Molecular signaling in zebrafish development and the vertebrate phylotypic period

SUMMARY During development vertebrate embryos pass through a stage where their morphology is most conserved between species, the phylotypic period (approximately the pharyngula). To explain the resistance to evolutionary changes of this period, one hypothesis suggests that it is characterized by a high level of interactions. Based on this hypothesis, we examined protein–protein interactions, signal transduction cascades and miRNAs over the course of zebrafish development, and the conservation of expression of these genes in mouse development. We also investigated the characteristics of genes highly expressed before or during the presumed phylotypic period. We show that while there is a high diversity of interactions during the phylotypic period (protein–DNA, RNA–RNA, cell–cell, and between tissues), which is well conserved with mouse, there is no clear difference with later, more morphologically divergent, stages. We propose that the phylotypic period may rather be the expression at the morphological level of strong conservation of molecular processes earlier in development.     

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.     

Inverting the hourglass: quantitative evidence against the phylotypic stage in vertebrate development

The concept of a phylotypic stage, when all vertebrate embryos show low phenotypic diversity, is an important cornerstone underlying modern developmental biology. Many theories involving patterns of development, developmental modules, mechanisms of development including developmental integration, and the action of natural selection on embryological stages have been proposed with reference to the phylotypic stage. However, the phylotypic stage has never been precisely defined, or conclusively supported or disproved by comparative quantitative data. We tested the predictions of the 'developmental hourglass' definition of the phylotypic stage quantitatively by looking at the pattern of developmental-timing variation across vertebrates as a whole and within mammals. For both datasets, the results using two different metrics were counter to the predictions of the definition: phenotypic variation between species was highest in the middle of the developmental sequence. This surprising degree of developmental character independence argues against the existence of a phylotypic stage in vertebrates. Instead, we hypothesize that numerous tightly delimited developmental modules exist during the mid-embryonic period. Further, the high level of timing changes (heterochrony) between these modules may be an important evolutionary mechanism giving rise to the diversity of vertebrates. The onus is now clearly on proponents of the phylotypic stage to present both a clear definition of it and quantitative data supporting its existence.     

Evolution of developmental roles of Pax2/5/8 paralogs after independent duplication in urochordate and vertebrate lineages

Background

Gene duplication provides opportunities for lineage diversification and evolution of developmental novelties. Duplicated genes generally either disappear by accumulation of mutations (nonfunctionalization), or are preserved either by the origin of positively selected functions in one or both duplicates (neofunctionalization), or by the partitioning of original gene subfunctions between the duplicates (subfunctionalization). The Pax2/5/8 family of important developmental regulators has undergone parallel expansion among chordate groups. After the divergence of urochordate and vertebrate lineages, two rounds of independent gene duplications resulted in the Pax2, Pax5, and Pax8 genes of most vertebrates (the sister group of the urochordates), and an additional duplication provided the pax2a and pax2b duplicates in teleost fish. Separate from the vertebrate genome expansions, a duplication also created two Pax2/5/8 genes in the common ancestor of ascidian and larvacean urochordates.

Results

To better understand mechanisms underlying the evolution of duplicated genes, we investigated, in the larvacean urochordate Oikopleura dioica, the embryonic gene expression patterns of Pax2/5/8 paralogs. We compared the larvacean and ascidian expression patterns to infer modular subfunctions present in the single pre-duplication Pax2/5/8 gene of stem urochordates, and we compared vertebrate and urochordate expression to infer the suite of Pax2/5/8 gene subfunctions in the common ancestor of olfactores (vertebrates + urochordates). Expression pattern differences of larvacean and ascidian Pax2/5/8 orthologs in the endostyle, pharynx and hindgut suggest that some ancestral gene functions have been partitioned differently to the duplicates in the two urochordate lineages. Novel expression in the larvacean heart may have resulted from the neofunctionalization of a Pax2/5/8 gene in the urochordates. Expression of larvacean Pax2/5/8 in the endostyle, in sites of epithelial remodeling, and in sensory tissues evokes like functions of Pax2, Pax5 and Pax8 in vertebrate embryos, and may indicate ancient origins for these functions in the chordate common ancestor.

Conclusion

Comparative analysis of expression patterns of chordate Pax2/5/8 duplicates, rooted on the single-copy Pax2/5/8 gene of amphioxus, whose lineage diverged basally among chordates, provides new insights into the evolution and development of the heart, thyroid, pharynx, stomodeum and placodes in chordates; supports the controversial conclusion that the atrial siphon of ascidians and the otic placode in vertebrates are homologous; and backs the notion that Pax2/5/8 functioned in ancestral chordates to engineer epithelial fusions and perforations, including gill slit openings.     

Wnt and Frizzled RNA expression in human mesenchymal and embryonic (H7) stem cells

Background

Wnt signals are important for embryonic stem cells renewal, growth and differentiation. Although 19 Wnt, 10 Frizzled genes have been identified in mammals, their expression patterns in stem cells were largely unknown.

Results

We conducted RNA expression profiling for the Wnt ligands, their cellular receptors "Frizzleds" and co-receptors LRP5/6 in human embryonic stem cells (H7), human bone marrow mesenchymal cells, as well as mouse totipotent F9 teratocarcinoma embryonal cells. Except failing to express Wnt2 gene, totipotent F9 cells expressed RNA for all other 18 Wnt genes as well as all 10 members of Frizzled gene family. H7 cells expressed RNA for each of the 19 Wnt genes. In contrast, human mesenchymal cells did not display detectable RNA expression of Wnt1, Wnt8a, Wnt8b, Wnt9b, Wnt10a, and Wnt11. Analysis of Frizzled RNAs in H7 and human mesechymal cells revealed expression of 9 members of the receptor gene family, except Frizzled8. Expression of the Frizzled co-receptor LRP5 and LRP6 genes were detected in all three cell lines. Human H7 and mouse F9 cells express nearly a full complement of both Wnts and Frizzleds genes. The human mesenchymal cells, in contrast, have lost the expression of six Wnt ligands, i.e. Wnt1, 8a, 8b, 9b, 10a and 11.

Conclusion

Puripotent human H7 and mouse F9 embryonal cells express the genes for most of the Wnts and Frizzleds. In contrast, multipotent human mesenchymal cells are deficient in expression of Frizzled-8 and of 6 Wnt genes.     

Origins of anteroposterior patterning and Hox gene regulation during chordate evolution

All chordates share a basic body plan and many common features of early development. Anteroposterior (AP) regions of the vertebrate neural tube are specified by a combinatorial pattern of Hox gene expression that is conserved in urochordates and cephalochordates. Another primitive feature of Hox gene regulation in all chordates is a sensitivity to retinoic acid during embryogenesis, and recent developmental genetic studies have demonstrated the essential role for retinoid signalling in vertebrates. Two AP regions develop within the chordate neural tube during gastrulation: an anterior 'forebrain-midbrain' region specified by Otx genes and a posterior 'hindbrain-spinal cord' region specified by Hox genes. A third, intermediate region corresponding to the midbrain or midbrain-hindbrain boundary develops at around the same time in vertebrates, and comparative data suggest that this was also present in the chordate ancestor. Within the anterior part of the Hox-expressing domain, however, vertebrates appear to have evolved unique roles for segmentation genes, such as Krox-20, in patterning the hindbrain. Genetic approaches in mammals and zebrafish, coupled with molecular phylogenetic studies in ascidians, amphioxus and lampreys, promise to reveal how the complex mechanisms that specify the vertebrate body plan may have arisen from a relatively simple set of ancestral developmental components.     

Three different noggin genes antagonize the activity of bone morphogenetic proteins in the zebrafish embryo.

The dorsoventral polarity of the vertebrate embryo is established through interactions between ventrally expressed bone morphogenetic proteins and their organizer-borne antagonists Noggin, Chordin, and Follistatin. While the opposing interactions between Short Gastrulation/Chordin and Decapentaplegic/BMP4 have been evolutionarily conserved in arthropods and vertebrates, there has been up to now no functional evidence of an implication of Noggin in the early patterning of organisms other than Xenopus. We have studied the contribution of Noggin to the embryonic development of the zebrafish. While single-copy noggin genes have been characterized in several vertebrate species, we report that the zebrafish genome harbors three noggin homologues. Overexpression experiments show that Noggin1, Noggin2, and Noggin3 can antagonize ventralizing BMPs. While all three factors have similar biological activities, their embryonic expression is different. The combined expression of the three genes recapitulates the different aspects of the expression of the single-copy noggin genes of other organisms. This suggests that the three zebrafish noggin genes and the single noggin genes of other vertebrates have evolved from a common ancestor and that subsequent differential loss of tissue-specific elements in the promoters of the different zebrafish genes accounts for their more restricted spatiotemporal expression. Finally we show that noggin1 is expressed in the fish organizer and able to dorsalize the embryo, suggesting its implication in the dorsoventral patterning of the zebrafish.     

Development of polyembryonic insects: a major departure from typical insect embryogenesis

 The parasitic wasp Copidosoma floridanum represents the most extreme form of polyembryonic development known, forming up to 2000 embryos from a single egg. To understand the mechanisms of embryonic patterning in polyembryonic wasps and the evolutionary changes that led to this form of development we have analyzed embryonic development at the cellular level using confocal and scanning electron microscopy. C. floridanum embryogenesis can be divided into three phases: (1) early cleavage that leads to formation of a primary morula, (2) a proliferative phase that involves partitioning of embryonic cells into thousands of morulae, and (3) morphogenesis whereby individual embryos develop into larvae. This developmental program represents a major departure from typical insect embryogenesis, and we describe several features of morphogenesis unusual for insects. The early development of polyembryonic wasps, which likely evolved in association with a shift in life history to endoparasitism, shows several analogies with mammalian embryogenesis, including early separation of extraembryonic and embryonic cell lineages, formation of a morula and embryonic compaction. However, the late morphogenesis of polyembryonic wasps proceeds in a fashion conserved in all insects. Collectively, this suggests a lack of developmental constraints in early development, but a strong conservation of the phylotypic stage. Received: 27 June 1997 / Accepted: 11 January 1998     

Early expressed genes showing a dichotomous developing pattern in the lancelet embryo

Lancelets (amphioxus), although showing the most similar anatomical features to vertebrates, never develop a vertebrate-like head but rather several structures specific to this animal. The lancelet anatomical specificity seems to be traceable to early developmental stages, such as the vertebrate dorsal and anterior-posterior determinations. The BMP and Wnt proteins play important roles in establishing the early basis of the dorsal structures and the head in vertebrates. The early behavior of BMP and Wnt may be also related to the specific body structures of lancelets. The expression patterns of a dpp-related gene, Bbbmp2/4, and two wnt-related genes, Bbwnt7 and Bbwnt8, have been studied in comparison with those of brachyury and Hnf-3beta class genes. The temporal expression patterns of these genes are similar to those of vertebrates; Bbbmp2/4 and Bbwnt8 are first expressed in the invaginating primitive gut and the equatorial region, respectively, at the initial gastrula stage. However, spatial expression pattern of Bbbmp2/4 differs significantly from the vertebrate cognates. It is expressed in the mid-dorsal inner layer of gastrulae and widely in the anterior region, in which vertebrates block BMP signaling. The present study suggests that the lancelet embryo may have two distinct developmental domains from the gastrula stage, the domains of which coincide later with the lateral diverticular and the somitocoelomic regions. The embryonic origin of the anterior-specific structures in lancelets corresponds to the anterior domain where Bbbmp2/4 is continuously expressed.