different blastomeres of the sea urchin embryo was re-examined. We have employed a new method to isolate substantial numbers of different kinds of blastomeres from 16-cell-stage embryos, and we have used newly available molecular markers to analyze possible vegetal differentiation. We have found that, while isolated mesomere pairs behave according to the classical expectations and develop into ectodermal vesicles, there is a clear effect of reaggregating two or more mesomere pairs. They survive better in long-term culture and, after prolonged periods, they display an astonishing ability to express vegetal differentiation. We also combined mesomeres with stained micromeres or macromeres from the vegetal hemisphere. Although induction of guts and spicules was observed, there was little if any effect of varying the ratio of different blastomeres on the kinds of differentiation obtained.

The chick's marginal zone and primitive streak formation. I. Coordinative effect of induction and inhibition.

A variety of transplantation experiments of posterior and lateral marginal zone fragments at stages X, XI, and XII have been carried out in order to test their relevance to the development of a primitive streak (PS). At the stages studied the marginal zone (MZ) was shown to behave as a ring-like gradient field, the maximal value of which was at the posterior end (PM). The PM was found to be capable at the same time of promoting the development of a PS and of suppressing the inductive potential of other regions of the MZ. By systematically evaluating inductive and suppressive capacities of PMs, at different developmental stages, it was found that both features are maximal at stage X. During stages XI and XII, both properties gradually decrease in the MZ and build up in the forming hypoblast.

The chick's marginal zone and primitive streak formation. II. Quantification of the marginal zone's potencies--temporal and spatial aspects.

When a posterior fragment of the chick's marginal zone (PM) was exchanged with equal sized lateral marginal zone fragment (LM), of the same blastoderm, its capacity to initiate an ectopic primitive streak (PS) was found to be both size and stage dependent. Good correlation was demonstrated between the areas of PM fragments and the number of cells they contained. In stage X blastoderms, PM fragments containing less than 1200 cells were incapable of initiating an ectopic PS. Transplanted PMs containing between 1200 and 1500 cells initiated a lateral ectopic PS in 50% of the cases, while in the other 50% a posterior PS developed from the original posterior side. PMs containing 1500 cells or more in all cases initiating an ectopic PS and inhibited the formation of a posterior PS. At stage XI, laterally transplanted PMs containing less than 1800 cells were not effective. Stage XI PMs containing 1800-2300 cells in some cases succeeded in initiating a lateral ectopic PS, in addition to the posterior one. Stage XI PMs containing 2300 cells or more invariably promoted the development of an ectopic PS, but were unable to suppress the formation of a posterior PS, so that two PSs developed in the same blastoderm, one posterior and one ectopic. When a stage XI PM fragment was laterally transplanted into a younger, stage X blastoderm, the minimal effective cell number needed to initiate an ectopic PS increased to at least 3000 cells, again without inhibiting the formation of a posterior PS. The inductive potential of a stage X PM is therefore at least twice that of a stage XI PM. The marginal zone belt of stage X blastoderms was checked for the decrease in its developmental potential from the posterior to the lateral side by evaluating its effect on the developmental expression of two competing stage X PMs, one located posteriorly and the other inserted laterally. The developmental expression of the laterally inserted PM was consistently inferior to that of the posterior PM. The developmental expression of each PM was not related to absolute size, but depended on the size ratio of lateral PM/posterior PM. When the ratio was 1.2 or less, only posterior PSs developed. When the ratio was 1.3-1.4, three different results were encountered: (1) only a posterior PS, (2) posterior plus lateral, and (3) only lateral PS. When the ratio was 1.5 or more, only a lateral PS developed, which suppressed the posterior PS.

The embryo-forming potency of the posterior marginal zone in stages X through XII of the chick.

At stage X a small posterior marginal zone (PM) fragment, when transplanted into similar-size hole in the lateral marginal zone, can initiate the development of an ectopic axis. The laterally transplanted PM, inhibits the regeneration of an axis at the original posterior side from the lateral section of the marginal zone (LM) inserted to replace it. At stage XI both the axis-forming and inhibitory capacities of the PM fragment become weaker and an axis-forming capacity starts to build up anterior to the PM, resulting in the formation of two primitive streaks at 90 degrees to each other. At stage XII the change of potencies exhibited at stage XI is more pronounced, the ability of the transplanted PM to promote axis formation at the new site is lost, and an axis is formed from the original posterior side of the blastoderm.

Developmental potencies of area opaca and marginal zone areas of early chick blastoderms.

The marginal zone, in pregastrulating chick blastoderms, has been defined as the intermediate ring between the epiblast proper and the most external region, the area opaca (Spratt & Haas, 1960). Azar & Eyal-Giladi (1979) have shown that the marginal zone of a stage XIII blastoderm has the capacity of regenerating an inductive layer which when in contact with a competent stage XIII epiblast can cause the formation of axial structures. The present work demonstrates that at stage X (Eyal-Giladi & Kochav, 1976) the marginal zone can both induce and respond to its own inductive stimulus by forming an embryonic axis. By stage XIII the marginal zone seems to have lost its competence to respond to an inductive stimulus and cannot form an embryonic axis. It is further shown that at stages X--XIII the area opaca is neither competent to develop any sort of axial structures nor has the capacity to generate an inductive layer.