15-zinc finger protein Bloody Fingers is required for zebrafish morphogenetic movements during neurulation

A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C(2)H(2) type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.     

Absence of Ca2+-induced mitochondrial permeability transition but presence of bongkrekate-sensitive nucleotide exchange in C. crangon and P. serratus

Mitochondria from the embryos of brine shrimp (Artemia franciscana) do not undergo Ca(2+)-induced permeability transition in the presence of a profound Ca(2+) uptake capacity. Furthermore, this crustacean is the only organism known to exhibit bongkrekate-insensitive mitochondrial adenine nucleotide exchange, prompting the conjecture that refractoriness to bongkrekate and absence of Ca(2+)-induced permeability transition are somehow related phenomena. Here we report that mitochondria isolated from two other crustaceans, brown shrimp (Crangon crangon) and common prawn (Palaemon serratus) exhibited bongkrekate-sensitive mitochondrial adenine nucleotide transport, but lacked a Ca(2+)-induced permeability transition. Ca(2+) uptake capacity was robust in the absence of adenine nucleotides in both crustaceans, unaffected by either bongkrekate or cyclosporin A. Transmission electron microscopy images of Ca(2+)-loaded mitochondria showed needle-like formations of electron-dense material strikingly similar to those observed in mitochondria from the hepatopancreas of blue crab (Callinectes sapidus) and the embryos of Artemia franciscana. Alignment analysis of the partial coding sequences of the adenine nucleotide translocase (ANT) expressed in Crangon crangon and Palaemon serratus versus the complete sequence expressed in Artemia franciscana reappraised the possibility of the 208-214 amino acid region for conferring sensitivity to bongkrekate. However, our findings suggest that the ability to undergo Ca(2+)-induced mitochondrial permeability transition and the sensitivity of adenine nucleotide translocase to bongkrekate are not necessarily related phenomena.     

A distinct sequence in the adenine nucleotide translocase from Artemia franciscana embryos is associated with insensitivity to bongkrekate and atypical effects of adenine nucleotides on Ca2+ uptake and sequestration

Mitochondria isolated from embryos of the crustacean Artemia franciscana lack the Ca(2+)-induced permeability transition pore. Although the composition of the pore described in mammalian mitochondria is unknown, the impacts of several effectors of the adenine nucleotide translocase (ANT) on pore opening are firmly established. Notably, ADP, ATP and bongkrekate delay, whereas carboxyatractyloside hastens, Ca(2+)-induced pore opening. Here, we report that adenine nucleotides decreased, whereas carboxyatractyloside increased, Ca(2+) uptake capacity in mitochondria isolated from Artemia embryos. Bongkrekate had no effect on either Ca(2+) uptake or ADP-ATP exchange rate. Transmission electron microscopy imaging of Ca(2+)-loaded Artemia mitochondria showed needle-like formations of electron-dense material in the absence of adenine nucleotides, and dot-like formations in the presence of adenine nucleotides or Mg(2+). Energy-filtered transmission electron microscopy showed the material to be rich in calcium and phosphorus. Sequencing of the Artemia mRNA coding for ANT revealed that it transcribes a protein with a stretch of amino acids in the 198-225 region with 48-56% similarity to those from other species, including the deletion of three amino acids in positions 211, 212 and 219. Mitochondria isolated from the liver of Xenopus laevis, in which the ANT shows similarity to that in Artemia except for the 198-225 amino acid region, demonstrated a Ca(2+)-induced bongkrekate-sensitive permeability transition pore, allowing the suggestion that this region of ANT may contain the binding site for bongkrekate.     

Xenopus frizzled 4 is a maternal mRNA and its zygotic expression is localized to the neuroectoderm and trunk lateral plate mesoderm

We describe the identification and expression pattern of Xenopus frizzled 4 (Xfz4) gene during early development. Xfz4 protein presents characteristic features of a frizzled family member. The mature protein sequence of Xfz4 is 93% identical to murine Mfz4. Xfz4 is a maternal mRNA, its expression level remains constant during early development. The mRNA is first localized during gastrulation to the dorsal presumptive neuroectoderm. At the end of gastrulation, Xfz4 mRNA is detected in the dorso-anterior neuroectoderm. During neurulation, Xfz4 mRNA is expressed as a band on both side of the forebrain, and in the trunk lateral plate mesoderm. As development proceeds, expression of Xfz4 mRNA in the trunk lateral plate mesoderm decreases but persists in the forebrain. It is also expressed in the posterior unsegmented somitic mesoderm from late tail-bud stage onward.     

An amphioxus snail gene: Expression in paraxial mesoderm and neural plate suggests a conserved role in patterning the chordate embryo

 Homologs of the Drosophila snail gene have been characterized in several vertebrates. In addition to being expressed in mesoderm during gastrulation, vertebrate snail genes are also expressed in presumptive neural crest and/or its derivatives. Given that neural crest is unique to vertebrates and is considered to be of fundamental importance in their evolution, we have cloned and characterized the expression of a snail gene from amphioxus, a cephalochordate widely accepted as the sister group of the vertebrates. We show that, at the amino acid sequence level, the amphioxus snail gene is a clear phylogenetic outgroup to all the characterized vertebrate snail genes. During embryogenesis snail expression initially becomes restricted to the paraxial or presomitic mesoderm of amphioxus. Later, snail is expressed at high levels in the lateral neural plate, where it persists during neurulation. Our results indicate that an ancestral function of snail genes in the lineage leading to vertebrates is to define the paraxial mesoderm. Furthermore, our results indicate that a cell population homologous to the vertebrate neural crest may be present in amphioxus, thus providing an important link in the evolution of this key vertebrate tissue. Received: 11 May 1998 / Accepted: 2 August 1998     

Apoptotic and anti-proliferative effects of all-trans retinoic acid. Adenine nucleotide translocase sensitizes HeLa cells to all-trans retinoic acid

We examined the apoptotic and anti-proliferative effects of all-trans retinoic acid (atRA) in HeLa cells. Our results demonstrated that HeLa cells were more sensitive to the anti-proliferative effects of atRA than to its apoptotic effects. Furthermore, we demonstrated that caspase inhibition attenuates cell death but does not alter the atRA-dependent reduction in cell proliferation, which suggests that atRA-induced apoptosis is independent of the arrest in cell proliferation. To check whether ANT proteins mediated these atRA effects, we transiently transfected cells with expression vectors encoding for individual ANT (adenine nucleotide translocase 1-3). Our results revealed that ANT1 and ANT3 over-expressing HeLa cells increased their atRA sensitivity. Thus, our results not only demonstrate the different functional activities of ANT isoforms, but also contribute to a better understanding of the properties of atRA as an anti-tumoral agent used in cancer therapy.     

Functional expression of human adenine nucleotide translocase 4 in Saccharomyces cerevisiae

The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31) was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4) in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells.     

IL-4-induced upregulation of adenine nucleotide translocase 3 and its role in Th cell survival from apoptosis

We have identified mitochondrial adenine nucleotide translocase (ANT)3 as a novel target up-regulated by IL-4 in human T cells. The IL-4-induced ANT3 expression is dependent on tyrosine kinase, NF-kappaB, PI3K/Akt, and Erk pathways. In fact, IL-4 induced specific activation of NF-kappaB, Akt, and Erk in Jurkat T cells and partially rescued these cells from dexamethasone-induced apoptosis. The IL-4-mediated T cell survival was blocked by inhibitors of tyrosine kinase, NF-kappaB, PI3K/Akt, and Erk. During the IL-4-induced T cell rescue, there was a concomitant increase in ANT3, nuclear NF-kappaB, and Bcl-2 and a decrease in ANT1, I-kappaB, and mitochondrial Bax-alpha levels. Importantly, overexpression of ANT3 effectively protected T cells from dexamethasone-induced apoptosis, while forced expression of ANT1 caused apoptosis. In contrast, siRNA knock-out of ANT3 expression induced T cell apoptosis and blocked the IL-4-mediated cell survival. Together these results suggest that ANT3 has a potential role in Th cell survival and immune cell homeostasis.     

Increase in the adenine nucleotide translocase content of duckling subsarcolemmal mitochondria during cold acclimation

Intermyofibrillar and subsarcolemmal mitochondria were isolated from duckling gastrocnemius muscle. The adenine nucleotide translocase (ANT) content of subsarcolemmal mitochondria was found to be half of that present in intermyofibrillar mitochondria. In addition, cold acclimation resulted in a 1.7-fold increase in subsarcolemmal mitochondrial ANT content, with intermyofibrillar mitochondrial ANT remaining constant. This change in mitochondrial ANT content correlates with the previously reported cold-induced change in the sensitivity of mitochondria to palmitate-inhibited ATP synthesis [Roussel et al. (1998) FEBS Lett. 439, 258-262]. It is suggested that the mitochondrial ANT content enhances or reduces the fatty acid uncoupling activity in tissue, depending on the energetic state of mitochondria.     

Discontinuity of primary and secondary neural tube in spina bifida induced by retinoic acid in mice

This report shows by light microscopy the appearance of secondary neurulation separated from primary neurulation and its developmental fate in the spinal cord of mice exposed to retinoic acid in utero. The embryos and fetuses were derived from pregnant mice (ICR strain) given 60, 40, or 0 mg/kg of retinoic acid in olive oil on day 8 of gestation orally and killed 1, 2, or 10 days later. Separation of the primary neural fold from the secondary neural tube was seen in 9- and 10-day-old embryos: the caudal part of the neuroepithelium of the primary neural fold was disarranged with non-closed posterior neuropore, and underneath it the secondary neural tissue extended caudally with abnormal notochord. At term, fetuses showed spina bifida, including myeloschisis, myelocele, and diplomyelia (diastematomyelia) with abnormal distribution of ganglionic cells. These cord lesions were located between the third lumbar and second coccygeal levels. The former two cord anomalies were associated with diplomyelia and split the dorsal and ventral portions of the spinal cord with an overlapping zone between the third lumbar and third sacral levels. These findings suggest that the separation from primary neurulation is due to the lesions in both primary neural folds and notochord induced by retinoic acid and that the spinal cord caudal to the third lumbar level originates from both neuroectoderm and mesenchyme-like cells while that caudal to the third sacral level originates from mesenchyme-like cells only.     

The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria.

Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.