Gap junctions are proteinaceous channels that reside at the plasma membrane and permit the exchange of ions, metabolites, and second messengers between neighboring cells. Connexin proteins are the subunits of gap junction channels. Mutations in zebrafish cx43 cause the short fin (sof b123) phenotype which is characterized by short fins due to defects in length of the bony fin rays. Previous findings from our lab demonstrate that Cx43 is required for both cell proliferation and joint formation during fin regeneration. Here we demonstrate that semaphorin3d (sema3d) functions downstream of Cx43. Semas are secreted signaling molecules that have been implicated in diverse cellular functions such as axon guidance, cell migration, cell proliferation, and gene expression. We suggest that Sema3d mediates the Cx43-dependent functions on cell proliferation and joint formation. Using both in situ hybridization and quantitative RT-PCR, we validated that sema3d expression depends on Cx43 activity. Next, we found that knockdown of Sema3d recapitulates all of the sof b123 and cx43-knockdown phenotypes, providing functional evidence that Sema3d acts downstream of Cx43. To identify the potential Sema3d receptor(s), we evaluated gene expression of neuropilins and plexins. Of these, nrp2a, plxna1, and plxna3 are expressed in the regenerating fin. Morpholino-mediated knockdown of plxna1 did not cause cx43-specific defects, suggesting that PlexinA1 does not function in this pathway. In contrast, morpholino-mediated knockdown of nrp2a caused fin overgrowth and increased cell proliferation, but did not influence joint formation. Moreover, morpholino-mediated knockdown of plxna3 caused short segments, influencing joint formation, but did not alter cell proliferation. Together, our findings reveal that Sema3d functions in a common molecular pathway with Cx43. Furthermore, functional evaluation of putative Sema3d receptors suggests that Cx43-dependent cell proliferation and joint formation utilize independent membrane-bound receptors to mediate downstream cellular phenotypes.

Joints are essential for skeletal flexibly and form, yet the process underlying joint morphogenesis is poorly understood. Zebrafish caudal fins are comprised of numerous segmented bony fin rays, where growth occurs by the sequential addition of new segments and new joints. Here, we evaluate joint gene expression during fin regeneration. First, we identify three genes that influence joint formation, evx1, dlx5a, and mmp9. We place these genes in a common molecular pathway by evaluating both their expression patterns along the distal-proximal axis (i.e. where the youngest tissue is always the most distal), and by evaluating changes in gene expression following gene knockdown. Prior studies from our lab indicate that the gap junction protein Cx43 suppresses joint formation. Remarkably, changes in Cx43 activity alter the expression of joint markers. For example, the reduced levels of Cx43 in the sof b123 mutant causes short fin ray segments/premature joints. We also find that the expression of evx1-dlx5a-mmp9 is shifted distally in sof b123, consistent with premature expression of these genes. In contrast, increased Cx43 in the alf dty86 mutant leads to stochastic joint failure and stochastic loss of evx1 expression. Indeed, reducing the level of Cx43 in alf dty86 rescues both the evx1 expression and joint formation. These results suggest that Cx43 influences the pattern of joint formation by influencing the timing of evx1 expression.

Endocardial and myocardial progenitors originate in distinct regions of the anterior lateral plate mesoderm and migrate to the midline where they coalesce to form the cardiac tube. Endocardial progenitors acquire a molecular identity distinct from other vascular endothelial cells and initiate expression of specific genes such as nfatc1. Yet the molecular pathways and tissue interactions involved in establishing endocardial identity are poorly understood. The endocardium develops in tight association with cardiomyocytes. To test for a potential role of the myocardium in endocardial morphogenesis, we used two different zebrafish models deficient in cardiomyocytes: the hand2 mutant and a myocardial-specific genetic ablation method. We show that in hand2 mutants endocardial progenitors migrate to the midline but fail to assemble into a cardiac cone and do not express markers of differentiated endocardium. Endocardial differentiation defects were rescued by myocardial but not endocardial-specific expression of hand2. In metronidazole-treated myl7:nitroreductase embryos, myocardial cells were targeted for apoptosis, which resulted in the loss of endocardial nfatc1 expression. However, endocardial cells were present and retained expression of general vascular endothelial markers. We further identified bone morphogenetic protein (BMP) as a candidate myocardium-derived signal required for endocardial differentiation. Chemical and genetic inhibition of BMP signaling at the tailbud stage resulted in severe inhibition of endocardial differentiation while there was little effect on myocardial development. Heat-shock-induced bmp2b expression rescued endocardial nfatc1 expression in hand2 mutants and in myocardiumdepleted embryos. Our results indicate that the myocardium is crucial for endocardial morphogenesis and differentiation, and identify BMP as a signal involved in endocardial differentiation.

Background How joints are correctly positioned in the vertebrate skeleton remains poorly understood. From our studies on the regenerating fin, we have evidence that the gap junction protein Cx43 suppresses joint formation by suppressing the expression of the evx1 transcription factor. Joint morphogenesis proceeds through at least two discrete stages. First, cells that will produce the joint condense in a single row on the bone matrix (“initiation”). Second, these cells separate coincident with articulation of the bone matrix. We propose that Cx43 activity is transiently reduced prior to joint initiation. Results We first define the timing of joint initiation with respect to regeneration. We next correlate reduced cx43 expression and increased evx1 expression with initiation. Through manipulation of cx43 expression we demonstrate that Cx43 negatively influences evx1 expression and joint formation. We further demonstrate that Cx43 activity in the dermal fibroblasts is required to rescue joint formation in the cx43 mutant, short fin b123. Conclusions We conclude that Cx43 activity in the dermal fibroblasts influences the expression of evx1, and therefore the differentiation of the precursor cells that give rise to the joint-forming osteoblasts.

Microtubules (MTs) or their subunits, tubulin dimers, interact with multiple components that contribute to intracellular metabolic pathways. MTs are required for insulin-dependent transport of glucose transporter 4 to the plasma membrane, they bind most glycolytic enzymes and are required for translation of the mRNA encoding hypoxia inducible factor-1a. Tubulin dimers bind the voltage-dependent anion channel of the mitochondrial outer membrane; this channel functions in metabolite transport in and out of mitochondria. We hypothesize that tubulin partitioning between dimer and polymer pools regulates multiple steps in metabolism, where metabolic output is greatest when both tubulin dimers and MT polymers are present and reduced by drug treatments that disrupt this normal balance. Experimental evidence from these drug-induced changes in tubulin dimer/polymer partitioning supports our model for several metabolic steps. Signal transduction pathways that stabilize or destabilize MTs can shift the normal ratio between unpolymerized and polymerized tubulin dimers, and one downstream consequence of this shift in tubulin partitioning could be a change in metabolic output. V C 2012 Wiley Periodicals, Inc Key Words: glycolysis, oxidative phosphorylation, glucose transport, voltage-dependent anion channel, tubulin IntroductionT he microtubule (MT) cytoskeleton has well-characterized roles in cell polarity, motility, mitosis, and vesicle traffic. The dynamic turnover of MTs is critical to most of these processes, allowing the cell to reorganize an array of MTs rapidly for specific functions. Here we posit an additional role for MTs in regulating metabolic processes and propose that metabolism is greatest when both MTs and their tubulin subunits are present in cells. Disruption of this normal balance, to either inhibit or promote MT polymerization, is expected to decrease metabolic output. Our hypothesis predicts that chemotherapies that stabilize or destabilize MTs will significantly impact metabolic pathways and reduce ATP levels. Importantly, even a small decrease in ATP production can slow cell proliferation. For example, a 19% decrease in ATP production was sufficient to induce senescence in mouse embryo fibroblasts [Kondoh et al., 2005], indicating that even a relatively small contribution of the MT cytoskeleton to metabolic output could have a significant impact on cell fate.The metabolic components and pathways we consider here include glucose transporters (GLUTs), glycolysis, the pentose phosphate pathway (PPP), and mitochondrial oxidative phosphorylation. Glucose enters cells through glucose transporters called GLUTs; the concentration of GLUTs at the plasma membrane can be regulated by insulin and requires trafficking of GLUT-containing vesicles along MTs to reach the plasma membrane. Within the cytoplasm, glucose is broken down to pyruvate during glycolysis, yielding ATP and NADH. Several intermediate products of glucose breakdown, including glucose-6-phosphate (G6P) and glyceraldehyde-3-phosphate (GAP), can also ...