Robert C. Angerer

Comparative aspects of DNA organization in metazoa

Data on sequence organization in metazoa are reviewed and tabulated. It is shown that the features of sequence organization previously observed in Xenopus DNA are extremely widespread. At least 70% of DNA fragments 2,000–3,000 nucleotides long contain both single copy and repetitive sequence in all the organisms examined except Drosophila.

4 Patterning the sea urchin embryo: Gene regulatory networks, signaling pathways, and cellular interactions

We discuss steps in the specification of major tissue territories of the sea urchin embryo that occur between fertilization and hatching blastula stage and the cellular interactions required to coordinate morphogenetic processes that begin after hatching. We review evidence that has led to new ideas about how this embryo is initially patterned: (1) Specification of most of the tissue territories is not direct, but proceeds gradually by progressive subdivision of broad, maternally specified domains that depend on opposing gradients in the ratios of animalizing transcription factors (ATFs) and vegetalizing (beta-catenin) transcription factors; (2) the range of maternal nuclear beta-catenin extends further than previously proposed, that is, into the animal hemisphere, where it programs many cells to adopt early aboral ectoderm characteristics; (3) cells at the extreme animal pole constitute a unique ectoderm region, lacking nuclear beta-catenin; (4) the pluripotential mesendoderm is created by the combined outputs of ATFs and nuclear beta-catenin, which initially overlap in the macromeres, and by an undefined early micromere signal; (5) later micromere signals, which activate Notch and Wnt pathways, subdivide mesendoderm into secondary mesenchyme and endoderm; and (6) oral ectoderm specification requires reprogramming early aboral ectoderm at about the hatching blastula stage. Morphogenetic processes that follow initial fate specification depend critically on continued interactions among cells in different territories. As illustrations, we discuss the regulation of (1) the ectoderm/endoderm boundary, (2) mesenchyme positioning and skeletal growth, (3) ciliated band formation, and (4) several suppressive interactions operating late in embryogenesis to limit the fates of multipotent cells.

A Wnt-FoxQ2-Nodal Pathway Links Primary and Secondary Axis Specification in Sea Urchin Embryos

The primary (animal-vegetal) (AV) and secondary (oral-aboral) (OA) axes of sea urchin embryos are established by distinct regulatory pathways. However, because experimental perturbations of AV patterning also invariably disrupt OA patterning and radialize the embryo, these two axes must be mechanistically linked. Here we show that FoxQ2, which is progressively restricted to the animal plate during cleavage stages, provides this linkage. When AV patterning is prevented by blocking the nuclear function of beta-catenin, the animal plate where FoxQ2 is expressed expands throughout the future ectoderm, and expression of nodal, which initiates OA polarity, is blocked. Surprisingly, nodal transcription and OA differentiation are rescued simply by inhibiting FoxQ2 translation. Therefore, restriction of FoxQ2 to the animal plate is a crucial element of canonical Wnt signaling that coordinates patterning along the AV axis with the initiation of OA specification.

Single copy DNA and structural gene sequence relationships among four sea urchin species

Measurements of the divergence of single copy DNA sequences among four sea urchin species are presented. At a standard criterion for reassociation (0.12 M phosphate buffer, 60° C, hydroxyapatite binding) we observe the following extents of reaction and reductions in thermal stability for single copy DNA reassociation between Strongylocentrotus purpuratus tracer and heterologous driver DNA: S. dröbachiensis 68% and 2.5°C; S. franciscanus 51% and 3.5° C; Lytechinus pictus 12% and 7.5° C. The implied extents of sequence relatedness are consistent with the phylogenetic relationships of these species. The rate of single copy sequence divergence in the evolutionary lines leading to the Strongylocentrotus species is estimated to be 0.06–0.35% per million years. The rate of divergence of total single copy sequence has been compared to that of structural gene sequences represented in S. purpuratus gastrula polysomal messenger RNA. When closely related species, S. purpuratus and S. franciscanus, are compared, these polysomal sequences are found to diverge at a lower rate than does the total single copy sequence. For two very distantly related species, S. purpuratus and L. pictus, a small fraction of the single copy DNA sequence is probably conserved. These conserved sequences are not enriched in their content of structural gene sequences.

The sea urchin animal pole domain is a Six3-dependent neurogenic patterning center

Two major signaling centers have been shown to control patterning of sea urchin embryos. Canonical Wnt signaling in vegetal blastomeres and Nodal signaling in presumptive oral ectoderm are necessary and sufficient to initiate patterning along the primary and secondary axes, respectively. Here we define and characterize a third patterning center, the animal pole domain (APD), which contains neurogenic ectoderm, and can oppose Wnt and Nodal signaling. The regulatory influence of the APD is normally restricted to the animal pole region, but can operate in most cells of the embryo because, in the absence of Wnt and Nodal, the APD expands throughout the embryo. We have identified many constituent APD regulatory genes expressed in the early blastula and have shown that expression of most of them requires Six3 function. Furthermore, Six3 is necessary for the differentiation of diverse cell types in the APD, including the neurogenic animal plate and immediately flanking ectoderm, indicating that it functions at or near the top of several APD gene regulatory networks. Remarkably, it is also sufficient to respecify the fates of cells in the rest of the embryo, generating an embryo consisting of a greatly expanded, but correctly patterned, APD. A fraction of the large group of Six3-dependent regulatory proteins are orthologous to those expressed in the vertebrate forebrain, suggesting that they controlled formation of the early neurogenic domain in the common deuterostome ancestor of echinoderms and vertebrates.

DNA Sequence Organization in the Mollusc Aplysia Californica

The sequence organization of the DNA of the mollusc Aplysia californica has been examined by a combination of techniques. Close-spaced interspersion of repetitive and single copy sequences occurs throughout the majority of the genome. Detailed examination of the DNA of this protostome reveals great similarities to the pattern observed in the two deuterostome organisms previously examined in detail in this laboratory, Xenopus laevis and Strongylocentrotus purpuratus. Labeled and unlabeled Aplysia DNA were prepared from developing embryos and sheared to a fragment length of 400 nucleotides. The kinetics of reassociation were studied by means of hydroxyapatite chromatography, single-strand-specific S1 nuclease, and optical methods of assay. Aplysia DNA of this fragment length contains at least five resolvable kinetic fractions. One classification of these fractions, listed with their reassociation rate constants (l M-1 sec-1) is: single copy (0.00057), slow (0.047), fast (2.58), very fast (4000), and foldback (greater than 10(5)). Sequence arrangement was deduced from: the kinetics of reassociation of DNA fragments of length 400 or 2000 nucleotides; the hyperchromicity of reassociated fragments containing duplex regions; the size of duplex regions resistant to S1 nuclease; and the reassociation of labeled fragments of various lengths with short driver fragments. More than 80% of the single copy DNA sequences are interspersed with repetitive sequences. The maximum spacing of the repeats is about 2000 nucleotides, and the average less than 1000. The very fast fraction does not show interspersion with single copy sequences or with other kinetic fractions. The foldback fraction sequences are fairly widely interspersed. The slow fraction sequences are interspersed with the fast fraction, and possibly also with the single copy DNA. The fast fraction is the dominant interspersed repetitive fraction. Its sequences are adjacent to the great majority of the single copy sequences and have an average length of about 300 nucleotides.

The evolution of nervous system patterning: insights from sea urchin development

Recent studies of the sea urchin embryo have elucidated the mechanisms that localize and pattern its nervous system. These studies have revealed the presence of two overlapping regions of neurogenic potential at the beginning of embryogenesis, each of which becomes progressively restricted by separate, yet linked, signals, including Wnt and subsequently Nodal and BMP. These signals act to specify and localize the embryonic neural fields – the anterior neuroectoderm and the more posterior ciliary band neuroectoderm – during development. Here, we review these conserved nervous system patterning signals and consider how the relationships between them might have changed during deuterostome evolution.

Integration of Canonical and Noncanonical Wnt Signaling Pathways Patterns the Neuroectoderm Along the Anterior-Posterior Axis of Sea Urchin Embryos

Three different Wnt signaling pathways function to restrict the anterior neuroectoderm state to the anterior end of the sea urchin embryo, a mechanism of anterior fate restriction that could be conserved among deuterostomes.

TGFβ signaling positions the ciliary band and patterns neurons in the sea urchin embryo

The ciliary band is a distinct region of embryonic ectoderm that is specified between oral and aboral ectoderm. Flask-shaped ciliary cells and neurons differentiate in this region and they are patterned to form an integrated tissue that functions as the principal swimming and feeding organ of the larva. TGFβ signaling, which is known to mediate oral and aboral patterning of the ectoderm, has been implicated in ciliary band formation. We have used morpholino knockdown and ectopic expression of RNA to alter TGFβ signaling at the level of ligands, receptors, and signal transduction components and assessed the differentiation and patterning of the ciliary band cells and associated neurons. We propose that the primary effects of these signals are to position the ciliary cells, which in turn support neural differentiation. We show that Nodal signaling, which is known to be localized by Lefty, positions the oral margin of the ciliary band. Signaling from BMP through Alk3/6, affects the position of the oral and aboral margins of the ciliary band. Since both Nodal and BMP signaling produce ectoderm that does not support neurogenesis, we propose that formation of a ciliary band requires protection from these signals. Expression of BMP2/4 and Nodal suppress neural differentiation. However, the response to receptor knockdown or dominant-negative forms of signal transduction components indicate signaling is not acting directly on unspecified ectoderm cells to prevent their differentiation as neurons. Instead, it produces a restricted field of ciliary band cells that supports neurogenesis. We propose a model that incorporates spatially regulated control of Nodal and BMP signaling to determine the position and differentiation of the ciliary band, and subsequent neural patterning.

Molecular paleoecology: using gene regulatory analysis to address the origins of complex life cycles in the late Precambrian

SUMMARY Molecular paleoecology is the application of molecular data to test hypotheses made by paleoecological scenarios. Here, we use gene regulatory analysis to test between two competing paleoecological scenarios put forth to explain the evolution of complex life cycles. The first posits that early bilaterians were holobenthic, and the evolution of macrophagous grazing drove the exploitation of the pelagos by metazoan eggs and embryos, and eventually larvae. The alternative hypothesis predicts that early bilaterians were holopelagic, and new adult stages were added on when these holopelagic forms began to feed on the benthos. The former hypothesis predicts that the larvae of protostomes and deuterostomes are not homologous, with the implication that larval‐specific structures, including the apical organ, are the products of convergent evolution, whereas the latter hypothesis predicts homology of larvae, specifically homology of the apical organ. We show that in the sea urchin, Strongylocentrotus purpuratus, the transcription factors NK2.1 and HNF6 are necessary for the correct spatial expression profiles of five different cilia genes. All of these genes are expressed exclusively in the apical plate after the mesenchyme‐blastula stage in cells that also express NK2.1 and HNF6. In addition, abrogation of SpNK2.1 results in embryos that lack the apical tuft. However, in the red abalone, Haliotis rufescens, NK2.1 and HNF6 are not expressed in any cells that also express these same five cilia genes. Nonetheless, like the sea urchin, the gastropod expresses both NK2.1 and FoxA around the stomodeum and foregut, and FoxA around the proctodeum. As we detected no similarity in the development of the apical tuft between the sea urchin and the abalone, these molecular data are consistent with the hypothesis that the evolution of mobile, macrophagous metazoans drove the evolution of complex life cycles multiple times independently in the late Precambrian.