Invertebrate Orthologs Research Articles

Identification and analysis of cabut orthologs in invertebrates and vertebrates

Cabut (cbt) is a Drosophila melanogaster gene involved in epidermal dorsal closure (DC). Its expression is dependent on the Jun N-terminal kinase (JNK) cascade, and it functions downstream of Jun regulating dpp expression in the leading edge cells. The Cbt protein contains three C(2)H(2)-type zinc fingers and a serine-rich domain, suggesting that it functions as a transcription factor. We have identified single cbt orthologs in other Drosophila species, as well as in other insects and invertebrate organisms like ascidians and echinoderms, but not in nematodes. Gene structure and protein sequence are highly conserved among Drosophilidae, but are more diverged in the other species of invertebrates analyzed. According to this, we demonstrate that cbt expression is detected in the embryonic lateral epidermis in several Drosophila species, as it occurs in D. melanogaster, thus suggesting that the cbt orthologs may have a conserved role in these species during DC. We have also analyzed the genomes of several vertebrate species, finding that the cbt orthologous genes in these organisms encode proteins that belong to the TIEG family of Sp1-like/Krüppel-like transcription factors. Phylogenetic analysis of the invertebrate and vertebrate proteins identified indicates that they mainly follow the expected phylogeny of the species, and that the cbt gene was duplicated during vertebrate evolution. Because we were not able to identify cbt orthologous genes neither in yeast nor in plants, our results suggest that this gene has been probably conserved throughout metazoans and that it may play a fundamental role in animal biology.

Developmental roles of the BMP1/TLD metalloproteinases

The astacin family (M12A) of the metzincin subclan MA(M) of metalloproteinases has been detected in developing and mature individuals of species that range from hydra to humans. Functions of this family of metalloproteinase vary from digestive degradation of polypeptides, to biosynthetic processing of extracellular proteins, to activation of growth factors. This review will focus on a small subgroup of the astacin family; the bone morphogenetic protein 1 (BMP1)/Tolloid (TLD)-like metalloproteinases. In vertebrates, the BMP1/TLD-like metalloproteinases play key roles in regulating formation of the extracellular matrix (ECM) via biosynthetic processing of various precursor proteins into mature functional enzymes, structural proteins, and proteins involved in initiating mineralization of the ECM of hard tissues. Roles in ECM formation include: processing of the C-propeptides of procollagens types I-III, to yield the major fibrous components of vertebrate ECM; proteolytic activation of the enzyme lysyl oxidase, necessary to formation of covalent cross-links in collagen and elastic fibers; processing of NH2-terminal globular domains and C-propeptides of types V and XI procollagen chains to yield monomers that are incorporated into and control the diameters of collagen type I and II fibrils, respectively; processing of precursors for laminin 5 and collagen type VII, both of which are involved in securing epidermis to underlying dermis; and maturation of small leucine-rich proteoglycans. The BMP1/TLD-related metalloproteinases are also capable of activating the vertebrate transforming growth factor-beta (TGF-beta)-like "chalones" growth differentiation factor 8 (GDF8, also known as myostatin), and GDF11 (also known as BMP11), involved in negative feedback inhibition of muscle and neural tissue growth, respectively; by freeing them from noncovalent latent complexes with their cleaved prodomains. BMP1/TLD-like proteinases also liberate the vertebrate TGF-beta-like morphogens BMP2 and 4 and their invertebrate ortholog decapentaplegic, from latent complexes with the vertebrate extracellular antagonist chordin and its invertebrate ortholog short gastrulation (SOG), respectively. The result is formation of the BMP signaling gradients that form the dorsal-ventral axis in embryogenesis. Thus, BMP1/TLD-like proteinases appear to be key to regulating and orchestrating formation of the ECM and signaling by various TGF-beta-like proteins in morphogenetic and homeostatic events.

Recycling to the Plasma Membrane is Delayed in EHD1 Knockout Mice

EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N-terminal domain that contains nucleotide-binding motifs, a central coiled-coil domain involved in oligomerization and a C-terminal region that harbors the EH domain. Studies in C. elegans and EHD1 depletion by RNA interference in human cells have demonstrated that it regulates recycling of membrane proteins. We addressed the physiological role of EHD1 through its inactivation in the mouse. Ehd1 knockout mice were indistinguishable from normal mice, had a normal life span and showed no histological abnormalities. Analysis of transferrin uptake in Ehd1(-/-) embryonic fibroblasts demonstrated delayed recycling to the plasma membrane with accumulation of transferrin in the endocytic recycling compartment. Our results corroborate the established role of EHD1 in the exit of membrane proteins from recycling endosomes in vivo in a mouse model.

An Essential Function of the C. elegans Ortholog of TPX2 Is to Localize Activated Aurora A Kinase to Mitotic Spindles

In vertebrates, the microtubule binding protein TPX2 is required for meiotic and mitotic spindle assembly. TPX2 is also known to bind to and activate Aurora A kinase and target it to the spindle. However, the relationship between the TPX2-Aurora A interaction and the role of TPX2 in spindle assembly is unclear. Here, we identify TPXL-1, a C. elegans protein that is the first characterized invertebrate ortholog of TPX2. We demonstrate that an essential role of TPXL-1 during mitosis is to activate and target Aurora A to microtubules. Our data suggest that this targeting stabilizes microtubules connecting kinetochores to the spindle poles. Thus, activation and targeting of Aurora A appears to be an ancient and conserved function of TPX2 that plays a central role in mitotic spindle assembly.

Hair-Trigger Responses

Underlying the sense of hearing and balance are hair cells that transduce the mechanical deflection of their hair bundles into electrical signals through mechanosensitive ion channels, which open and trigger an inward transduction current. Sidi et al. have identified a transient receptor potential cation channel called NompC in zebrafish sensory hair cells as a core component of this mechanosensory machinery. Mutants lacking functional NompC no longer responded to acoustic stimuli. Invertebrate orthologs of NompC were previously implicated in hearing, but a vertebrate mechanosensor has remained elusive. S. Sidi, R. W. Friedrich, T. Nicolson, NompC TRP channel required for vertebrate sensory hair cell mechanotransduction. Science 301 , 96-99 (2003). [Abstract] [Full Text]

Structural gene organization and evolutionary aspects of the V-ATPase accessory subunit Ac45

The vacuolar H +-ATPase (V-ATPase) is a multisubunit enzyme that couples ATP hydrolysis to proton pumping across membranes. The intracellular targeting and activity of the V-ATPase may be regulated via proteins that interact with the pump such as the accessory subunit Ac45. Here we report the isolation and characterization of the gene encoding Ac45. This single-copy gene is located in a gene-dense region of chromosome Xq and consists of 10 exons spanning ∼8 kb in the mouse and human genomes. The gene structure is poorly conserved in that its invertebrate orthologs of Caenorhabditis elegans and Drosophila melanogaster encompass only six and four exons extending over 4.1 and 2.1 kb, respectively. Furthermore, the overall degree of amino acid sequence identity between the mammalian and invertebrate Ac45 proteins is very low (<18%), except for a surprisingly highly conserved putative targeting motif in the carboxy-terminal region. Primer extension analysis revealed that the mouse Ac45 gene contains two major transcription initiation sites. The start sites are not preceded by a clear CAAT-box and are located in a CpG island. The most downstream start site contains a TATA-box and transcriptional regulatory elements such as PEA-3, F2F, Maz and Sp1. The limited number of regulatory DNA elements common in the genes encoding Ac45 and V-ATPase subunits suggests a differential regulation of these genes. Together with the finding that Ac45 appears to occur only in multicellular organisms, these results indicate that this accessory subunit directs the V-ATPase to specialized and complex vacuolar systems.

Cloning and Characterization of Hepp, a Novel Gene Expressed Preferentially in Hematopoietic Progenitors and Mature Blood Cells

ABSTRACTThrough differential screening of mouse hematopoietic stem cell (HSC) and progenitor-subtracted cDNA libraries we have identified a progenitor cell-specific transcript that represents a novel gene, named Hepp (hematopoietic progenitor protein). The mouse Hepp gene encodes a protein of 237 amino acids with no detectable known functional domains or motifs. Lack of invertebrate orthologs and a high degree of evolutionary conservation of the peptide sequence in vertebrate species (zebrafish, mouse, human) suggest that the Hepp gene could have conserved although as yet unknown function in vertebrates. Mouse Hepp shows a restricted expression pattern in adult tissues and is transcribed at a very low level in heart, lung, spleen, and thymus and at a higher level in muscle. During embryonic hematopoiesis Hepp is not expressed in mouse fetal liver HSC (Sca-1+c-kit+AA4.1+Lin− cells), but is abundantly transcribed in the population of hematopoietic progenitors (AA4.1− cells). Similarly, during adult hematopoiesis Hepp is not transcribed in the highly enriched population of bone marrow HSC (Rh-123lowSca-1+c-kit+Lin− cells), but its expression is upregulated as a greater heterogeneous population of bone marrow HSC (Lin−Sca-1+ cells) differentiates into progenitors (Lin−Sca-1− cells) and more mature lymphoid and myeloid cell types. A restricted pattern of expression in adult tissues and preferential expression in both fetal and adult hematopoietic progenitors and mature blood cells suggest that Hepp could be involved in molecular regulation of HSC and progenitor cell lineage commitment and differentiation.

Dorsoventral axis inversion: Enteropneust anatomy links invertebrates to chordates turned upside down

The relationships between chordates with their dorsal nerve cord and other animal groups remain unclear. The hemichordata, specifically the enteropneusta (acorn worms), have been considered a sister group to the chordata. Enteropneusts combine various chordate features (e.g. lateral gill openings, dorsal nerve cord) with features that are usually associated with gastroneuralian invertebrates (e.g. dorsal heart, circumenteric nerve ring, ventral nerve cord). Here we analyse various morphological and functional characteristics that enteropneusts share with either invertebrates or chordates in the light of our recent proposal that the chordata may derive – by bodily dorsoventral inversion – from a gastroneuralian ancestor. We show that many seemingly non-chordate features of enteropneusts will align with similar features in the chordates – provided that we compare the ventral side of an enteropneust to the dorsal side of a chordate. This inversion proposes several interesting and new putative homologies between enteropneusts and acranian chordates, such as between their epibranchial ridge/endostyle (later thyroid gland), their postanal tails, atrial walls, and also between the chordates’ dorsal notochord and the enteropneusts’ posteroventral pygochord. Significantly, positional homology between notochord and pygochord is also supported by the expression domains of Brachyury orthologs in vertebrates and invertebrates: a Brachyury ortholog is active in the posteroventral mesoderm in Drosophila and in the dorsal mesoderm in chordates. In conclusion, we propose that the anatomy of enteropneusts may serve as a conceptual ‘missing link’ between gastroneuralian invertebrates and notoneuralian chordates. We discuss whether the enteropneust’s dorsoanterior nervous centre plus their ventral trunk cord then corresponds to brain and dorsal nerve cord in the chordata.

C-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor.

Ligand-induced down-regulation of two growth factor receptors, EGF receptor (ErbB-1) and ErbB-3, correlates with differential ability to recruit c-Cbl, whose invertebrate orthologs are negative regulators of ErbB. We report that ligand-induced degradation of internalized ErbB-1, but not ErbB-3, is mediated by transient mobilization of a minor fraction of c-Cbl into ErbB-1-containing endosomes. This recruitment depends on the receptor's tyrosine kinase activity and an intact carboxy-terminal region. The alternative fate is recycling of internalized ErbBs to the cell surface. Cbl-mediated receptor sorting involves covalent attachment of ubiquitin molecules, and subsequent lysosomal and proteasomal degradation. The oncogenic viral form of Cbl inhibits down-regulation by shunting endocytosed receptors to the recycling pathway. These results reveal an endosomal sorting machinery capable of controlling the fate, and, hence, signaling potency, of growth factor receptors.

SVOP, an Evolutionarily Conserved Synaptic Vesicle Protein, Suggests Novel Transport Functions of Synaptic Vesicles

We describe a novel synaptic vesicle protein called SVOP that is distantly related to the synaptic vesicle proteins SV2A, SV2B, and SV2C (20-22% sequence identity). Both SVOP and SV2 contain 12 transmembrane regions. However, SV2 is highly glycosylated, whereas SVOP is not. Databank searches revealed that closely related homologs of SVOP are present in Caenorhabditis elegans and Drosophila (48% sequence identity), suggesting that SVOP is evolutionarily ancient. In contrast, no invertebrate orthologs of SV2 were detected. The sequences of SVOP and SV2 exhibit homology with transport proteins, in particular with mammalian organic cation and anion transporters. SVOP and SV2 are more distantly related to eukaryotic and bacterial phosphate, sugar, and organic acid transporters. SVOP is expressed at detectable levels only in brain and endocrine cells where it is primarily localized to synaptic vesicles and microvesicles. SVOP is present in all brain regions, with particularly high levels in large pyramidal neurons of the cerebral cortex. Immunocytochemical staining of adjacent rat brain sections for SVOP and SV2 demonstrated that SVOP and SV2 are probably coexpressed in most neurons. Although the functions of SV2 and SVOP remain obscure, the evolutionary conservation of SVOP, its hydrophobic nature, and its homology to transporters strongly support a role in the uptake of a novel, as yet unidentified component of synaptic vesicles. Thus synaptic vesicles contain two classes of abundant proteins with 12 transmembrane regions that are related to transporters, nonglycosylated SVOP and highly glycosylated SV2, suggesting that the transport functions of synaptic vesicles may be more complex than currently envisioned.