Heterochronic development of pelvic fins in zebrafish: possible involvement of temporal regulation of pitx1 expression.

Anterior and posterior paired appendages of vertebrates are notable examples of heterochrony in the relative timing of their development. In teleosts, posterior paired appendages (pelvic fin buds) emerge much later than their anterior paired appendages (pectoral fin buds). Pelvic fin buds of zebrafish (Danio rerio) appear at 3 weeks post-fertilization (wpf) during the larva-to-juvenile transition (metamorphosis), whereas pectoral fin buds arise from the lateral plate mesoderm on the yolk surface at the embryonic stage. Here we explored the mechanism by which presumptive pelvic fin cells maintain their fate, which is determined at the embryonic stage, until the onset of metamorphosis. Expression analysis revealed that transcripts of pitx1, one of the key factors for the development of posterior paired appendages, became briefly detectable in the posterior lateral plate mesoderm at early embryonic stages. Further analysis indicated that the pelvic fin-specific pitx1 enhancer was in the poised state at the larval stage and is activated at the juvenile stage. We discuss the implications of these findings for the heterochronic development of pelvic fin buds.

Regulation of the limb shape during the development of the Chinese softshell turtles.

Interdigital cell death is an important mechanism employed by amniotes to shape their limbs; inhibiting this process leads to the formation of webbed fingers, as seen in bats and ducks. The Chinese softshell turtle Pelodiscus sinensis (Reptilia: Testudines: Trionychidae) has a distinctive limb morphology: the anterior side of the limbs has partially webbed fingers with claw-like protrusions, while the posterior fingers are completely enclosed in webbings. Here, P. sinensis embryos were investigated to gain insights on the evolution of limb-shaping mechanisms in amniotes. We found cell death and cell senescence in their interdigital webbings. Spatial or temporal modulation of these processes were correlated with the appearance of indentations in the webbings, but not a complete regression of this tissue. No differences in interdigital cell proliferation were found. In subsequent stages, differential growth of the finger cartilages led to a major difference in limb shape. While no asymmetry in bone morphogenetic protein signaling was evident during interdigital cell death stages, some components of this pathway were expressed exclusively in the clawed digit tips, which also had earlier ossification. In addition, a delay and/or truncation in the chondrogenesis of the posterior digits was found in comparison with the anterior digits of P. sinensis, and also when compared with the previously published pattern of digit skeletogenesis of turtles without posterior webbings. In conclusion, modulation of cell death, as well as a heterochrony in digit chondrogenesis, may contribute to the formation of the unique limbs of the Chinese softshell turtles.

Evolution of the avian digital pattern.

Variation in digit number has occurred multiple times in the history of archosaur evolution. The five digits of dinosaur limbs were reduced to three in bird forelimbs, and were further reduced in the vestigial forelimbs of the emu. Regulation of digit number has been investigated previously by examining genes involved in anterior-posterior patterning in forelimb buds among emu (Dromaius novaehollandiae), chicken (Gallus gallus) and zebra finch (Taeniopygia guttata). It was described that the expression of posterior genes are conserved among these three birds, whereas expression of anterior genes Gli3 and Alx4 varied significantly. Here we re-examined the expression pattern of Gli3 and Alx4 in the forelimb of emu, chicken and zebra finch. We found that Gli3 is expressed in the anterior region, although its range varied among species, and that the expression pattern of Alx4 in forelimb buds is broadly conserved in a stage-specific manner. We also found that the dynamic expression pattern of the BMP antagonist Gremlin1 (Grem1) in limb buds, which is critical for autopodial expansion, was consistent with the digital pattern of emu, chicken and zebra finch. Furthermore, in emu, variation among individuals was observed in the width of Grem1 expression in forelimb buds, as well as in the adult skeletal pattern. Our results support the view that the signalling system that regulates the dynamic expression of Grem1 in the limb bud contributes substantially to variations in avian digital patterns.

Cux2 refines the forelimb field by controlling expression of Raldh2 and Hox genes.

In vertebrates, two pairs of buds that give rise to the fore- and hindlimbs form at discrete positions along the rostral-caudal axis of the body. The mechanism responsible for the positioning of the limb buds is still largely unknown. Here we show a novel function for Cut homeobox transcription factor 2 (Cux2), the ortholog of Drosophila cut, in refining the forelimb field during chick development. Cux2 is expressed in the forelimb field before the emergence of the limb buds. Knocking down the expression of Cux2 using small interfering RNA (siRNA) resulted in a caudal shift of the forelimb bud, whereas misexpression of Cux2 or the constitutively active Cux2-VP16 caused a rostral shift of the forelimb bud or reduction of the forelimb field along the anterior-posterior axis. Further functional analyses revealed that expression of Hoxb genes and retinaldehyde dehydrogenase 2 (Raldh2), which are involved in limb positioning, are directly activated by Cux2 in the lateral plate mesoderm. Our data suggest that Cux2 in the lateral plate mesoderm refines the forelimb field via regulation of Raldh2 and Hoxb genes in chicken embryos.

Anterior migration of lateral plate mesodermal cells during embryogenesis of the pufferfish Takifugu niphobles: insight into the rostral positioning of pelvic fins.

In vertebrates, paired appendages (limbs and fins) are derived from the somatic mesoderm subsequent to the separation of the lateral plate mesoderm into somatic and splanchnic layers. This is less clear for teleosts, however, because the developmental processes of separation into two layers and of extension over the yolk have rarely been studied. During teleost evolution, the position of pelvic fins has generally shifted rostrally (Rosen; Nelson, 1982, 1994), although at the early embryonic stage the presumptive pelvic fin cells are initially located near the future anus region - the anterior border of hoxc10a expression in the spinal cord - regardless of their final destination. Our previous studies in zebrafish (abdominal pelvic fins) and Nile tilapia (thoracic pelvic fins) showed that the presumptive pelvic fin cells shift their position with respect to the body trunk after its protrusion from the yolk surface. Furthermore, in Nile tilapia, presumptive pelvic fin cells migrate anteriorly on the yolk surface. Here, we examined the embryonic development of the lateral plate mesoderm at histological levels in the pufferfish Takifugu niphobles, which belongs to the highly derived teleost order Tetraodontiformes, and lacks pelvic fins. Our results show that, in T. niphobles, the lateral plate mesoderm bulges out as two separate layers of cells alongside the body trunk prior to its further extension to cover the yolk sphere. Once the lateral plate mesoderm extends laterally, it rapidly covers the surface of the yolk. Furthermore, cells located near the anterior border of hoxc10a expression in the spinal cord reach the anterior-most region of the yolk surface. In light of our previous and current studies, we propose that anterior migration of presumptive pelvic fin cells might be required for them to reach the thoracic or more anterior positions as is seen in other highly derived teleost groups.

pfaB products determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis.

When pDHA4, a vector carrying all five pfaA-pfaE genes responsible for docosahexaenoic acid (DHA; 22:6) biosynthesis in Moritella marina MP-1, was coexpressed in Escherichia coli with the individual pfaA-pfaD genes for eicosapentaenoic acid (EPA; 20:5) biosynthesis from Shewanella pneumatophori SCRC-2738, both polyunsaturated fatty acids were synthesized only in the recombinant carrying pfaB for EPA synthesis. Escherichia coli coexpressing a deleted construct comprising pfaA, pfaC, pfaD and pfaE for EPA and pfaB for DHA produced EPA and DHA. Both EPA and DHA were detected in bacteria that inherently contained pfa genes for DHA. These results suggest that PfaB is the key enzyme determining the final product in EPA or DHA biosynthesis.

Recombinant production of docosahexaenoic acid in a polyketide biosynthesis mode in Escherichia coli.

The docosahexaenoic acid (DHA) biosynthesis gene cluster (pDHA3) from the DHA-producing Moritella marina strain MP-1 includes the genes pfaA, pfaB, pfaC, and pfaD, which are similar to the genes of polyketide biosynthesis. When this cluster was co-expressed in Escherichia coli with M. marina MP-1 pfaE, which encodes phosphopantetheinyl transferase, DHA was biosynthesized. The maximum production of DHA (5% of total fatty acids) was observed at 15 degrees C. This is the first report of the recombinant production of DHA in a polyketide biosynthesis mode.

LuxA gene of light organ symbionts of the bioluminescent fish Acropoma japonicum (Acropomatidae) and Siphamia versicolor (Apogonidae) forms a lineage closely related to that of Photobacterium leiognathi ssp. mandapamensis.

A molecular phylogenetic analysis of luxA gene sequences of light organ symbionts of the fish Acropoma japonicum (Acropomatidae) and Siphamia versicolor (Apogonidae) revealed that the sequences were related to those of Photobacterium leiognathi ssp. mandapamensis, which is not known to occur as a light organ symbiont among bioluminescent P. leiognathi clades. The presence of another lux gene element, luxF, coding for nonfluorescent protein, provided additional support for the identity of the light organ symbionts of the fish. Cladogenesis of the light organ symbiont P. leiognathi may be influenced by the radiation of host fishes.

Escherichia coli engineered to produce eicosapentaenoic acid becomes resistant against oxidative damages.

The colony-forming ability of Escherichia coli genetically engineered to produce eicosapentaenoic acid (EPA) grown in 3mM hydrogen peroxide (H(2)O(2)) was similar to that of untreated cells. It was rapidly lost in the absence of EPA. H(2)O(2)-induced protein carbonylation was enhanced in cells lacking EPA. The fatty acid composition of the transformants was unaffected by H(2)O(2) treatment, but the amount of fatty acids decreased in cultures of cells lacking EPA and increased in cultures of cells producing EPA, suggesting that cellular EPA is stable in the presence of H(2)O(2) in vivo and may protect cells directly against oxidative damage. We discuss the possible role of EPA in partially blocking the penetration of H(2)O(2) into cells through membranes containing EPA.