Discovery of genes expressed in Hydra embryogenesis

Hydra's remarkable capacity to regenerate, to proliferate asexually by budding, and to form a pattern de novo from aggregates allows studying complex cellular and molecular processes typical for embryonic development. The underlying assumption is that patterning in adult hydra tissue relies on factors and genes which are active also during early embryogenesis. Previously, we reported that in Hydra the timing of expression of conserved regulatory genes, known to be involved in adult patterning, differs greatly in adults and embryos (Fr?bius, A.C., Genikhovich, G., Kürn, U., Anton-Erxleben, F. and Bosch, T.C.G., 2003. Expression of developmental genes during early embryogenesis of Hydra. Dev. Genes Evol. 213, 445-455). Here, we describe an unbiased screening strategy to identify genes that are relevant to Hydra vulgaris embryogenesis. The approach yielded two sets of differentially expressed genes: one set was expressed exclusively or nearly exclusively in the embryos, while the second set was upregulated in embryos in comparison to adult polyps. Many of the genes identified in hydra embryos had no matches in the database. Among the conserved genes upregulated in embryos is the Hydra orthologue of Embryonic Ectoderm Development (HyEED). The expression pattern of HyEED in developing embryos suggests that interstitial stem cells in Hydra originate in the endoderm. Importantly, the observations uncover previously unknown differences in genes expressed by embryos and polyps and indicate that not only the timing of expression of developmental genes but also the genetic context is different in Hydra embryos compared to adults.     

Conserved and novel gene expression between regeneration and asexual fission in <Emphasis Type="Italic">Nematostella vectensis</Emphasis>

Due to work in model systems (e.g., flies and mice), the molecular mechanisms of embryogenesis are known in exquisite detail. However, these organisms are incapable of asexual reproduction and possess limited regenerative abilities. Thus, the mechanisms of alternate developmental trajectories and their relation to embryonic mechanisms remain understudied. Because these developmental trajectories are present in a diverse group of animal phyla spanning the metazoan phylogeny, including cnidarians, annelids, and echinoderms, they are likely to have played a major role in animal evolution. The starlet sea anemone Nematostella vectensis, an emerging model system, undergoes larval development, asexual fission, and complete bi-directional regeneration in the field and laboratory. In order to investigate to what extent embryonic patterning mechanisms are utilized during alternate developmental trajectories, we examined expression of developmental regulatory genes during regeneration and fission. When compared to previously reported embryonic expression patterns, we found that all genes displayed some level of expression consistent with embryogenesis. However, five of seven genes investigated also displayed striking differences in gene expression between one or more developmental trajectory. These results demonstrate that alternate developmental trajectories utilize distinct molecular mechanisms upstream of major developmental regulatory genes such as fox, otx, and Hox-like.     

The wings ofBombyx mori develop from larval discs exhibiting an early differentiated state: a preliminary report

Lepidopteran insects present a complex organization of appendages which develop by various mechanisms. In the mulberry silkworm,Bombyx mori a pair of meso- and meta-thoracic discs located on either side in the larvae gives rise to the corresponding fore- and hind-wings of the adult. These discs do not experience massive cell rearrangements during metamorphosis and display the adult wing vein pattern. We have analysed wing development inB. mori by two approaches, viz., expression of patterning genes in larval wing discs, and regulatory capacities of larval discs following explantation or perturbation. Expression of Nubbin is seen all over the presumptive wing blade domains unlike inDrosophila, where it is confined to the hinge and the wing pouch. Excision of meso- and meta-thoracic discs during the larval stages resulted in emergence of adult moths lacking the corresponding wings without any loss of thoracic tissues suggesting independent origin of wing and thoracic primordia. The expression of wingless and distal-less along the dorsal/ventral margin in wing discs correlated well with their expression profile in adultDrosophila wings. Partially excised wing discs did not showin situ regeneration or duplication suggesting their early differentiation. The presence of adult wing vein patterns discernible in larval wing discs and the patterns of marker gene expression as well as the inability of these discs to regulate growth suggested that wing differentiation is achieved early inB. mori. The timings of morphogenetic events are different and the wing discs behave like presumptive wing buds opening out as wing blades inB. mori unlike evagination of only the pouch region as wing blades seen inDrosophila.     

The effect of manipulating ecdysteroid signaling on embryonic eye development in the locust<Emphasis Type="Italic"> Schistocerca americana</Emphasis>

Adult body plan differentiation in holometabolous insects depends on global induction and control by ecdysteroid hormones during the final phase of postembryogenesis. Studies in Drosophila melanogaster and Manduca sexta have shown that this pertains also to the development of the compound eye retina. It is unclear whether the hormonal control of postembryonic eye development in holometabolous insects represents evolutionary novelty or heritage from hemimetabolous insects, which develop compound eyes during embryogenesis. We therefore investigated the effect of manipulating ecdysteroid signaling in cultured embryonic eye primordia of the American desert locust Schistocerca americana, in which ecdysteroid level changes are known to induce three rounds of embryonic molt. Although at a considerably reduced rate compared to in vivo development, early differentiation and terminal maturation of the embryonic retina was observed in culture even if challenged with the ecdysteroid antagonist cucurbitacin B. Supplementing cultures with 20-hydroxyecdysone (20E) accelerated differentiation and maturation, and enhanced cell proliferation. Considering these results, and the relation between retina differentiation and ecdysteroid level changes during locust embryogenesis, we conclude that ecdysteroids are not an essential but possibly a modulatory component of embryonic retina development in S. americana. We furthermore found evidence that 20E initiated precocious epithelial morphogenesis of the posterior retinal margin indicating a more general role of ecdysteroids in insect embryogenesis.Electronic Supplementary Material Supplementary material is available in the online version of this article at Edited by C. Desplan     

A<Emphasis Type="Italic"> Dickkopf</Emphasis>-<Emphasis Type="Italic">3</Emphasis>-related gene is expressed in differentiating nematocytes in the basal metazoan<Emphasis Type="Italic"> Hydra</Emphasis>

In vertebrate development the Dickkopf protein family carries out multiple functions and is represented by at least four different genes with distinct biological activities. In invertebrates such as Drosophila and Caenorhabditis, Dickkopf genes have so far not been identified. Here we describe the identification and characterization of a Dickkopf gene with a deduced amino acid sequence closely related to that of chicken Dkk-3 in the basal metazoan Hydra. HyDkk-3 appears to be the only Dickkopf gene in Hydra. The gene is expressed in the gastric region in nematocytes at a late differentiation stage. In silico searches of EST and genome databases indicated the absence of Dkk genes from the protostomes Drosophila and Caenorhabditis, whereas within the deuterostomes, a Dkk-3 gene could be identified in the genome of the urochordate Ciona intestinalis. The results indicate that at an early stage of evolution of multicellularity Dickkopf proteins have already played important roles as developmental signals. They also suggest that vertebrate Dkk-1, 2 and 4 may have originated from a common ancestor gene of Dkk-3.H. Fedders and R. Augustin contributed equally to this workEdited by D. Tautz     

Expression of photosynthesis-related genes and their regulation by light during somatic embryogenesis in<Emphasis Type="Italic"> Daucus carota</Emphasis>

To clarify the spatial and temporal pattern of gene expression for photosynthesis-associated proteins during somatic embryogenesis in Daucus carota L., the localization of mRNAs for three genes, rbcL, Lhcb and por, was examined in dark-grown and light-irradiated somatic embryos by in situ hybridization. The three mRNAs were expressed in common in the mesophyll precursor cells of light-irradiated embryos at the late torpedo and plantlet stages, but characteristic expression patterns of each photosynthesis-related gene were also observed. Expression of rbcL mRNA first occurred throughout the embryo but gradually became localized in the mesophyll precursor cells and cortex during early embryogenesis. Localization of Lhcb mRNA in the mesophyll precursor cells and shoot apical meristem became clear in the early torpedo stage. Expression of Lhcb mRNA was not affected by light during early embryogenesis, but could be induced by light in the torpedo stage, suggesting that light-inducible expression of Lhcb mRNA arises within the torpedo stage. At the late torpedo stage, clear localization of por mRNA started in mesophyll precursor cells of the cotyledon in light-irradiated embryos. Greening potency of the embryo also appeared first at this stage. Therefore, greening and initial differentiation of photosynthetic tissues during somatic embryogenesis seem to be associated with coordinated expression of mRNA for rbcL, Lhcb and por in late torpedo-shaped embryos.Abbreviations DIG Digoxigenin - Lhcb3 Gene encoding a type-III light-harvesting chlorophyll a/b-binding protein of photosystem II - LHCII Light-harvesting chlorophyll a/b-binding protein of photosystem II - POR Protochlorophyllide oxidoreductase - rbcL Gene encoding the large subunit of Rubisco - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase     

MOLECULAR MECHANISMS IN DEVELOPMENTAL BIOLOGY

Some general molecular mechanisms underlying development are described. Namely: those involved in the differentiation of the R7 receptor inDrosophilaembryonic retina; those involved in the determination of embryonic axes and in polar cell differentiation, inDrosophila; those involved in the determination of the AB and P cell lineage and in vulva differentiation inCaenorhabditisembryos.     

Sex determination in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> and<Emphasis Type="Italic"> Musca domestica</Emphasis> converges at the level of the terminal regulator<Emphasis Type="Italic"> doublesex</Emphasis>

Sex-determining cascades are supposed to have evolved in a retrograde manner from bottom to top. Wilkins 1995 hypothesis finds support from our comparative studies in Drosophila melanogaster and Musca domestica, two dipteran species that separated some 120 million years ago. The sex-determining cascades in these flies differ at the level of the primary sex-determining signal and their targets, Sxl in Drosophila and F in Musca. Here we present evidence that they converge at the level of the terminal regulator, doublesex (dsx), which conveys the selected sexual fate to the differentiation genes. The dsx homologue in Musca, Md-dsx, encodes male-specific (MdDSX^M) and female-specific (MdDSX^F) protein variants which correspond in structure to those in Drosophila. Sex-specific regulation of Md-dsx is controlled by the switch gene F via a splicing mechanism that is similar but in some relevant aspects different from that in Drosophila. MdDSX^F expression can activate the vitellogenin genes in Drosophila and Musca males, and MdDSX^M expression in Drosophila females can cause male-like pigmentation of posterior tergites, suggesting that these Musca dsx variants are conserved not only in structure but also in function. Furthermore, downregulation of Md-dsx activity in Musca by injecting dsRNA into embryos leads to intersexual differentiation of the gonads. These results strongly support a role of Md-dsx as the final regulatory gene in the sex-determining hierarchy of the housefly.Edited by D. Tautz     

Genes controlling posterior gut development in theDrosophila embryo

Our present detailed understanding of the genetic mechanisms controlling segmentation has been made possible, in large part, by comprehensive screens of cuticular morphology that identified genes involved in epidermal patterning. To systematically identify genes involved in internal morphogenesis, specifically development of the gut, we have screened mutant embryos produced by a collection of 53 embryonic lethal mutations affecting embryonic pattern formation or differentiation, and a collection of 161 deficiencies covering, in aggregate, approximately 70% of the genome. Staining with the anti-crumbs antibody was used to characterize the Malpighian tubules and hindgut, as well as other internal organs. The geneshuckebein, tailless andwingless, and two previously undescribed loci at 24C/D and 68D/E, are required to establish the primordia for the posterior midgut and hindgut/Malpighian tubules. A locus in region 30A/C is required for extension of the midgut epithelium to surround the yolk, and region 36E/37F is required for outbudding of the Malpighian tubule primordia. Several deficiencies were identified that uncover loci with specific effects on the morphogenesis (elongation, lumen formation) of the hindgut and Malpighian tubules and on the formation of constrictions in the midgut.     

<Emphasis Type="Italic">Rab11</Emphasis> is required for embryonic nervous system development in <Emphasis Type="Italic">Drosophila</Emphasis>

In eukaryotes, membrane trafficking is regulated by the small monomeric GTPases of Rab protein family. Rab11, an evolutionary conserved, ubiquitously expressed subfamily of the Rab GTPases, has been implicated in the regulation of vesicular trafficking through the recycling of endosomes. To dissect out the role of this protein during embryonic nervous system development, we have studied the expression pattern of Rab11 in the ventral nerve cord during neuronal differentiation in the Drosophila embryo. When the dominant-negative or constitutively-active mutant DRab11 proteins are expressed in neurons, or when homozygous mutant Rab11 embryos are analyzed, defects are found in the developing central nervous system, along with disorganization and misrouting of embryonic axons. Our results provide the first in vivo evidence that Rab11 is involved in the development of the nervous system during Drosophila embryogenesis. This work was supported by the DST (to J.K.R.) and SRF from ICMR, New Delhi (to T.B.).     

Hox genes: Downstream “effectors” of retinoic acid signaling in vertebrate embryogenesis

One of the major regulatory challenges of animal development is to precisely coordinate in space and time the formation, specification, and patterning of cells that underlie elaboration of the basic body plan. How does the vertebrate plan for the nervous and hematopoietic systems, heart, limbs, digestive, and reproductive organs derive from seemingly similar population of cells? These systems are initially established and patterned along the anteroposterior axis (AP) by opposing signaling gradients that lead to the activation of gene regulatory networks involved in axial specification, including the Hox genes. The retinoid signaling pathway is one of the key signaling gradients coupled to the establishment of axial patterning. The nested domains of Hox gene expression, which provide a combinatorial code for axial patterning, arise in part through a differential response to retinoic acid (RA) diffusing from anabolic centers established within the embryo during development. Hence, Hox genes are important direct effectors of retinoid signaling in embryogenesis. This review focuses on describing current knowledge on the complex mechanisms and regulatory processes, which govern the response of Hox genes to RA in several tissue contexts including the nervous system during vertebrate development.