Development Genes and Evolution

Miyazaki, Yuriko; Nishida, Hiroki; Kumano, Gaku

doi: 10.1007/s00427-006-0129-9pmid: 17216525

Coordinated regulation of inductive events, both spatially and temporally, during animal development ensures that tissues are induced at their specific positions within the embryo. The ascidian brain is induced in cells at the anterior edge of the animal hemisphere by fibroblast growth factor (FGF) signals secreted from vegetal cells. To clarify how this process is spatially regulated, we first identified the sources of the FGF signal by examining the expression of brain markers Hr-Otx and Hr-ETR-1 in embryos in which FGF signaling is locally inhibited by injecting individual blastomeres with morpholino oligonucleotide against Hr-FGF9/16/20, which encodes an endogenous brain inducer. The blastomeres identified as the inducing sources are A5.1 and A5.2 at the 16-cell stage and A6.2 and A6.4 at the 24-cell stage, which are juxtaposed with brain precursors at the anterior periphery of the embryo at the respective stages. We also showed that all the cells of the animal hemisphere are capable of expressing Hr-Otx in response to the FGF signal. These results suggest that the position of inducers, rather than competence, plays an important role in determining which animal cells are induced to become brain tissues during ascidian embryogenesis. This situation in brain induction contrasts with that in mesoderm induction, where the positions at which the notochord and mesenchyme are induced are determined mainly by intrinsic competence factors that are inherited by signal-receiving cells.

Maleszka, J.; Forêt, S.; Saint, R.; Maleszka, R.

doi: 10.1007/s00427-006-0127-ypmid: 17216269

Small chemosensory proteins (CSPs) belong to a conserved, but poorly understood, protein family found in insects and other arthropods. They exhibit both broad and restricted expression patterns during development. In this paper, we used a combination of genome annotation, transcriptional profiling and RNA interference to unravel the functional significance of a honeybee gene (csp5) belonging to the CSP family. We show that csp5 expression resembles the maternal-zygotic pattern that is characterized by the initiation of transcription in the ovary and the replacement of maternal mRNA with embryonic mRNA. Blocking the embryonic expression of csp5 with double-stranded RNA causes abnormalities in all body parts where csp5 is highly expressed. The treated embryos show a “diffuse”, often grotesque morphology, and the head skeleton appears to be severely affected. They are ‘unable-to-hatch’ and cannot progress to the larval stages. Our findings reveal a novel, essential role for this gene family and suggest that csp5 (unable-to-hatch) is an ectodermal gene involved in embryonic integument formation. Our study confirms the utility of an RNAi approach to functional characterization of novel developmental genes uncovered by the honeybee genome project and provides a starting point for further studies on embryonic integument formation in this insect.

Krattinger, Anne; Gendre, Nanaë; Ramaekers, Ariane; Grillenzoni, Nicola; Stocker, Reinhard F.

doi: 10.1007/s00427-007-0134-7pmid: 17323106

In this paper, we study DmOAZ, the unique Drosophila melanogaster homologue of the OAZ zinc finger protein family. We show partial conservation of the zinc finger organization between DmOAZ and the vertebrate members of this family. We determine the exon/intron structure of the dmOAZ gene and deduce its open reading frame. Reverse transcriptase-polymerase chain reaction analysis shows that dmOAZ is transcribed throughout life. In the embryo, strongest DmOAZ expression is observed in the posterior spiracles. We suggest that dmOAZ acts as a secondary target of the Abd-B gene in posterior spiracle development, downstream of cut and ems. In a newly created loss-of-function mutant, dmOAZ93, the “filzkörper” part of the posterior spiracles, is indeed structurally abnormal. The dmOAZ93 mutant is a larval lethal, a phenotype that may be linked to the spiracular defect. Given the dmOAZ93 mutant as a new tool, the fruit fly may provide an alternative model for analyzing in vivo the functions of OAZ family members.

Grillenzoni, Nicola; de Vaux, Véronique; Meuwly, Jocelyne; Vuichard, Séverine; Jarman, Andrew; Holohan, Eimear; Gendre, Nanaë; Stocker, Reinhard F.

doi: 10.1007/s00427-007-0135-6pmid: 17260155

In this paper, we address the role of proneural genes in the formation of the dorsal organ in the Drosophila larva. This organ is an intricate compound comprising the multineuronal dome—the exclusive larval olfactory organ—and a number of mostly gustatory sensilla. We first determine the numbers of neurons and of the different types of accessory cells in the dorsal organ. From these data, we conclude that the dorsal organ derives from 14 sensory organ precursor cells. Seven of them appear to give rise to the dome, which therefore may be composed of seven fused sensilla, whereas the other precursors produce the remaining sensilla of the dorsal organ. By a loss-of-function approach, we then analyze the role of atonal, amos, and the achaete-scute complex (AS-C), which in the adult are the exclusive proneural genes required for chemosensory organ specification. We show that atonal and amos are necessary and sufficient in a complementary way for four and three of the sensory organ precursors of the dome, respectively. AS-C, on the other hand, is implicated in specifying the non-olfactory sensilla, partially in cooperation with atonal and/or amos. Similar links for these proneural genes with olfactory and gustatory function have been established in the adult fly. However, such conserved gene function is not trivial, given that adult and larval chemosensory organs are anatomically very different and that the development of adult olfactory sensilla involves cell recruitment, which is unlikely to play a role in dome formation.

Jud, Molly; Razelun, Jamie; Bickel, Jeremy; Czerwinski, Mike; Schisa, Jennifer A.

doi: 10.1007/s00427-006-0130-3pmid: 17216268

Within the rhabditid phylogeny of nematodes, the great majority of species are gonochoristic, having evolved as obligate male/female species. In contrast, the well-studied nematode model system, Caenorhabditis elegans, is androdioecious, utilizing a hermaphroditic/male reproductive system. We have previously determined that in the arrested oocytes of old-aged C. elegans hermaphrodites with depleted sperm, large cytoplasmic ribonucleoprotein foci form. The formation of these foci is reversible, as they dissociate within 3 h after a male mates with the hermaphrodite, resupplying it with sperm. The functional significance of these oocyte foci is not known and previously has not been clear for a hermaphroditic species in which oocytes of young adults wait only approximately 23 min to be fertilized. One hypothesis is that the foci function to maintain maternal mRNAs in oocytes while fertilization is delayed. In this paper, we examine four gonochoristic rhabditid species: Caenorhabditis remanei, Caenorhabditis sp. CB5161, Caenorhabditis sp. PS1010, and Rhabditella axei DF5006. We demonstrate that in three of these four species, ovulation arrests in unmated females until mating occurs and large cytoplasmic foci develop in arrested oocytes. The oocyte foci contain nuclear pore proteins and, in C. remanei at least, the RNA-binding protein MEX-3 as well as RNA. We speculate that these foci maintain the integrity of ooctyes, possibly maintaining the stability or translational repression of maternal mRNAs in unmated females. We further speculate that their presence in oocytes of old-aged C. elegans hermaphrodites is due to conservation from an ancestral gonochoristic state.

Bredenkamp, Nicholas; Seoighe, Cathal; Illing, Nicola

doi: 10.1007/s00427-006-0128-xpmid: 17260156

Comparative analysis of orthologues from diverse vertebrates can be used to identify molecular signatures that are important for gene function and which may predict novel regulatory mechanisms or explain morphological diversity. The forkhead box G1 (FoxG1) transcription factor is potentially a strong candidate gene for determining forebrain size in vertebrates due to its role in the development of the telencephalon, where it promotes progenitor proliferation and suppresses premature neurogenesis. To investigate the role of FoxG1 in forebrain evolution, we cloned and analyzed the cDNA sequences for nine new FoxG1 orthologues, including six mammals and three reptiles, and show that there is an extended proline and glutamine region in the N-terminal domain that is specific to mammals. In contrast to some previous studies of other potential determinants of brain size, we find no evidence that the coding sequence of FoxG1 has evolved under positive selection in vertebrates. Previously published work has indicated that FOXG1 was duplicated in humans, and two forms, FOXG1A and FOXG1B, are present in the Entrez Gene database. We report that FOXG1 has not been duplicated in humans and that FOXG1A is likely to be an artifact. Our comparative analysis of FOXG1B and its orthologues has revealed a very high level of conservation in the 3′ untranslated region (UTR). Using available computational tools, we find evidence for conserved recognition sites for the miR-9 and miR-33 microRNAs in the FoxG1 3′ UTR and hypothesize that these brain-expressed microRNAs may regulate FoxG1 post-transcriptionally during forebrain development.

Sullivan, James C.; Ryan, Joseph F.; Mullikin, James C.; Finnerty, John R.

doi: 10.1007/s00427-007-0136-5pmid: 17310352

Evolutionarily conserved gene clusters are interesting for two reasons: (1) they may illuminate ancient events in genome evolution and (2) they may reveal ongoing stabilizing selection; that is, the conservation of gene clusters may have functional significance. To test if the Wnt family of signaling factors exhibits conserved clustering in basal metazoans and if those clusters are of functional importance, we searched the genomic sequence of the sea anemone Nematostella vectensis for Wnt clusters and correlated the clustering we observed with published expression patterns. Our results indicate that the Wnt1–Wnt6–Wnt10 cluster observed in Drosophila melanogaster is partially conserved in the cnidarian lineage; Wnt6 and Wnt10 are separated by less than 4,500 nucleotides in Nematostella. A novel cluster comprised of Wnt5–Wnt7/Wnt7b was observed in Nematostella. Clustered Wnt genes do not exhibit Hox-like colinearity nor is the expression of linked Wnt genes more similar than the expression of nonlinked Wnt genes. Wnt6 and Wnt10 are not expressed in a spatially or temporally contiguous manner, and Wnt5 and Wnt7 are expressed in different germ layers.

Khila, Abderrahman; Grbić, Miodrag

doi: 10.1007/s00427-007-0132-9pmid: 17262226

A major prerequisite to understanding the evolution of developmental programs includes an appreciation of gene function in a comparative context. RNA interference (RNAi) represents a powerful method for reverse genetics analysis of gene function. However, RNAi protocols exist for only a handful of arthropod species. To extend functional analysis in basal arthropods, we developed a RNAi protocol for the two-spotted spider mite Tetranychus urticae focusing on Distal-less (Dll), a conserved gene involved in appendage specification in metazoans. First, we describe limb morphogenesis in T. urticae using confocal and scanning electron microscopy. Second, we examine T. urticae Dll (Tu-Dll) mRNA expression patterns and correlate its expression with appendage development. We then show that fluorescently labeled double-stranded RNA (dsRNA) and short interfering RNA (siRNA) molecules injected into the abdomen of adult females are incorporated into the oviposited eggs, suggesting that dsRNA reagents can be systemically distributed in spider mites. Injection of longer dsRNA as well as siRNA induced canonical limb truncation phenotypes as well as the fusion of leg segments. Our data suggest that Dll plays a conserved role in appendage formation in arthropods and that such conserved genes can serve as reliable starting points for the development of functional protocols in nonmodel organisms.