Expression of zebrafish aldh1a3 (raldh3) and absence of aldh1a1 in teleosts

The vitamin A-derived morphogen retinoic acid (RA) plays important roles during the development of chordate animals. The Aldh1a-family of RA-synthesizing enzymes consists of three members, Aldh1a1-3 (Raldh1-3), that are dynamically expressed throughout development. We have searched the known teleost genomes for the presence of Raldh family members and have found that teleost fish possess orthologs of Aldh1a2 and Aldh1a3 only. Here we describe the expression of aldh1a3 in the zebrafish, Danio rerio. Whole mount in situ hybridization shows that aldh1a3 is expressed during eye development in the retina flanking the optic stalks and later is expressed ventrally, opposite the expression domain of aldh1a2. During inner ear morphogenesis, aldh1a3 is expressed in developing sensory epithelia of the cristae and utricular macula and is specifically up-regulated in epithelial projections throughout the formation of the walls of the semicircular canals and endolymphatic duct. In contrast to the mouse inner ear, which expresses all three Raldhs, we find that only aldh1a3 is expressed in the zebrafish otocyst, while aldh1a2 is present in the periotic mesenchyme. During larval stages, additional expression domains of aldh1a3 appear in the anterior pituitary and the swim bladder. Our analyses provide a starting point for genetic studies to examine the role of RA in these organs and emphasize the suitability of the zebrafish inner ear in dissecting the contribution of RA signaling to the development of the vestibular system.     

aldh7a1 Regulates Eye and Limb Development in Zebrafish

Uveal coloboma is a potentially blinding congenital ocular malformation caused by failure of the optic fissure to close during development. Although mutations in numerous genes have been described, these account for a minority of cases, complicating molecular diagnosis and genetic counseling. Here we describe a key role of aldh7a1 as a gene necessary for normal eye development. We show that morpholino knockdown of aldh7a1 in zebrafish causes uveal coloboma and misregulation of nlz1, another known contributor to the coloboma phenotype, as well as skeletal abnormalities. Knockdown of aldh7a1 leads to reduced cell proliferation in the optic cup of zebrafish, delaying the approximation of the edges of the optic fissure. The aldh7a1 morphant phenotype is partially rescued by co-injection of nlz1 mRNA suggesting that nlz1 is functionally downstream of aldh7a1 in regulating cell proliferation in the optic cup. These results support a role of aldh7a1 in ocular development and skeletal abnormalities in zebrafish.     

Quantitative staging of embryonic development of the tobacco hawkmoth,Manduca sexta

Summary A complete timetable of embryonic development of the tobacco hawkmoth,Manduca sexta (Lepidoptera: Sphingidae), is presented. Using living embryos, 20 developmental stages from oviposition to hatching are described with respect to their morphological and physiological maturation. This staging series provides a simple method to identify the stage ofManduca development during all phases of embryogenesis.     

Efficient embryonic culture method for the Japanese striped snake,Elaphe quadrivirgata,and its early developmental stages

The morphogenesis of snake embryos is an elusive yet fascinating research target for developmental biologists. However, few data exist on development of early snake embryo due to limited availability of pregnant snakes, and the need to harvest early stage embryos directly from pregnant snakes before oviposition without knowing the date of fertilization. We established an ex vivo culture method for early snake embryos using the Japanese striped snake, Elaphe quadrivirgata. This method, which we named “sausage‐style (SS) culture”, allows us to harvest snake embryos at specific stages for each experiment. Using this SS culture system, we calculated somite formation rate at early stages before oviposition. The average somite formation rate between 6/7 and 12/13 somite stages was 145.9 min, between 60/70 and 80/91 somite stages 42.4 min, and between 113–115 and 126/127 somite stages 71 min. Thus, somite formation rate that we observed during early snake embryogenesis was changed over time. We also describe a developmental staging series for E. quadrivirgata. This is the first report of a developmental series of early snake embryogenesis prior to oviposition by full‐color images with high‐resolution. We propose that the SS culture system is an easy method for treating early snake embryos ex vivo.     

Duplication and distinct expression patterns of two thrombospondin-1 isoforms in teleost fishes

Two types of thrombospondin-1 (named TSP-1a and TSP-1b) were cloned from two species of teleosts, the Nile tilapia and medaka. Phylogenetic analysis of these TSP-1 sequences, together with those available from other vertebrates further demonstrated that two types of TSP-1 exist only in teleosts, extending the finding in fugu and tetraodon to two additional fish species. The expression of both genes was examined using tilapia at various developmental stages. Tilapia TSP-1a and TSP-1b were each expressed in a wide range of tissues examined. The early expression of TSP-1b in both XX and XY gonads from 5 dah (day after hatching) onwards suggested an important role in the formation of gonads, while the expression of TSP-1a only in ovaries during later stages of development (from 120 dah onwards) may suggest that TSP-1a is involved in oogenesis. During the 14-day spawning cycle, the expression of both types of TSP-1 exhibited distinct peaks at day 5 (peak of vitellogenesis) and day 12 (oocyte maturation). In situ hybridization analyses revealed differential expression, with TSP-1a occurring in granulosa cells and TSP-1b in theca cells. Furthermore, both TSP-1a and -1b were expressed in skeletal tissues but with clear temporal and spatial differences. In contrast, only TSP-1b was found in the myosepta. The positive signals of both TSP-1a and TSP-1b were also detected in the heart and spleen, and TSP-1a in brain and intestine by both RT-PCR and in situ hybridization.     

Structural characterization of glycosaminoglycans from zebrafish in different ages

The zebrafish (Danio rerio) is a popular model organism for the study of developmental biology, disease mechanisms, and drug discovery. Glycosaminoglycans (GAGs), located on animal cell membranes and in the extracellular matrix, are important molecules in cellular communication during development, in normal physiology and pathophysiology. Vertebrates commonly contain a variety of GAGs including chondroitin/dermatan sulfates, heparin/heparan sulfate, hyaluronan and keratan sulfate. Zebrafish might represent an excellent experimental organism to study the biological roles of GAGs. A recent study showing the absence of heparan sulfate in adult zebrafish, suggested a more detailed evaluation of the GAGs present in this important model organism needed to be undertaken. This report aimed at examining the structural alterations of different GAGs at the molecular level at different developmental stages. GAGs were isolated and purified from zebrafish in different stages in development ranging from 0.5 days to adult. The content and disaccharide composition of chondroitin sulfate and heparan sulfate were determined using chemical assays, liquid chromotography and mass spectrometry. The presence of HS in adult fish was also confirmed using ¹H-NMR.     

Molecular cloning and expression of a second zebrafish aldehyde dehydrogenase 2 gene (aldh2b).

Aldehyde dehydrogenase 2 (ALDH2) is primarily responsible for detoxification of short-chain aldehydes in vivo. Previously it was reported that zebrafish has an aldh2 gene. Here we report the presence of a second aldh2 gene (aldh2b) in zebrafish. Zebrafish aldh2b locates adjacently to aldh2 on Chromosome 5 and the two genes share the same genomic organizations. aldh2b was predicted to encode a protein comprising 516 amino acids. The protein exhibits 95% amino acid identity with zebrafish ALDH2 and more than 76% identity with other vertebrate ALDH2s, respectively. Employing RT-PCR analysis, we demonstrated that both aldh2 and aldh2b mRNAs were present in embryos at cleavage stage (2 hpf: hour post fertilization) throughout protruding-mouth stage (72 hpf) and in different adult tissues of zebrafish. Taken together, our results reveal that zebrafish has two orthologues of aldh2 gene and the two genes share similar expression patterns during early development and in adult tissues.     

Opened brood chamber with embryos of different stages from the demosponge Amphimedon queenslandica.

Opened brood chamber with embryos of different stages from the demosponge Amphimedon queenslandica. This poriferan species has its genome sequenced, assembled, and annotated and serves as a model organism for sponge developmental biology. Image supplied by Bernie Degnan (University of Queensland).     

Development of the embryo, larva and early juvenile of Nile tilapia Oreochromis niloticus (Pisces: Cichlidae). Developmental staging system

We described the developmental stages for the embryonic, larval and early juvenile periods of Nile tilapia Oreochromis niloticus to elucidate sequential events of craniofacial development. Craniofacial development of cichlids, especially differentiation and morphogenesis of the pharyngeal skeleton, progresses until about 30 days postfertilization (dpf). Because there is no comprehensive report describing the sequential processes of craniofacial development up to 30 dpf, we newly defined 32 stages using a numbered staging system. For embryonic development, we defined 18 stages (stages 1-18), which were grouped into seven periods named the zygote, cleavage, blastula, gastrula, segmentation, pharyngula and hatching periods. For larval development, we defined seven stages (stages 19-25), which were grouped into two periods, early larval and late larval. For juvenile development until 30 dpf, we defined seven stages (stages 26-32) in the early juvenile period. This developmental staging system for Nile tilapia O. niloticus will benefit researchers investigating skeletogenesis throughout tilapia ontogeny and will also facilitate comparative evolutionary developmental biology studies of haplochromine cichlids, which comprise the species flocks of Lakes Malawi and Victoria.     

Molecular cloning and expression of retinoic-acid synthesizing enzyme raldh2 from Takifugu rubripes

Retinaldehyde dehydrogenases (raldhs) synthesize retinoic acid (RA), which is required for pattern formation and organogenesis during embryogenesis. To elucidate the common role of RA on vertebrate embryos, we first sought to clone a homologous gene to human raldh2 from fugu, Takifugu rubripes. We cloned a 1837 bp cDNA that encodes fugu raldh. The deduced amino acid sequence of the fugu raldh comprises 502 amino acids. The fugu Raldh showed highest sequence identity to zebrafish, Danio rerio, Raldh2 (79.9%). The fugu Raldh also showed high sequence identity to other vertebrate Raldh2: Xenopus laevis (77.2%), human (77.4%), mouse (74.3%) and chick (73.9%). Comparative genomic analysis showed that the gene arrangement around fugu raldh agreed with that of human raldh2. Fugu raldh mRNA was expressed through embryogenesis similarly to raldh2 in other vertebrates. These results and phylogenetic analyses suggest that pufferfish raldh is a fugu orthologue of other species' raldh2.     

Miple1 and miple2 encode a family of MK/PTN homologues in Drosophila melanogaster

Midkine (MK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. To date, this family of proteins has been implicated in multiple processes, such as growth, survival, and migration of various cells, and has roles in neurogenesis and epithelial–mesenchymal interaction during organogenesis. In this report, we have characterized two members of the MK/PTN family of proteins in Drosophila, named Miple1 and Miple2, from Midkine and Pleiotrophin. Drosophila miple1 and miple2 encode secreted proteins which are expressed in spatially restricted, nonoverlapping patterns during embryogenesis. Expression of miple1 can be found at high levels in the central nervous system, while miple2 is strongly expressed in the developing midgut endoderm. The identification of homologues of the MK/PTN family in this genetically tractable model organism should allow an analysis of their function during complex developmental processes. C. Englund, A. Birve, and L. Falileeva contributed equally to this work.     

Zebrafish CTH1, a C3H zinc finger protein, is expressed in ovarian oocytes and embryos

 The Zfcth1 gene is, as the previously cloned carp cth1 gene, related to the mammalian TIS 11 family of primary response genes and encodes a protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during oogenesis and early embryogenesis up to gastrulation in the zebrafish (Danio rerio). Maternal cth1 message is present in the ovary of 1-month-old fish and of adult fish in oocytes at all stages of maturation. In the youngest oocytes the message is localized in the cytoplasm all around the nucleus, in larger oocytes the message becomes restricted to the future animal pole of the embryo, and in mature oocytes the expression is sharply localized in the cortical layer under the micropyle. After ovulation the cth1 messenger spreads over the cytoplasmic cap and is distributed over the blastomeres during subsequent cleavages. In subsequent stages maternal expression of cth1 gradually disappears. From early epiboly stages onward embryonic cth1 expression is localized to the germ ring and the hypoblast cells in the central part of the embryonic shield. In the shield, cth1 expression largely overlaps with the area of gooscoid expression in the first involuting cells. In stages after 70% of epiboly cth1 expression diminishes and soon can no longer be detected in the embryo. Next to a developmental role in cell fate determination we propose a function for cth1 during oocyte maturation. Received: 19 October 1998 / Accepted: 16 February 1999     

Morphological and temporal variation in early embryogenesis contributes to species divergence in Malawi cichlid fishes

The cichlid fishes comprise the largest extant vertebrate family and are the quintessential example of rapid “explosive” adaptive radiations and phenotypic diversification. Despite low genetic divergence, East African cichlids harbor a spectacular intra- and interspecific morphological diversity, including the hyper-variable, neural crest (NC)-derived traits such as coloration and craniofacial skeleton. Although the genetic and developmental basis of these phenotypes has been investigated, understanding of when, and specifically how early, in ontogeny species-specific differences emerge, remains limited. Since adult traits often originate during embryonic development, the processes of embryogenesis could serve as a potential source of species-specific variation. Consequently, we designed a staging system by which we compare the features of embryogenesis between three Malawi cichlid species—Astatotilapia calliptera, Tropheops sp. ‘mauve’ and Rhamphochromis sp. “chilingali”—representing a wide spectrum of variation in pigmentation and craniofacial morphologies. Our results showed fundamental differences in multiple aspects of embryogenesis that could underlie interspecific divergence in adult adaptive traits. First, we identified variation in the somite number and signatures of temporal variation, or heterochrony, in the rates of somite formation. The heterochrony was also evident within and between species throughout ontogeny, up to the juvenile stages. Finally, the identified interspecific differences in the development of pigmentation and craniofacial cartilages, present at the earliest stages of their overt formation, provide compelling evidence that the species-specific trajectories begin divergence during early embryogenesis, potentially during somitogenesis and NC development. Altogether, our results expand our understanding of fundamental cichlid biology and provide new insights into the developmental origins of vertebrate morphological diversity.     

Stages and other aspects of the embryology of the parthenogenetic Marmorkrebs (Decapoda, Reptantia, Astacida)

The early development of the parthenogenetic Marmorkrebs (marbled crayfish) is described with respect to external morphology, cell lineage, and segment formation. Due to its parthenogenetic reproduction mode, the question arises whether or not the marbled crayfish is a suitable model organism for developmental approaches. To address this question, we describe several aspects of the embryonic development until hatching. We establish ten stages based on characteristic external changes in the living eggs such as blastoderm formation, gastrulation process, formation and differentiation of the naupliar and post-naupliar segments, limb bud differentiation, and eye differentiation. The study of the post-naupliar cell division patterns, segment formation, and engrailed expression reveals distinct similarities to that of other freshwater crayfish. On this basis, we evaluate the possibility of a generalization of ontogenetic processes in the Marmorkrebs for either freshwater crayfish or other crustacean developmental systems.     

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.     

Identification and characterization of the zebrafish glutathione S‐transferase Pi‐1

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S‐transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi‐1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3‐month‐old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione‐conjugating activity toward 1‐chloro‐2,4‐dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH‐dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined.     

Proteomic Characterization of Caenorhabditis elegans Larval Development

The nematode Caenorhabditis elegans is widely used as a model organism to study cell and developmental biology. Quantitative proteomics of C. elegans is still in its infancy and, so far, most studies have been performed on adult worm samples. Here, we used quantitative mass spectrometry to characterize protein level changes across the four larval developmental stages (L1–L4) of C. elegans. In total, we identified 4130 proteins, and quantified 1541 proteins that were present across all four stages in three biological replicates from independent experiments. Using hierarchical clustering and functional ontological analyses, we identified 21 clusters containing proteins with similar protein profiles across the four stages, and highlighted the most overrepresented biological functions in each of these protein clusters. In addition, we used the dataset to identify putative larval stage‐specific proteins in each individual developmental stage, as well as in the early and late developmental stages. In summary, this dataset provides system‐wide analysis of protein level changes across the four C. elegans larval developmental stages, which serves as a useful resource for the C. elegans research community. MS data were deposited in ProteomeXchange ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the primary accession identifier PXD006676.     

Consequences of Lineage-Specific Gene Loss on Functional Evolution of Surviving Paralogs: ALDH1A and Retinoic Acid Signaling in Vertebrate Genomes

Genome duplications increase genetic diversity and may facilitate the evolution of gene subfunctions. Little attention, however, has focused on the evolutionary impact of lineage-specific gene loss. Here, we show that identifying lineage-specific gene loss after genome duplication is important for understanding the evolution of gene subfunctions in surviving paralogs and for improving functional connectivity among human and model organism genomes. We examine the general principles of gene loss following duplication, coupled with expression analysis of the retinaldehyde dehydrogenase Aldh1a gene family during retinoic acid signaling in eye development as a case study. Humans have three ALDH1A genes, but teleosts have just one or two. We used comparative genomics and conserved syntenies to identify loss of ohnologs (paralogs derived from genome duplication) and to clarify uncertain phylogenies. Analysis showed that Aldh1a1 and Aldh1a2 form a clade that is sister to Aldh1a3-related genes. Genome comparisons showed secondarily loss of aldh1a1 in teleosts, revealing that Aldh1a1 is not a tetrapod innovation and that aldh1a3 was recently lost in medaka, making it the first known vertebrate with a single aldh1a gene. Interestingly, results revealed asymmetric distribution of surviving ohnologs between co-orthologous teleost chromosome segments, suggesting that local genome architecture can influence ohnolog survival. We propose a model that reconstructs the chromosomal history of the Aldh1a family in the ancestral vertebrate genome, coupled with the evolution of gene functions in surviving Aldh1a ohnologs after R1, R2, and R3 genome duplications. Results provide evidence for early subfunctionalization and late subfunction-partitioning and suggest a mechanistic model based on altered regulation leading to heterochronic gene expression to explain the acquisition or modification of subfunctions by surviving ohnologs that preserve unaltered ancestral developmental programs in the face of gene loss.     

The viability of the first generation roach (<Emphasis Type="Italic">Rutilus rutilus</Emphasis> L.), bream (<Emphasis Type="Italic">Abramis brama</Emphasis> L.), and blue bream (<Emphasis Type="Italic">Abramis ballerus</Emphasis> L.) hybrids at early stages of development

The viability of bream, roach, and blue bream F ₁hybrids at early stages of their development is analyzed. Viability is controlled according to parameters of the survival rate at stages from fertilized eggs to yearlings. During embryogenesis, significant stages (blastula—gastrula and hatching) are revealed by the amount of losses. The viability of hybrids of the first generation (compared to pure species) from the hatching stage and in the subsequent development constantly increases. At the stage of fingerlings, the viability of F ₁hybrids significantly exceeds that of pure species, which points to the heterozygous effect according to the parameters of hybrid survival for the first generation, which is absent in embryogenesis.