Mitochondrial activity, embryogenesis, and the dialogue between the big and little brains of the cell.

While it is clear that mitochondria play integral roles in cellular homeostasis, adaptation, cellular and survival, recent studies suggest possible roles for mitochondria as modulators of what were previously considered cytoplasmic/nuclear signaling systems. Embryonic patterning has been linked to asymmetries in mitochondria-based respiratory activity. As outlined by Coffman (2009), defining the role of mitochondria as modulators of embryonic patterning is inherently difficult, given their essential metabolic roles. This review attempts to place mitochondrial-transcription factor interactions in the context of the early development of the tetrapod Xenopus laevis, where a number of the proteins and signaling systems known to play critical roles in embryonic patterning, e.g. ß-catenin, NF-κB, p53, and STAT3, have been found to localize to mitochondria.

Thinking about the conceptual foundations of the biological sciences.

Not until the core concepts of biology are clearly defined and become the focus of instruction and assessment can we expect meaningful improvements in biological literacy and the removal of unnecessary barriers to student engagement.

Teaching without a textbook: strategies to focus learning on fundamental concepts and scientific process.

Textbooks are ubiquitous. They are available for almost every conceivable subdiscipline of biology, and few of us would consider teaching a course without using a textbook. Over the years, they have become more colorful, more encyclopedic, and accompanied by more ancillary materials such as CD-ROMs, study guides, and websites. With all these tools to assist our students, it seems reasonable that they are able to learn more and better than ever. Thus, the question most instructors ask themselves is most likely which textbook to use, not whether to use a textbook. But does the use of textbooks really help students learn better? In this Point of View, I invited a commentary on this question from a faculty member who has decided to abandon the use of a textbook in an introductory level cell and molecular biology course.

SOX7 is an immediate-early target of VegT and regulates Nodal-related gene expression in Xenopus.

In zebrafish, the divergent F-type SOX casanova acts downstream of Nodal signaling to specify endoderm. While no casanova orthologs have been identified in tetrapods, the F-type SOX, SOX7, is supplied maternally in Xenopus (Fawcett and Klymkowsky, 2004. GER 4, 29). Subsequent RT-PCR and section-based in situ hybridization analyses indicate that SOX7 mRNA is localized to the vegetal region of the blastula-stage embryo. Overexpression and maternal depletion studies reveal that the T-box transcription factor VegT, which initiates mesoendodermal differentiation, directly regulates SOX7 expression. SOX7, but not SOX17 (another F-type SOX), binds to sites within the Xnr5 promoter and SOX7, but not SOX17, induces expression of the Nodal-related genes Xnr1, Xnr2, Xnr4, Xnr5, and Xnr6, the homeodomain transcription factor Mixer, and the endodermal marker SOX17beta; both SOX7 and SOX17 induce expression of the pan-endodermal marker endodermin. SOX7's induction of Xnr expression in animal caps is independent of Mixer and Nodal signaling. In animal caps, VegT's ability to induce Mixer and Edd appears to depend upon SOX7 activity. Whole embryo experiments suggests that vegetal factors partially compensate for the absence of SOX7. Based on the antagonistic effects of SOX7 and SOX3 (Zhang et al., 2004. Dev. Biol. 273, 23) and their common binding sites in the Xnr5 promoter, we propose a model in which competitive interactions between these two proteins are involved in refining the domain of endodermal differentiation.

Embryonic expression of Xenopus laevis SOX7.

SOX7, first described in Xenopus laevis by Shiozawa et al. [Biochim. Biophys. Acta 1309(1996)73], is a member, along with SOXs 17 and 18, of the F subgroup of SOX-type transcription factors. As part of a study of maternal SOX proteins that may modulate beta-catenin signaling, we isolated a XSOX7 cDNA from oocyte RNA and examined the pattern of XSOX7 expression during early development. While present maternally cell-type specific expression was first observed in the ciliated cells within the epidermis of early neurula stage embryos. As development proceeds, the pattern of XSOX7 expression becomes increasingly complex. XSOX7 is expressed in the aortic arch, the olfactory pit, the stomodeal depression, the procardiac tube, within cells of the developing embryonic vasculature, in the notochord, and within the hindbrain. XSOX7 expression continues within the hindbrain in 3-day old ( approximately stage 40) larvae. Given its widespread expression, XSOX7 is likely to be involved in a number of developmental processes.

The beta-catenin/VegT-regulated early zygotic gene Xnr5 is a direct target of SOX3 regulation.

In Xenopus laevis, beta-catenin-mediated dorsal axis formation can be suppressed by overexpression of the HMG-box transcription factor XSOX3. Mutational analysis indicates that this effect is due not to the binding of XSOX3 to beta-catenin nor to its competition with beta-catenin-regulated TCF-type transcription factors for specific DNA binding sites, but rather to SOX3 binding to sites within the promoter of the early VegT- and beta-catenin-regulated dorsal-mesoderm-inducing gene Xnr5. Although B1-type SOX proteins, such as XSOX3, are commonly thought to act as transcriptional activators, XSOX3 acts as a transcriptional repressor of Xnr5 in both the intact embryo and animal caps injected with VegT RNA. Expression of a chimeric polypeptide composed of XSOX3 and a VP16 transcriptional activation domain or morpholino-induced decrease in endogenous XSOX3 polypeptide levels lead to an increase in Xnr5 expression, as does injection of an anti-XSOX3 antibody that inhibits XSOX3 DNA binding. These observations indicate that maternal XSOX3 acts in a novel manner to restrict Xnr5 expression to the vegetal hemisphere.

Limb development in a "nonmodel" vertebrate, the direct-developing frog Eleutherodactylus coqui.

Mechanisms that mediate limb development are regarded as highly conserved among vertebrates, especially tetrapods. Yet, this assumption is based on the study of relatively few species, and virtually none of those that display any of a large number of specialized life-history or reproductive modes, which might be expected to affect developmental pattern or process. Direct development is an alternative life history found in many anuran amphibians. Many adult features that form after hatching in metamorphic frogs, such as limbs, appear during embryogenesis in direct-developing species. Limb development in the direct-developing frog Eleutherodactylus coqui presents a mosaic of apparently conserved and novel features. The former include the basic sequence and pattern of limb chondrogenesis, which are typical of anurans generally and appear largely unaffected by the gross shift in developmental timing; expression of Distal-less protein (Dlx) in the distal ectoderm; expression of the gene Sonic hedgehog (Shh) in the zone of polarizing activity (ZPA); and the ability of the ZPA to induce supernumerary digits when transplanted to the anterior region of an early host limb bud. Novel features include the absence of a morphologically distinct apical ectodermal ridge, the ability of the limb to continue distal outgrowth and differentiation following removal of the distal ectoderm, and earlier cessation of the inductive ability of the ZPA. Attempts to represent tetrapod limb development as a developmental "module" must allow for this kind of evolutionary variation among species.

Paranemin and the organization of desmin filament networks.

De novo expression of vimentin, GFAP or peripherin leads to the assembly of an extended intermediate filament network in intermediate filament-free SW13/cl.2 cells. Desmin, in contrast, does not form extended filament networks in either SW13/cl.2 or intermediate filament-free mouse fibroblasts. Rather, desmin formed short thickened filamentous structures and prominent spot-like cytoplasmic aggregates that were composed of densely packed 9-11 nm diameter filaments. Analysis of stably transfected cell lines indicates that the inability of desmin to form extended networks is not due to a difference in the level of transgene expression. Nestin, paranemin and synemin are large intermediate filament proteins that coassemble with desmin in muscle cells. Although each of these large intermediate filament proteins colocalized with desmin when coexpressed in SW-13 cells, expression of paranemin, but not synemin or nestin, led to the formation of an extended desmin network. A similar rescue of desmin network organization was observed when desmin was coexpressed with vimentin, which coassembles with desmin, or with keratins, which formed a distinct filament network. These studies demonstrate that desmin filaments differ in their organizational properties from the other vimentin-like intermediate filament proteins and appear to depend upon coassembly with paranemin, at least when they are expressed in non-muscle cells, in order to form an extended filament network.

Cadherins and catenins, Wnts and SOXs: embryonic patterning in Xenopus.

Wnt signaling plays a critical role in a wide range of developmental and oncogenic processes. Altered gene regulation by the canonical Wnt signaling pathway involves the cytoplasmic stabilization of beta-catenin, a protein critical to the assembly of cadherin-based cell-cell adherence junctions. In addition to binding to cadherins, beta-catenin also interacts with transcription factors of the TCF-subfamily of HMG box proteins and regulates their activity. The Xenopus embryo has proven to be a particularly powerful experimental system in which to study the role of Wnt signaling components in development and differentiation. We review this literature, focusing on the role of Wnt signaling and interacting components in establishing patterns within the early embryo.

A comparative evaluation of beta-catenin and plakoglobin signaling activity.

Vertebrates have two Armadillo-like proteins, beta-catenin and plakoglobin. Mutant forms of beta-catenin with oncogenic activity are found in many human tumors, but plakoglobin mutations are not commonly found. In fact, plakoglobin has been proposed to suppress tumorigenesis. To assess differences between beta-catenin and plakoglobin, we compared several of their biochemical properties. After transient transfection of 293T cells with an expression vector encoding either of the two proteins, soluble wild type beta-catenin does not significantly accumulate, whereas soluble wild type plakoglobin is readily detected. As anticipated, beta-catenin is stabilized by the oncogenic mutation S37A; however, the analogous mutation in plakoglobin (S28A) does not alter its half-life. S37A-beta-catenin activates a TCF/LEF-dependent reporter 20-fold more potently than wild type beta-catenin, and approximately 5-fold more potently than wild type or S28A plakoglobin. These differences may be attributable to an enhanced affinity of S37A beta-catenin for LEF1 and TCF4, as observed here by immunoprecipitation assays. We show that the carboxyl-terminal domain is largely responsible for the difference in signaling and that the Armadillo repeats account for the remainder of the difference. The relatively weak signaling by plakoglobin and the failure of the S28A mutation to enhance its stability, may explain why plakoglobin mutations are infrequent in malignancies.

Regulation of Wnt signaling by Sox proteins: XSox17 alpha/beta and XSox3 physically interact with beta-catenin.

Using a functional screen in Xenopus embryos, we identified a novel function for the HMG box protein XSox17 beta. Ectopic expression of XSox17 beta ventralizes embryos by inhibiting the Wnt pathway downstream of beta-catenin but upstream of the Wnt-responsive gene Siamois. XSox17 beta also represses transactivation of a TCF/LEF-dependent reporter construct by Wnt and beta-catenin. In animal cap experiments, it both activates transcription of endodermal genes and represses beta-catenin-stimulated expression of dorsal genes. The inhibition activity of XSox17 beta maps to a region C-terminal to the HMG box; this region of XSox17 beta physically interacts with the Armadillo repeats of beta-catenin. Two additional Sox proteins, XSox17 alpha and XSox3, likewise bind to beta-catenin and inhibit its TCF-mediated signaling activity. These results reveal an unexpected mechanism by which Sox proteins can modulate Wnt signaling pathways.

Membrane-anchored plakoglobins have multiple mechanisms of action in Wnt signaling.

In Wnt signaling, beta-catenin and plakoglobin transduce signals to the nucleus through interactions with TCF-type transcription factors. However, when plakoglobin is artificially engineered to restrict it to the cytoplasm by fusion with the transmembrane domain of connexin (cnxPg), it efficiently induces a Wnt-like axis duplication phenotype in Xenopus. In Xenopus embryos, maternal XTCF3 normally represses ventral expression of the dorsalizing gene Siamois. Two models have been proposed to explain the Wnt-like activity of cnxPg: 1) that cnxPg inhibits the machinery involved in the turnover of cytosolic beta-catenin, which then accumulates and inhibits maternal XTCF3, and 2) that cnxPg directly acts to inhibit XTCF3 activity. To distinguish between these models, we created a series of N-terminal deletion mutations of cnxPg and examined their ability to induce an ectopic axis in Xenopus, activate a TCF-responsive reporter (OT), stabilize beta-catenin, and colocalize with components of the Wnt signaling pathway. cnxPg does not colocalize with the Wnt pathway component Dishevelled, but it does lead to the redistribution of APC and Axin, two proteins involved in the regulation of beta-catenin turnover. Expression of cnxPg increases levels of cytosolic beta-catenin; however, this effect does not completely explain its signaling activity. Although cnxPg and Wnt-1 stabilize beta-catenin to similar extents, cnxPg activates OT to 10- to 20-fold higher levels than Wnt-1. Moreover, although LEF1 and TCF4 synergize with beta-catenin and plakoglobin to activate OT, both suppress the signaling activity of cnxPg. In contrast, XTCF3 suppresses the signaling activity of both beta-catenin and cnxPg. Both exogenous XLEF1 and XTCF3 are sequestered in the cytoplasm of Xenopus cells by cnxPg. Based on these data, we conclude that, in addition to its effects on beta-catenin, cnxPg interacts with other components of the Wnt pathway, perhaps TCFs, and that these interactions contribute to its signaling activity.

Inhibition of neural crest migration in Xenopus using antisense slug RNA.

Based primarily on studies in the chick, it has been assumed that the zinc finger transcription factor Slug is required for neural crest migration. In the mouse, however, Slug is not expressed in the premigratory neural crest, which forms normally in Slug -/- animals. To study the role of Slug in Xenopus laevis, we used the injection of XSlug antisense RNA and tissue transplantation. Injection of Slug antisense RNA did not suppress the early expression of the related gene XSnail, but led to reduced expression of both XSlug and XSnail in later stage embryos, whereas the expression of another neural crest marker, XTwist, was not affected. Down-regulation of XSlug and XSnail was associated with the inhibition of neural crest cell migration and the reduction or loss of many neural crest derivatives. In particular, the formation of rostral cartilages was often highly aberrant, whereas the posterior cartilages were less frequently affected. The effects of Slug antisense RNA on neural crest migration and cartilage formation were rescued by the injection of either XSlug or XSnail mRNA. These studies indicate that XSlug is required for neural crest migration, that XSlug and XSnail may be functionally redundant, and that both genes are required to maintain each other's expression in the neural crest development of xenopus laevis.

Plakophilin, armadillo repeats, and nuclear localization.

Plakophilins are armadillo-repeat containing proteins, identified through their localization to desmosomes. Expressed in a wide range of tissues, plakophilins are largely nuclear in most cell types [Schmidt et al. (1997) Cell Tissue Res 290:481; Mertens et al. (1996) J. Cell Biol 135:1009]. Using Xenopus embryos and cultured A6 cells, together with myc- and green fluorescent protein (GFP)-tags, we found that both the N-terminal, non-armadillo repeat "head" and the C-terminal armadillo repeat-containing regions can enter nuclei. The "arm" repeat domain is predominantly cytoplasmic and concentrated at the cell cortex, whereas the head and full-length polypeptides are concentrated in the nucleus. The head domain can also be seen to decorate and disrupt keratin filament network organization in some cells. In the course of these studies, we found that the distribution of the myc-epitope and green fluorescence differed in fixed cells, e.g., while the green fluorescence of a myc- and GFP-tagged head domain polypeptide was usually exclusively nuclear, a substantial fraction of the myc-immunoreactivity was cytoplasmic. Treating cells with the translation inhibitor cycloheximide reduces the cytoplasmic myc-signal, suggesting that it represented nascent polypeptides awaiting folding and nuclear import. Based on these types of experiments, GFP can be seen as a marker of the distribution of the mature form of the tagged polypeptide.

Jaw muscle development as evidence for embryonic repatterning in direct-developing frogs.

The Puerto Rican direct-developing frog Eleutherodactylus coqui (Leptodactylidae) displays a novel mode of jaw muscle development for anuran amphibians. Unlike metamorphosing species, several larval-specific features never form in E. coqui; embryonic muscle primordia initially assume an abbreviated, mid-metamorphic configuration that is soon remodelled to form the adult morphology before hatching. Also lacking are both the distinct population of larval myofibres and the conspicuous, larval-to-adult myofibre turnover that are characteristic of muscle development in metamorphosing species. These modifications are part of a comprehensive alteration in embryonic cranial patterning that has accompanied life history evolution in this highly speciose lineage. Embryonic 'repatterning' in Eleutherodactylus may reflect underlying developmental mechanisms that mediate the integrated evolution of complex structures. Such mechanisms may also facilitate, in organisms with a primitively complex life cycle, the evolutionary dissociation of embryonic, larval, and adult features.

Cytoplasmically anchored plakoglobin induces a WNT-like phenotype in Xenopus.

Plakoglobin is one of two vertebrate proteins closely related to the Drosophila segment polarity gene product armadillo. Overexpression of plakoglobin induces neural axis duplication in Xenopus and the exogenous plakoglobin is localized to nuclei (Karnovsky, A., and Klymkowsky, M. W., Proc. Natl. Acad. Sci. USA 92, 4255, 1995; Rubenstein, A., et al., Dev. Genet., 1997, in press). We have carried out a series of experiments to test whether the nuclear localization of plakoglobin is required for its inductive effects. Prior to the midblastula transition exogenous plakoglobin is cytoplasmic and concentrated in the cortical regions of blastomeres; after the midblastula transition exogenous plakoglobin accumulates in embryonic nuclei. The addition of a "nuclear localization sequence" does not change the timing of plakoglobin's nuclear localization, suggesting that it is anchored in the cytoplasm prior to the midblastula transition. Next, we constructed two "membrane-anchored" forms of plakoglobin. These are exclusively cytoplasmic; yet both were as effective at producing a "Wnt-like" axis duplication as were "free," unfettered forms of plakoglobin. Moreover, expression of anchored plakoglobins had no apparent effect on the cytoplasmic or nuclear levels of beta-catenin. These data indicate that plakoglobin can act cytoplasmically to generate a WNT-like phenotype. Taken together with the ventralizing effects of a mutant from of the XTcf-3 transcription factor, described by Molenaar et al. Cell 86, 391, 1996, we speculate that in the early Xenopus embryo, activation of plakoglobin (or beta-catenin) inhibits the activity of XTcf-3 or a XTcf-3-like factor.

Localizing the adhesive and signaling functions of plakoglobin.

Plakoglobin (PKG) is a major component of cell-cell adhesive junctions. It is also closely related to the Drosophila segment polarity gene product armadillo and can induce a WNT-like neural axis duplication (NAD) phenotype in Xenopus [Kamovsky and Klymkowsky, 1995.] To define the regions of PKG involved in cell adhesion and inductive signaling, we examined the behavior of mutated forms of PKG in Xenopus. Deletion of amino acids 22 through 39 (in the Xenopus PKG sequence increased the apparent stability of the polypeptide within the embryo and increased its ability to induce a WNT-like, NAD phenotype when expressed in the vegetal hemisphere. The N-terminal "head" and first 6 "ARM" repeats of PKG, or the C-terminal "tail" and the last 3 "ARM" repeats, could be removed without destroying the remaining polypeptide's ability to induce a NAD phenotype. The nuclear localization of mutant PKGs, however, was not strictly correlated with the ability to induce a NAD phenotype, i.e., some inactive polypeptides still accumulate in nuclei. Removal of PKG's head and first ARM repeat, which includes its alpha-catenin binding site, resulted in a polypeptide that, when expressed in the embryo, generated alpha dramatic cell adhesion defect. Removal of the next three ARM repeats abolished this adhesion defect, suggesting that the polypeptide no longer competes effectively with endogenous catenins for binding to cadherins. Expression of a form of PKG truncated after the 5th ARM repeat produced a milder cell adhesion defect, whereas expression of a polypeptide truncated after the 8th ARM repeat had little apparent effect on cellular adhesion. Based on these observations, we conclude that functions related to stability and cellular adhesion reside in the N-terminal region of the polypeptide, whereas the ability to induce a NAD phenotype lies within repeats 6-10 of the central region. The function(s) of the C-terminal domain of PKG remain uncertain at this time.