Evolution of the TGF-β signaling pathway and its potential role in the ctenophore, Mnemiopsis leidyi

The TGF-β signaling pathway is a metazoan-specific intercellular signaling pathway known to be important in many developmental and cellular processes in a wide variety of animals. We investigated the complexity and possible functions of this pathway in a member of one of the earliest branching metazoan phyla, the ctenophore Mnemiopsis leidyi. A search of the recently sequenced Mnemiopsis genome revealed an inventory of genes encoding ligands and the rest of the components of the TGF-β superfamily signaling pathway. The Mnemiopsis genome contains nine TGF-β ligands, two TGF-β-like family members, two BMP-like family members, and five gene products that were unable to be classified with certainty. We also identified four TGF-β receptors: three Type I and a single Type II receptor. There are five genes encoding Smad proteins (Smad2, Smad4, Smad6, and two Smad1s). While we have identified many of the other components of this pathway, including Tolloid, SMURF, and Nomo, notably absent are SARA and all of the known antagonists belonging to the Chordin, Follistatin, Noggin, and CAN families. This pathway likely evolved early in metazoan evolution as nearly all components of this pathway have yet to be identified in any non-metazoan. The complement of TGF-β signaling pathway components of ctenophores is more similar to that of the sponge, Amphimedon, than to cnidarians, Trichoplax, or bilaterians. The mRNA expression patterns of key genes revealed by in situ hybridization suggests that TGF-β signaling is not involved in ctenophore early axis specification. Four ligands are expressed during gastrulation in ectodermal micromeres along all three body axes, suggesting a role in transducing earlier maternal signals. Later expression patterns and experiments with the TGF-β inhibitor SB432542 suggest roles in pharyngeal morphogenesis and comb row organization.     

The evolution of metazoan axial properties

Renewed interest in the developmental basis of organismal complexity, and the emergence of new molecular tools, is improving our ability to study the evolution of metazoan body plans. The most substantial changes in body-plan organization occurred early in metazoan evolution; new model systems for studying basal metazoans are now being developed, and total-genome-sequencing initiatives are underway for at least three of the four most important taxa. The elucidation of how the gene networks that are involved in axial organization, germ-layer formation and cell differentiation are used differently during development is generating a more detailed understanding of the events that have led to the current diversity of multicellular life.     

vasa and nanos expression patterns in a sea anemone and the evolution of bilaterian germ cell specification mechanisms

Most bilaterians specify primordial germ cells (PGCs) during early embryogenesis using either inherited cytoplasmic germ line determinants (preformation) or induction of germ cell fate through signaling pathways (epigenesis). However, data from nonbilaterian animals suggest that ancestral metazoans may have specified germ cells very differently from most extant bilaterians. Cnidarians and sponges have been reported to generate germ cells continuously throughout reproductive life, but previous studies on members of these basal phyla have not examined embryonic germ cell origin. To try to define the embryonic origin of PGCs in the sea anemone Nematostella vectensis, we examined the expression of members of the vasa and nanos gene families, which are critical genes in bilaterian germ cell specification and development. We found that vasa and nanos family genes are expressed not only in presumptive PGCs late in embryonic development, but also in multiple somatic cell types during early embryogenesis. These results suggest one way in which preformation in germ cell development might have evolved from the ancestral epigenetic mechanism that was probably used by a metazoan ancestor.     

Alterations in oxidative phosphorylation complex proteins in the hearts of transgenic mice that overexpress the p38 MAP kinase activator, MAP kinase kinase 6

Ischemia-reperfusion (I/R) has critical consequences in the heart. Recent studies on the functions of I/R-activated kinases, such as p38 mitogen-activated protein kinase (MAPK), showed that I/R injury is reduced in the hearts of transgenic mice that overexpress the p38 MAPK activator MAPK kinase 6 (MKK6). This protection may be fostered by changes in the levels of many proteins not currently known to be regulated by p38. To examine this possibility, we employed the multidimensional protein identification technology MudPIT to characterize changes in levels of proteins in MKK6 transgenic mouse hearts, focusing on proteins in mitochondria, which play key roles in mediating I/R injury in the heart. Of the 386 mitochondrial proteins identified, the levels of 58 were decreased, while only 2 were increased in the MKK6 transgenic mouse hearts. Among those that were decreased were 21 mitochondrial oxidative phosphorylation complex proteins, which was unexpected because p38 is not known to mediate such decreases. Immunoblotting verified that proteins in each of the five oxidative phosphorylation complexes were reduced in MKK6 mouse hearts. On assessing functional consequences of these reductions, we found that MKK6 mouse heart mitochondria exhibited 50% lower oxidative respiration and I/R-mediated reactive oxygen species (ROS) generation, both of which are predicted consequences of decreased oxidative phosphorylation complex proteins. Thus the cardioprotection observed in MKK6 transgenic mouse hearts may be partly due to decreased electron transport, which is potentially beneficial, because damaging ROS are known to be generated by mitochondrial complexes I and III during reoxygenation.     

Developmental origin of segmental differences in the leech ectoderm: survival and differentiation of the distal tubule cell is determined by the host segment

The body plan of the adult leech is metameric, with each hemisegmental complement of ectodermal and mesodermal tissues being produced from a set of seven serially repeated embryonic blast cells. Previous studies have shown that homologous o blast cells give rise to an almost identical complement of descendant cells in each of the 21 abdominal segments, but that one o blast cell derivative--the distalmost cell of the nephridial tubule--is only present in 15 abdominal segments in the mature leech. Here we show that all o blast cells generate a presumptive distal tubule cell and that this cell migrates to its normal position in all abdominal segments. However, in segments which normally do not contain the mesodermal portion of the nephridium, the distal tubule cell dies before undergoing its terminal morphological differentiation. To ascertain whether the fate of the distal tubule cell is determined by its lineage history or by the segmental environment into which it is born, we utilized a previously described procedure for altering the segmental register between different embryonic cell lines. This procedure allowed us to effectively transplant o blast cells into more posterior segments prior to the cell divisions which generate their descendant clones. The results indicate that the survival or death of the distal tubule cell is determined by the identity of the host segment and that a given distal tubule cell could be effectively murdered or rescued by slipping its blast cell precursor into an appropriate segment. These findings suggest that the segment-specific pattern of distal tubule cell survival is not inherent to the O cell line, but arises from interactions with surrounding tissues.     

Direct,Differential Effects of Tamoxifen, 4-Hydroxytamoxifen,and Raloxifene on Cardiac Myocyte Contractility and Calcium Handling

Tamoxifen (Tam), a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. High Tam doses have been used for treatment of gliomas and cancers with multiple drug resistance, but long QT Syndrome is a side effect. Tam is also used experimentally in mice for inducible gene knockout in numerous tissues, including heart; however, the potential direct effects of Tam on cardiac myocyte mechanical function are not known. The goal of this study was to determine the direct, acute effects of Tam, its active metabolite 4-hydroxytamoxifen (4OHT), and related drug raloxifene (Ral) on isolated rat cardiac myocyte mechanical function and calcium handling. Tam decreased contraction amplitude, slowed relaxation, and decreased Ca²⁺ transient amplitude. Effects were primarily observed at 5 and 10 μM Tam, which is relevant for high dose Tam treatment in cancer patients as well as Tam-mediated gene excision in mice. Myocytes treated with 4OHT responded similarly to Tam-treated cells with regard to both contractility and calcium handling, suggesting an estrogen-receptor independent mechanism is responsible for the effects. In contrast, Ral increased contraction and Ca²⁺ transient amplitudes. At 10 μM, all drugs had a time-dependent effect to abolish cellular contraction. In conclusion, Tam, 4OHT, and Ral adversely and differentially alter cardiac myocyte contractility and Ca²⁺ handling. These findings have important implications for understanding the Tam-induced cardiomyopathy in gene excision studies and may be important for understanding effects on cardiac performance in patients undergoing high-dose Tam therapy.