Identification and characterization of PlAlix,the Alix homologue from the Mediterranean sea urchin Paracentrotus lividus

The sea urchin provides a relatively simple and tractable system for analyzing the early stages of embryo development. Here, we use the sea urchin species, Paracentrotus lividus, to investigate the role of Alix in key stages of embryogenesis, namely the egg fertilization and the first cleavage division. Alix is a multifunctional protein involved in different cellular processes including endocytic membrane trafficking, filamentous (F)‐actin remodeling, and cytokinesis. Alix homologues have been identified in different metazoans; in these organisms, Alix is involved in oogenesis and in determination/differentiation events during embryo development. Herein, we describe the identification of the sea urchin homologue of Alix, PlAlix. The deduced amino acid sequence shows that Alix is highly conserved in sea urchins. Accordingly, we detect the PlAlix protein cross‐reacting with monoclonal Alix antibodies in extracts from P. lividus, at different developmental stages. Focusing on the role of PlAlix during early embryogenesis we found that PlAlix is a maternal protein that is expressed at increasingly higher levels from fertilization to the 2‐cell stage embryo. In sea urchin eggs, PlAlix localizes throughout the cytoplasm with a punctuated pattern and, soon after fertilization, accumulates in larger puncta in the cytosol, and in microvilli‐like protrusions. Together our data show that PlAlix is structurally conserved from sea urchin to mammals and may open new lines of inquiry into the role of Alix during the early stages of embryo development.     

VEB4: Early zygotic mRNA expressed asymmetrically along the animal-vegetal axis of the sea urchin embryo

We have analyzed a gene, designated VEB4 , that is expressed transiently in very early blastulae of the sea urchin, Strongylocentrotus purpuratus . Sequence analysis of the complete open reading frame shows that VEB4 encodes an unusual, highly charged protein with a pl of 9.55. We show here that VEB4 mRNA accumulate in a spatial pattern that is indistinguishable from that of two other recently described genes encoding metallo-endoproteases, SpAN , related to astacin and SpHE , the hatching enzyme (Reynolds et al . 1992). VEB4 and other members of this gene set encode the earliest strictly zygotic gene products that have been identified. The asymmetric accumulation of VEB4 mRNA in non-vegetal blastomeres of the 16 cell embryo and their descendants reflects the animal-vegetal maternal developmental axis.     

Identification and developmental expression of the ets gene family in the sea urchin (Strongylocentrotus purpuratus)

A systematic search in the available scaffolds of the Strongylocentrotus purpuratus genome has revealed that this sea urchin has 11 members of the ets gene family. A phylogenetic analysis of these genes showed that almost all vertebrate ets subfamilies, with the exception of one, so far found only in mammals, are each represented by one orthologous sea urchin gene. The temporal and spatial expression of the identified ETS factors was also analyzed during embryogenesis. Five ets genes (Sp-Ets1/2, Sp-Tel, Sp-Pea, Sp-Ets4, Sp-Erf) are also maternally expressed. Three genes (Sp-Elk, Sp-Elf, Sp-Erf) are ubiquitously expressed during embryogenesis, while two others (Sp-Gabp, Sp-Pu.1) are not transcribed until late larval stages. Remarkably, five of the nine sea urchin ets genes expressed during embryogenesis are exclusively (Sp-Ets1/2, Sp-Erg, Sp-Ese) or additionally (Sp-Tel, Sp-Pea) expressed in mesenchyme cells and/or their progenitors. Functional analysis of Sp-Ets1/2 has previously demonstrated an essential role of this gene in the specification of the skeletogenic mesenchyme lineage. The dynamic, and in some cases overlapping and/or unique, developmental expression pattern of the latter five genes suggests a complex, non-redundant function for ETS factors in sea urchin mesenchyme formation and differentiation.     

Characterization of sea urchin unconventional myosins and analysis of their patterns of expression during early embryogenesis

Early sea urchin development requires a dynamic reorganization of both the actin cytoskeleton and cytoskeletal interactions with cellular membranes. These events may involve the activities of multiple members of the superfamily of myosin motor proteins. Using RT-PCR with degenerate myosin primers, we identified 11 myosin mRNAs expressed in unfertilized eggs and coelomocytes of the sea urchin Strongylocentrotus purpuratus. Seven of these sea urchin myosins belonged to myosin classes Igamma, II, V, VI, VII, IX, and amoeboid-type I, and the remaining four may be from novel classes. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I were either completely or partially cloned and their molecular structures characterized. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I shared a high degree of sequence identity with their respective family members from vertebrates and they retained their class-specific structure and domain organization. Analysis of expression of myosin-V, -VI, -VII, and amoeboid-type-I mRNAs during development revealed that each myosin mRNA displayed a distinct temporal pattern of expression, suggesting that myosins might be involved in specific events of early embryogenesis. Interestingly, the onset of gastrulation appeared to be a pivotal point in modulation of myosin mRNA expression. The presence of multiple myosin mRNAs in eggs and embryos provides insight into the potential involvement of multiple specific motor proteins in the actin-dependent events of embryo development.     

hch-1, a gene required for normal hatching and normal migration of a neuroblast in C. elegans, encodes a protein related to TOLLOID and BMP-1.

Proteins of the tolloid/bone morphogenetic protein (BMP)-1 family play important roles in the differentiation of cell fates. Among those proteins are BMP-1, which plays a role in cartilage and bone formation in mammals, the TOLLOID protein, which is required for the establishment of the dorsoventral axis of Drosophila embryos and BP10/SpAN, which are thought to act in the morphogenesis of sea urchins. These proteins have some properties in common. First, they contain the astacin metalloprotease domain, the CUB domain and the epidermal growth factor-like domain. Second, they are expressed in embryos at stages expected for their role in cell differentiation. Third, at least BMP-1 and TOLLOID are thought to interact with proteins of the transforming growth factor-beta family. We report that the hch-1 gene of the nematode Caenorhabditis elegans encodes a tolloid/BMP-1 family protein. The protein has the characteristic domains common to the tolloid/ BMP-1 family. Like other members of the family, it is expressed in embryos. However, the phenotype of hch-1 mutants shows that it is required for normal hatching and normal migration of a post-embryonic neuroblast. Furthermore, in spite of its expression in embryogenesis, it is not required for the viability of embryos. These results show new functions of the tolloid/BMP-1 family proteins and give insight into their evolution.     

Apoptosis: focus on sea urchin development

It has been proposed that the apoptosis is an essential requirement for the evolution of all animals, in fact the apoptotic program is highly conserved from nematodes to mammals. Throughout development, apoptosis is employed by multicellular organisms to eliminate damaged or unnecessary cells. Here, we will discuss both developmental programmed cell death (PCD) under normal conditions and stress induced apoptosis, in sea urchin embryos. Sea urchin represent an excellent model system for studying embryogenesis and cellular processes involved in metamorphosis. PCD plays an essential role in sculpting and remodelling the embryos and larvae undergoing metamorphosis. Moreover, this marine organism directly interacts with its environment, and is susceptible to effects of several aquatic contaminants. Apoptosis can be adopted as a defence mechanism against any environmental chemical, physical and mechanical stress, for removing irreversibly damaged cells. This review, while not comprehensive in its reporting, aims to provide an overview of current knowledge on mechanisms to regulate physiological and the induced apoptotic program in sea urchin embryos.     

A synthetic derivative of plant allylpolyalkoxybenzenes induces selective loss of motile cilia in sea urchin embryos.

Polyalkoxybenzenes are plant components displaying a wide range of biological activities. In these studies, we synthesized apiol and dillapiol isoxazoline analogues of combretastatins and evaluated their effect on sea urchin embryos. We have shown that p-methoxyphenyl isoxazoline caused sea urchin embryo immobilization due to the selective excision of motile cilia, whereas long immotile sensory cilia of apical tuft remained intact. This effect was completely reversed by washing the embryos. The compound did not alter cell division, blastulae hatching, and larval morphogenesis. In our hands, the molecule would serve as a convenient tool for in vivo studying morphogenetic processes in the sea urchin embryo. We anticipate that both the assay and the described derivative could be used for studies in ciliary function in embryogenesis.     

A conserved role for the nodal signaling pathway in the establishment of dorso-ventral and left-right axes in deuterostomes

Nodal factors play crucial roles during embryogenesis of chordates. They have been implicated in a number of developmental processes, including mesoderm and endoderm formation and patterning of the embryo along the anterior-posterior and left-right axes. We have analyzed the function of the Nodal signaling pathway during the embryogenesis of the sea urchin, a non-chordate organism. We found that Nodal signaling plays a central role in axis specification in the sea urchin, but surprisingly, its first main role appears to be in ectoderm patterning and not in specification of the endoderm and mesoderm germ layers as in vertebrates. Starting at the early blastula stage, sea urchin nodal is expressed in the presumptive oral ectoderm where it controls the formation of the oral-aboral axis. A second conserved role for nodal signaling during vertebrate evolution is its involvement in the establishment of left-right asymmetries. Sea urchin larvae exhibit profound left-right asymmetry with the formation of the adult rudiment occurring only on the left side. We found that a nodal/lefty/pitx2 gene cassette regulates left-right asymmetry in the sea urchin but that intriguingly, the expression of these genes is reversed compared to vertebrates. We have shown that Nodal signals emitted from the right ectoderm of the larva regulate the asymmetrical morphogenesis of the coelomic pouches by inhibiting rudiment formation on the right side of the larva. This result shows that the mechanisms responsible for patterning the left-right axis are conserved in echinoderms and that this role for nodal is conserved among the deuterostomes. We will discuss the implications regarding the reference axes of the sea urchin and the ancestral function of the nodal gene in the last section of this review.     

Transforming growth factor‐β signal regulates gut bending in the sea urchin embryo

During gastrulation, one of the most important morphogenetic events in sea urchin embryogenesis, the gut bends toward the ventral side to form an open mouth. Although the involvement of transforming growth factor‐β (TGF‐β) signals in the cell‐fate specification of the ectoderm and endoderm along the dorsal–ventral axis has been well reported, it remains unclear what controls the morphogenetic behavior of gut bending. Here, using two sea urchin species, Hemicentrotus pulcherrimus and Temnopleurus reevesii, we show that TGF‐β signals are required for gut bending toward the ventral side. To search for the common morphogenetic cue in these two species, we initially confirmed the expression patterns of the dorsal–ventral regulatory TGF‐β members, nodal, lefty, bmp2/4, and chordin, in T. reevesii because these factors are appropriate candidates to investigate the cue that starts gut bending, although genetic information about the body axes is entirely lacking in this species. Based on their expression patterns and a functional analysis of Nodal, the dorsal–ventral axis formation of T. reevesii is likely regulated by these TGF‐β members, as in other sea urchins. When the Alk4/5/7 signal was inhibited by its specific inhibitor, SB431542, before the late gastrula stage of T. reevesii, the gut was extended straight toward the anterior tip region, although the ectodermal dorsal–ventral polarity was normal. By contrast, H. pulcherrimus gut bending was sensitive to SB431542 until the prism stage. These data clearly indicate that gut bending is commonly dependent on a TGF‐β signal in sea urchins, but the timing of the response varies in different species.     

Gypsy/Ty3-class retrotransposons integrated in the DNA of herring,tunicate, and echinoderms

Eight new examples of retrotransposons of the Gypsy/Ty3 class have been identified in marine species. A 525-nt pol gene-coding region was amplified using degenerate primers from highly conserved regions and has extended the range of recognition of Gypsy/Ty3 far beyond those previously known. The following matrix shows the percentage AA divergence of the translations of this segment of the pol gene coding region. Spr2 Strongylocentrotus purpuratus, sea urchin 39 Por2 Pisaster ochraceus, starfish 46 45 Cprl Clupea pallasi, herring 51 52 41 Cirl Ciona intestinalis, tunicate bar52 49 49 55 P. orchraceus, starfish 55 60 60 62 62 Spr3 S. purpuratus, sea urchin 55 61 60 63 61 24 Tgrl^* Tripneustes gratilla, sea urchin 56 61 60 63 58 26 27 Lvrl^* Lytechinus variegatus, sea urchin 57 62 60 64 62 27 10 29 Sprl^* S. purpuratus 58 61 62 65 61 15 27 30 31 Spr4 S. purpuratus 72 72 74 75 72 73 72 72 73 72 Por3 P. ochraceus The underlines separate three groups of retrotransposons that can be recognized on the basis of this amino acid sequence. The new upper group shows surprising amino acid sequence similarity among members from the DNA of herring, sea urchin, starfish, and a tunicate. For example, the herring element differs by only 41 % from the Ciona element and 46% from the sea urchin element. The group between the lines includes members close to previously known elements (marked by asterisks) and has so far been found only in sea urchins. The two upper groups differ from each other by 55–60% and yet members of both groups (e.g., Sprl and Spr2) are integrated into the DNA of one species-S. purpuratus. Below the lower underline is listed the only known representative of a very distant group, which occurs in starfish DNA. In spite of large divergence, amino acid sequence comparisons indicate that all of the elements shown in the array are members of the LTR-containing class of retrotransposons that includes Gypsy of Drosophila and Ty3 of yeast. Of all known mobile elements this class shows the closest sequence similarity to retroviruses and has the same arrangement of genes as simpler retroviruses.Correspondence to: R.J. Britten     

Chordin is required for neural but not axial development in sea urchin embryos

The oral-aboral (OA) axis in the sea urchin is specified by the TGFβ family members Nodal and BMP2/4. Nodal promotes oral specification, whereas BMP2/4, despite being expressed in the oral territory, is required for aboral specification. This study explores the role of Chordin (Chd) during sea urchin embryogenesis. Chd is a secreted BMP inhibitor that plays an important role in axial and neural specification and patterning in Drosophila and vertebrate embryos. In Lytechinus variegatus embryos, Chd and BMP2/4 are functionally antagonistic. Both are expressed in overlapping domains in the oral territory prior to and during gastrulation. Perturbation shows that, surprisingly, Chd is not involved in OA axis specification. Instead, Chd is required both for normal patterning of the ciliary band at the OA boundary and for development of synaptotagmin B-positive (synB) neurons in a manner that is reciprocal with BMP2/4. Chd expression and synB-positive neural development are both downstream from p38 MAPK and Nodal, but not Goosecoid. These data are summarized in a model for synB neural development.     

dCMP-aminohydrolase activity during early sea urchin development. An example of negative enzyme control during embryogenesis

In sea urchin, unfertilized eggs have a very high level of dCMP-aminohydrolase (dCMPase) activity, which decreases gradually and at the pluteus stage it is only about a quarter of that found in the unfertilized egg. But in abnormal embryos and in disaggregated cells from embryos, no decrease in the dCMPase activity takes place. To understand the control mechanism involved in this enzyme activity during development, we have analyzed the effect of various drugs which interfere with information transfer, such as actinomycin C, puromycin, 5-azacytidine, 2-thio-uracil and p-fluoro-DL-phenylalanine on dCMPase activity in embryos of Paracentrotus lividus and Sphaerechinus granularis. Among these drugs only actinomycin induces a remarkable increase of the dCMPase activity in embryos with respect to unfertilized eggs. Puromycin has a differential and dose-dependent effect. Other drugs, although they affect normal development and macromolecular synthesis, do not significantly alter the dCMPase activity. On the basis of these results we suggest the presence of a repressor mechanism in the control of dCMP-aminohydrolase level during early embryogenesis of sea urchin.