Sox21 homeologs autoregulate expression levels to control progression through neurogenesis.

The SRY HMG box transcription factor Sox21 plays multiple critical roles in neurogenesis, with its function dependent on concentration and developmental stage. In the allotetraploid Xenopus laevis, there are two homeologs of sox21, namely sox21.S and sox21.L. Previous studies focused on Sox21.S, but its amino acid sequence is divergent, lacking conserved poly-A stretches and bearing more similarity with ancestral homologs. In contrast, Sox21.L shares higher sequence similarity with mouse and chick Sox21. To determine if Sox21.S and Sox21.L have distinct functions, we conducted gain and loss-of-function studies in Xenopus embryos. Our studies revealed that Sox21.S and Sox21.L are functionally redundant, but Sox21.L is more effective at driving changes than Sox21.S. These results also support our earlier findings in ectodermal explants, demonstrating that Sox21 function is dose-dependent. While Sox21 is necessary for primary neuron formation, high levels prevent their formation. Strikingly, these proteins autoregulate, with high levels of Sox21.L reducing sox21.S and sox21.L mRNA levels, and decreased Sox21.S promoting increased expression of sox21.L. Our findings shed light on the intricate concentration-dependent roles of Sox21 homeologs in Xenopus neurogenesis.

Xenopus Sox11 Partner Proteins and Functional Domains in Neurogenesis.

Sox11, a member of the SoxC family of transcription factors, has distinct functions at different times in neural development. Studies in mouse, frog, chick, and zebrafish show that Sox11 promotes neural fate, neural differentiation, and neuron maturation in the central nervous system. These diverse roles are controlled in part by spatial and temporal-specific protein interactions. However, the partner proteins and Sox11-interaction domains underlying these diverse functions are not well defined. Here, we identify partner proteins and the domains of Xenopus laevis Sox11 required for protein interaction and function during neurogenesis. Our data show that Sox11 co-localizes and interacts with Pou3f2 and Neurog2 in the anterior neural plate and in early neurons, respectively. We also demonstrate that Sox11 does not interact with Neurog1, a high-affinity partner of Sox11 in the mouse cortex, suggesting that Sox11 has species-specific partner proteins. Additionally, we determined that the N-terminus including the HMG domain of Sox11 is necessary for interaction with Pou3f2 and Neurog2, and we established a novel role for the N-terminal 46 amino acids in the specification of placodal progenitors. This is the first identification of partner proteins for Sox11 and of domains required for partner-protein interactions and distinct roles in neurogenesis.

Saccoglossus kowalevskii: Evo-devo insights from the mud.

Hemichordates have long been recognized as a critical group for addressing hypotheses of chordate origins. Historically this was due to anatomical traits that resembled those of chordates, most strikingly the dorsolateral gill slits. As molecular data and phylogenetic analyses were found to support a close phylogenetic relationship between hemichordates and chordates within the deuterostomes, interest was revived in hemichordates. In particular, Saccoglossus kowalevskii has been developed as a molecular model to represent hemichordate developmental biology. Herein, we highlight the considerations when choosing a particular species to study and the challenges we encountered when developing S. kowalevskii. We discuss our findings and how method and tool development enabled them, and how we envision expanding our repertoire of molecular tools in the future. Establishing a new model organism comes with many obstacles-from identifying a reliable season to collect animals, to developing modern molecular techniques. The Saccoglossus research community has benefited greatly from the collaborations and teamwork established over the years. As a result, Saccoglossus is well positioned to contribute to a new century of evolutionary developmental (evo-devo) research.

Sox21 regulates the progression of neuronal differentiation in a dose-dependent manner.

Members of the SoxB transcription factor family play critical roles in the regulation of neurogenesis. The SoxB1 proteins are required for the induction and maintenance of a proliferating neural progenitor population in numerous vertebrates, however the role of the SoxB2 protein, Sox21, is less clear due to conflicting results. To clarify the role of Sox21 in neurogenesis, we examined its function in the Xenopus neural plate. Here we report that misexpression of Sox21 expands the neural progenitor domain, and represses neuron formation by binding to Neurogenin (Ngn2) and blocking its function. Conversely, we found that Sox21 is also required for neuron formation, as cells lacking Sox21 undergo cell death and thus are unable to differentiate. Together our data indicate that Sox21 plays more than one role in neurogenesis, where a threshold level is required for cell viability and normal differentiation of neurons, but a higher concentration of Sox21 inhibits neuron formation and instead promotes progenitor maintenance.

Spatiotemporal development of the embryonic nervous system of Saccoglossus kowalevskii.

Defining the organization and temporal onset of key steps in neurogenesis in invertebrate deuterostomes is critical to understand the evolution of the bilaterian and deuterostome nervous systems. Although recent studies have revealed the organization of the nervous system in adult hemichordates, little attention has been paid to neurogenesis during embryonic development in this third major phylum of deuterostomes. We examine the early events of neural development in the enteropneust hemichordate Saccoglossus kowalevskii by analyzing the expression of 11 orthologs of key genes associated with neurogenesis in an expansive range of bilaterians. Using in situ hybridization (ISH) and RT-PCR, we follow the course of neural development to track the transition of the early embryonic diffuse nervous system to the more regionalized midline nervous system of the adult. We show that in Saccoglossus, neural progenitor markers are expressed maternally and broadly encircle the developing embryo. An increase in their expression and the onset of pan neural markers, indicate that neural specification occurs in late blastulae - early gastrulae. By mid-gastrulation, punctate expression of markers of differentiating neurons encircling the embryo indicate the presence of immature neurons, and at the end of gastrulation when the embryo begins to elongate, markers of mature neurons are expressed. At this stage, expression of a subset of neuronal markers is concentrated along the trunk ventral and dorsal midlines. These data indicate that the diffuse embryonic nervous system of Saccoglossus is transient and quickly reorganizes before hatching to resemble the adult regionalized, centralized nervous system. This regionalization occurs at a much earlier developmental stage than anticipated indicating that centralization is not linked in S. kowalevskii to a lifestyle change of a swimming larva metamorphosing to a crawling worm-like adult.

Lipooligosaccharide is required for the generation of infectious elementary bodies in Chlamydia trachomatis.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are the main lipid components of bacterial outer membranes and are essential for cell viability in most Gram-negative bacteria. Here we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, block the synthesis of LOS in the obligate intracellular bacterial pathogen Chlamydia trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication. However, bacteria grown under these conditions were no longer infectious. In the presence of LpxC inhibitors, replicative reticulate bodies accumulated in enlarged inclusions but failed to express selected late-stage proteins and transition to elementary bodies, a Chlamydia developmental form that is required for invasion of mammalian cells. These findings suggest the presence of an outer membrane quality control system that regulates Chlamydia developmental transition to infectious elementary bodies and highlights the potential application of LpxC inhibitors as unique class of antichlamydial agents.

Xenopus Sox3 activates sox2 and geminin and indirectly represses Xvent2 expression to induce neural progenitor formation at the expense of non-neural ectodermal derivatives.

The SRY-related, HMG box SoxB1 transcription factors are highly homologous, evolutionarily conserved proteins that are expressed in neuroepithelial cells throughout neural development. SoxB1 genes are down-regulated as cells exit the cell-cycle to differentiate and are considered functionally redundant in maintaining neural precursor populations. However, little is known about Sox3 function and its mode of action during primary neurogenesis. Using gain and loss-of-function studies, we analyzed Sox3 function in detail in Xenopus early neural development and compared it to that of Sox2. Through these studies we identified the first targets of a SoxB1 protein during primary neurogenesis. Sox3 functions as an activator to induce expression of the early neural genes, sox2 and geminin in the absence of protein synthesis and to indirectly inhibit the Bmp target Xvent2. As a result, Sox3 increases cell proliferation, delays neurogenesis and inhibits epidermal and neural crest formation to expand the neural plate. Our studies indicate that Sox3 and 2 have many similar functions in this process including the ability to activate expression of geminin in naïve ectodermal explants. However, there are some differences; Sox3 activates the expression of sox2, while Sox2 does not activate expression of sox3 and sox3 is uniquely expressed throughout the ectoderm prior to neural induction suggesting a role in neural competence. With morpholino-mediated knockdown of Sox3, we demonstrate that it is required for induction of neural tissue by BMP inhibition. Together these data indicate that Sox3 has multiple roles in early neural development including as a factor required for nogginmediated neural induction.

Cloning and developmental expression of the soxB2 genes, sox14 and sox21, during Xenopus laevis embryogenesis.

The Sox family of transcription factors is thought to regulate gene expression in a wide variety of developmental processes. Here we describe the cloning of the X. laevis orthologs of the SoxB2 family of transcription factors, sox14 and sox21. In situ hybridization revealed that sox14 expression is restricted to the hypothalamus, dorsal thalamus, the optic tectum, a region of the somatic motornucleus in the midbrain and hindbrain, the vestibular nuclei in the hindbrain and a discrete ventral domain in the developing spinal cord. In contrast to the limited expression domain of sox14, sox21 is found throughout the developing central nervous system, including the olfactory placodes, with strongest expression at the boundary between the midbrain and hindbrain.

Sox3 expression is maintained by FGF signaling and restricted to the neural plate by Vent proteins in the Xenopus embryo.

The formation of the nervous system is initiated when ectodermal cells adopt the neural fate. Studies in Xenopus demonstrate that inhibition of BMP results in the formation of neural tissue. However, the molecular mechanism driving the expression of early neural genes in response to this inhibition is unknown. Moreover, controversy remains regarding the sufficiency of BMP inhibition for neural induction. To address these questions, we performed a detailed analysis of the regulation of the soxB1 gene, sox3, one of the earliest genes expressed in the neuroectoderm. Using ectodermal explant assays, we analyzed the role of BMP, Wnt and FGF signaling in the regulation of sox3 and the closely related soxB1 gene, sox2. Our results demonstrate that both sox3 and sox2 are induced in response to BMP antagonism, but by distinct mechanisms and that the activation of both genes is independent of FGF signaling. However, both require FGF for the maintenance of their expression. Finally, sox3 genomic elements were identified and characterized and an element required for BMP-mediated repression via Vent proteins was identified through the use of transgenesis and computational analysis. Interestingly, none of the elements required for sox3 expression were identified in the sox2 locus. Together our data indicate that two closely related genes have unique mechanisms of gene regulation at the onset of neural development.

Biological and biochemical functions of RNA in the tetrahymena telomerase holoenzyme.

Telomerase extends chromosome ends by the synthesis of tandem simple-sequence repeats. Studies of minimal recombinant telomerase ribonucleoprotein (RNP) reconstituted in vitro have revealed sequences within the telomerase RNA subunit (TER) that are required to establish its internal template and other unique features of enzyme activity. Here we test the significance of these motifs following TER assembly into telomerase holoenzyme in vivo. We established a method for stable expression of epitope-tagged TER and TER variants in place of wild-type Tetrahymena TER. We found that sequence substitutions in nontemplate regions of TER altered telomere length maintenance in vivo, with an increase or decrease in the set point for telomere length homeostasis. We also characterized the in vitro activity of the telomerase holoenzymes reconstituted with TER variants, following RNA-based RNP affinity purification from cell extracts. We found that nontemplate sequence substitutions imposed specific defects in the fidelity and processivity of template use. These findings demonstrate nontemplate functions of TER that are critical for the telomerase holoenzyme catalytic cycle and for proper telomere length maintenance in vivo.