Helen P. Makarenkova

HDAC6 regulates mitochondrial transport in hippocampal neurons.

Background Tubulin is a major substrate of the cytoplasmic class II histone deacetylase HDAC6. Inhibition of HDAC6 results in higher levels of acetylated tubulin and enhanced binding of the motor protein kinesin-1 to tubulin, which promotes transport of cargoes along microtubules. Microtubule-dependent intracellular trafficking may therefore be regulated by modulating the activity of HDAC6. We have shown previously that the neuromodulator serotonin increases mitochondrial movement in hippocampal neurons via the Akt-GSK3β signaling pathway. Here, we demonstrate a role for HDAC6 in this signaling pathway. Methodology/Principal Findings We found that the presence of tubacin, a specific HDAC6 inhibitor, dramatically enhanced mitochondrial movement in hippocampal neurons, whereas niltubacin, an inactive tubacin analog, had no effect. Compared to control cultures, higher levels of acetylated tubulin were found in neurons treated with tubacin, and more kinesin-1 was associated with mitochondria isolated from these neurons. Inhibition of GSK3β decreased cytoplasmic deacetylase activity and increased tubulin acetylation, whereas blockade of Akt, which phosphorylates and down-regulates GSK3β, increased cytoplasmic deacetylase activity and decreased tubulin acetylation. Concordantly, the administration of 5-HT, 8-OH-DPAT (a specific 5-HT1A receptor agonist), or fluoxetine (a 5-HT reuptake inhibitor) increased tubulin acetylation. GSK3β was found to co-localize with HDAC6 in hippocampal neurons, and inhibition of GSK3β resulted in decreased binding of antibody to phosphoserine-22, a potential GSK3β phosphorylation site in HDAC6. GSK3β may therefore regulate HDAC6 activity by phosphorylation. Conclusions/Significance This study demonstrates that HDAC6 plays an important role in the modulation of mitochondrial transport. The link between HDAC6 and GSK3β, established here, has important implications for our understanding of neurodegenerative disorders. In particular, abnormal mitochondrial transport, which has been observed in such disorders as Alzheimers disease and Parkinsons disease, could result from the misregulation of HDAC6 by GSK3β. HDAC6 may therefore constitute an attractive target in the treatment of these disorders.

Differential Interactions of FGFs with Heparan Sulfate Control Gradient Formation and Branching Morphogenesis

Manipulation of the interaction of a morphogen with the extracellular matrix changes its biological activities by altering its diffusion. Branch or Elongate The graded distribution of morphogens, such as fibroblast growth factors (FGFs), is critically important for the patterning of tissues in the developing embryo. Binding of morphogens to heparan sulfate glycosaminoglycans (HSGAGs) controls their diffusion through the extracellular matrix (ECM); however, the extent to which these interactions modulate the activities of morphogens is unclear. Makarenkova et al. studied the differential effects of FGF7 and FGF10, two closely related FGFs with different biological activities, in the context of branching morphogenesis of epithelia from mouse embryonic lacrimal and submandibular glands. Whereas FGF7 induces branching of epithelial buds, FGF10 induces their elongation. Replacement of a single residue in the heparan-binding site of FGF10 with the corresponding residue of FGF7 resulted in a mutant FGF10 that acted as a functional mimic of FGF7; it diffused more readily into the ECM than did wild-type FGF10 and it induced branching rather than elongation of epithelial buds. Thus, not only are the gradients of morphogens established by their interactions with HSGAGs, but these interactions can also modulate their biological activities. The developmental activities of morphogens depend on the gradients that they form in the extracellular matrix. Here, we show that differences in the binding of fibroblast growth factor 7 (FGF7) and FGF10 to heparan sulfate (HS) underlie the formation of different gradients that dictate distinct activities during branching morphogenesis. Reducing the binding affinity of FGF10 for HS by mutating a single residue in its HS-binding pocket converted FGF10 into a functional mimic of FGF7 with respect to gradient formation and regulation of branching morphogenesis. In particular, the mutant form of FGF10 caused lacrimal and salivary gland epithelium buds to branch rather than to elongate. In contrast, mutations that reduced the affinity of the FGF10 for its receptor affected the extent, but not the nature, of the response. Our data may provide a general model for understanding how binding to HS regulates other morphogenetic gradients.

Embryonic Submandibular Gland Morphogenesis: Stage-Specific Protein Localization of FGFs, BMPs, Pax6 and Pax9 in Normal Mice and Abnormal SMG Phenotypes in FgfR2-IIIc+/Δ, BMP7–/– and Pax6–/–Mice

Embryonic submandibular salivary gland (SMG) initiation and branching morphogenesis are dependent on cell-cell communications between and within epithelium and mesenchyme. Such communications are typically mediated in other organs (teeth, lung, lacrimal glands) by growth factors in such a way as to translate autocrine, juxtacrine and paracrine signals into specific gene responses regulating cell division and histodifferentiation. Using Wnt1-Cre/R26R transgenic mice, we demonstrate that embryonic SMG mesenchyme is derived exclusively from cranial neural crest. This origin contrasts to that known for tooth mesenchyme, previously shown to be derived from both neural crest and nonneural crest cells. Thus, although both SMGs and teeth are mandibular derivatives, we can expect overlap and differences in the details of their early inductive interactions. In addition, since embryonic SMG branching morphogenesis is analogous to that seen in other branching organs, we also expect similarities of expression regarding those molecules known to be ubiquitous regulators of morphogenesis. In this study, we performed an analysis of the distribution of specific fibroblast growth factors (FGFs), FGF receptors, bone morphogenetic proteins (BMPs) and Pax transcription factors, previously shown to be important for tooth development and/or branching morphogenesis, from the time of initiation of embryonic SMG development until early branching morphogenesis. In addition, we report abnormal SMG phenotypes in FgfR2- IIIc+/Δ, BMP7–/–and Pax6–/– mice. Our results, in comparison with functional studies in other systems, suggest that FGF-2/FGFR-1, FGF-8/FGFR-2(IIIc) and FGF-10/FGFR-2(IIIb) signaling have different paracrine and juxtacrine functions during SMG initial bud formation and branching. Finally, our observations of abnormal SMGs in BMP7–/– and Pax6–/–indicate that both BMP7 and Pax6 play important roles during embryonic SMG branching morphogenesis.

The role of long range, local and direct signalling molecules during chick feather bud development involving the BMPs, Follistatin and the Eph receptor tyrosine kinase Eph-A4

The development of the feather buds during avian embryogenesis is a classic example of a spacing pattern. The regular arrangement of feather buds is achieved by a process of lateral inhibition whereby one developing feather bud prevents the formation of similar buds in the immediate vicinity. Lateral inhibition during feather formation implicates a role of long range signalling during this process. Recent work has shown that BMPs are able to enforce lateral inhibition during feather bud formation. However these results do not explain how the feather bud escapes the inhibition itself. We show that this could be achieved by the expression of the BMP antagonist, Follistatin. Furthermore we show that local application of Follistatin leads to the development of ectopic feather buds. We suggest that Follistatin locally antagonises the action of the BMPs and so permits the cellular changes associated with feather placode formation. We also provide evidence for the role of short range signalling during feather formation. We have correlated changes in cellular morphology in feather placodes with the expression of the gene Eph-A4 which encodes a receptor tyrosine kinase that requires direct cell-cell contact for activation. We show that the expression of this gene precedes cellular reorganisation required for feather bud formation.

Reactive Oxygen Species Modulate the Differentiation of Neurons in Clonal Cortical Cultures.

Reactive oxygen species (ROS) are important regulators of intracellular signaling. We examined the expression of ROS during rat brain development and explored their role in differentiation using cortical cultures. High levels of ROS were found in newborn neurons. Neurons produced ROS, not connected with cell death, throughout embryogenesis and postnatal stages. By P20, ROS-producing cells were found only in neurogenic regions. Cells with low levels of ROS, isolated from E15 brains by FACS, differentiated into neurons, oligodendrocytes, and astrocytes in clonal cultures. Neurons produced high ROS early in culture and later differentiated into two types: large pyramidal-like neurons that fired no or only a single action potential and smaller neurons that expressed nuclear calretinin and fired repeated action potentials. Antioxidant treatment did not alter neuron number but increased the ratio of small to large neurons. These findings suggest that modulation of ROS levels influences multiple aspects of neuronal differentiation.

Basic FGF Increases Communication between Cells of the Developing Neocortex

We have found that basic fibroblast growth factor (bFGF), applied to cortical progenitor cells in vitro, produces an increase in the expression of the gap junction protein connexin (Cx) 43 and in the mRNA encoding Cx 43. This effect was evident in both proliferating and nonproliferating cells. The elevated levels of mRNA suggest that bFGF is likely to exert its effect by upregulating the rate of transcription of the Cx 43 gene. We have further shown that the increase in Cx 43 expression is mediated through the receptor tyrosine kinase pathway and is associated with enhanced intercellular dye-coupling mediated by gap junctions. These results suggest that gap junction channels provide a direct conduit for mitogens released in response to bFGF to effectively regulate proliferation during corticogenesis.

Matrix metalloproteinase (MMP)-9 induced by Wnt signaling increases the proliferation and migration of embryonic neural stem cells at low O2 levels.

Recent studies have shown that various neural and embryonic stem cells cultured in 1–8% oxygen (O2), levels lower than those typically used in cell culture (20.9%), displayed increased rates of proliferation; however, the molecular mechanisms underlying these changes are largely undefined. In this study, using rigorously controlled O2 levels, we found that neural stem cells (NSCs) from embryonic day 15 rat cortex increased their rate of proliferation and migration in 1% O2 relative to 20% O2 without changes in viability. We sought to identify molecular changes in NSCs grown in 1% O2 that might account for these increases. In 1% O2, levels of the hypoxia-inducible transcription factor HIF-1α were transiently increased. Reduced adherence of NSCs in 1% O2 to basement membrane-coated plates was observed, and quantitative RT-PCR analysis confirmed that the levels of mRNA for an assortment of cell adhesion and extracellular matrix molecules were altered. Most notable was a 5-fold increase in matrix metalloproteinase (MMP)-9 mRNA. Specific inhibition of MMP-9 activity, verified using a fluorescent substrate assay, prevented the increase in proliferation and migration in 1% O2. The canonical Wnt pathway was recently shown to be activated in stem cells in low O2 via HIF-1α. Inhibition of Wnt signaling by DKK-1 also prevented the increase in proliferation, migration, and MMP-9 expression. Thus, MMP-9 is a key molecular effector, downstream of HIF-1α and Wnt activation, responsible for increased rates of NSC proliferation and migration in 1% O2.

Misexpression of IGF-I in the mouse lens expands the transitional zone and perturbs lens polarization

Insulin-like growth factor-I (IGF-I) has been implicated as a regulator of lens development. Experiments performed in the chick have indicated that IGF-I can stimulate lens fiber cell differentiation and may be involved in controlling lens polarization. To assess IGF-I activity on mammalian lens cells in vivo, we generated transgenic mice in which this factor was overexpressed from the alphaA-crystallin promoter. Interestingly, we observed no premature differentiation of lens epithelial cells. The pattern of lens polarization was perturbed, with an apparent expansion of the epithelial compartment towards the posterior lens pole. The distribution of immunoreactivity for MIP26 and p57(KIP2) and a modified pattern of proliferation suggested that this morphological change was best described as an expansion of the germinative and transitional zones. The expression of IGF-I signaling components in the normal transitional zone and expansion of the transitional zone in the transgenic lens both suggest that endogenous IGF-I may provide a spatial cue that helps to control the normal location of this domain.

The homeobox transcription factor Barx2 regulates chondrogenesis during limb development

Among the many factors involved in regulation of chondrogenesis, bone morphogenetic proteins (BMPs) and members of the Sox and homeobox transcription factor families have been shown to have crucial roles. Of these regulators, the homeobox transcription factors that function during chondrogenesis have been the least well defined. We show here that the homeobox transcription factor Barx2 is expressed in primary mesenchymal condensations, digital rays, developing joints and articular cartilage of the developing limb, suggesting that it plays a role in chondrogenesis. Using retroviruses and antisense oligonucleotides to manipulate Barx2 expression in limb bud micromass cultures, we determined that Barx2 is necessary for mesenchymal aggregation and chondrogenic differentiation. In accordance with these findings, Barx2 regulates the expression of several genes encoding cell-adhesion molecules and extracellular matrix proteins, including NCAM and collagen II (Col2a1) in the limb bud. Barx2 bound to elements within the cartilage-specific Col2a1 enhancer, and this binding was reduced by addition of Barx2 or Sox9 antibodies, or by mutation of a HMG box adjacent to the Barx2-binding element, suggesting cooperation between Barx2 and Sox proteins. Moreover, both Barx2 and Sox9 occupy Col2a1 enhancer during chondrogenesis in vivo. We also found that two members of the BMP family that are crucial for chondrogenesis, GDF5 and BMP4, regulate the pattern of Barx2 expression in developing limbs. Based on these data, we suggest that Barx2 acts downstream of BMP signaling and in concert with Sox proteins to regulate chondrogenesis.

Bmp7 regulates branching morphogenesis of the lacrimal gland by promoting mesenchymal proliferation and condensation

The lacrimal gland provides an excellent model with which to study the epithelial-mesenchymal interactions that are crucial to the process of branching morphogenesis. In the current study, we show that bone morphogenetic protein 7 (Bmp7) is expressed with a complex pattern in the developing gland and has an important role in regulating branching. In loss-of-function analyses, we find that Bmp7-null mice have distinctive reductions in lacrimal gland branch number, and that inhibition of Bmp activity in gland explant cultures has a very similar consequence. Consistent with this, exposure of whole-gland explants to recombinant Bmp7 results in increased branch number. In determining which cells of the gland respond directly to Bmp7, we have tested isolated mesenchyme and epithelium. We find that, as expected, Bmp4 can suppress bud extension in isolated epithelium stimulated by Fgf10, but interestingly, Bmp7 has no discernible effect. Bmp7 does, however, stimulate a distinct response in mesenchymal cells. This manifests as a promotion of cell division and formation of aggregates, and upregulation of cadherin adhesion molecules, the junctional protein connexin 43 and ofα -smooth muscle actin. These data suggest that in this branching system, mesenchyme is the primary target of Bmp7 and that formation of mesenchymal condensations characteristic of signaling centers may be enhanced by Bmp7. Based on the activity of Bmp7 in promoting branching, we also propose a model suggesting that a discrete region of Bmp7-expressing head mesenchyme may be crucial in determining the location of the exorbital lobe of the gland.