Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics.

During tissue development, multipotent progenitors differentiate into specific cell types in characteristic spatial and temporal patterns. We addressed the mechanism linking progenitor identity and differentiation rate in the neural tube, where motor neuron (MN) progenitors differentiate more rapidly than other progenitors. Using single cell transcriptomics, we defined the transcriptional changes associated with the transition of neural progenitors into MNs. Reconstruction of gene expression dynamics from these data indicate a pivotal role for the MN determinant Olig2 just prior to MN differentiation. Olig2 represses expression of the Notch signaling pathway effectors Hes1 and Hes5. Olig2 repression of Hes5 appears to be direct, via a conserved regulatory element within the Hes5 locus that restricts expression from MN progenitors. These findings reveal a tight coupling between the regulatory networks that control patterning and neuronal differentiation and demonstrate how Olig2 acts as the developmental pacemaker coordinating the spatial and temporal pattern of MN generation.

A novel small molecule that disrupts a key event during the oocyte-to-embryo transition in C. elegans.

The complex cellular events that occur in response to fertilization are essential for mediating the oocyte-to-embryo transition. Here, we describe a comprehensive small-molecule screen focused on identifying compounds that affect early embryonic events in Caenorhabditis elegans We identify a single novel compound that disrupts early embryogenesis with remarkable stage and species specificity. The compound, named C22, primarily impairs eggshell integrity, leading to osmotic sensitivity and embryonic lethality. The C22-induced phenotype is dependent upon the upregulation of the LET-607/CREBH transcription factor and its candidate target genes, which primarily encode factors involved in diverse aspects of protein trafficking. Together, our data suggest that in the presence of C22, one or more key components of the eggshell are inappropriately processed, leading to permeable, inviable embryos. The remarkable specificity and reversibility of this compound will facilitate further investigation into the role and regulation of protein trafficking in the early embryo, as well as serve as a tool for manipulating the life cycle for other studies such as those involving aging.

Targeted germ line disruptions reveal general and species-specific roles for paralog group 1 hox genes in zebrafish.

BACKGROUND: The developing vertebrate hindbrain is transiently segmented into rhombomeres by a process requiring Hox activity. Hox genes control specification of rhombomere fates, as well as the stereotypic differentiation of rhombomere-specific neuronal populations. Accordingly, germ line disruption of the paralog group 1 (PG1) Hox genes Hoxa1 and Hoxb1 causes defects in hindbrain segmentation and neuron formation in mice. However, antisense-mediated interference with zebrafish hoxb1a and hoxb1b (analogous to murine Hoxb1 and Hoxa1, respectively) produces phenotypes that are qualitatively and quantitatively distinct from those observed in the mouse. This suggests that PG1 Hox genes may have species-specific functions, or that anti-sense mediated interference may not completely inactivate Hox function in zebrafish. RESULTS: Using zinc finger and TALEN technologies, we disrupted hoxb1a and hoxb1b in the zebrafish germ line to establish mutant lines for each gene. We find that zebrafish hoxb1a germ line mutants have a more severe phenotype than reported for Hoxb1a antisense treatment. This phenotype is similar to that observed in Hoxb1 knock out mice, suggesting that Hoxb1/hoxb1a have the same function in both species. Zebrafish hoxb1b germ line mutants also have a more severe phenotype than reported for hoxb1b antisense treatment (e.g. in the effect on Mauthner neuron differentiation), but this phenotype differs from that observed in Hoxa1 knock out mice (e.g. in the specification of rhombomere 5 (r5) and r6), suggesting that Hoxa1/hoxb1b have species-specific activities. We also demonstrate that Hoxb1b regulates nucleosome organization at the hoxb1a promoter and that retinoic acid acts independently of hoxb1b to activate hoxb1a expression. CONCLUSIONS: We generated several novel germ line mutants for zebrafish hoxb1a and hoxb1b. Our analyses indicate that Hoxb1 and hoxb1a have comparable functions in zebrafish and mouse, suggesting a conserved function for these genes. In contrast, while Hoxa1 and hoxb1b share functions in the formation of r3 and r4, they differ with regards to r5 and r6, where Hoxa1 appears to control formation of r5, but not r6, in the mouse, whereas hoxb1b regulates formation of r6, but not r5, in zebrafish. Lastly, our data reveal independent regulation of hoxb1a expression by retinoic acid and Hoxb1b in zebrafish.

Getting an A with the 3Cs: Chromosome Conformation Capture for Undergraduates.

Chromosome conformation capture (3C) is a powerful tool that has spawned a family of similar techniques (e.g., Hi-C, 4C, and 5C, referred to here as 3C techniques) that provide detailed information of the three-dimensional organization of chromatin. The 3C techniques have been used in a wide range of studies, from monitoring the changes in chromatin organization in cancer cells to identifying enhancer contacts made with gene promoters. While many of the studies using these techniques are asking big genome-wide questions with intricate sample types (i.e., single-cell analysis), what is often lost is that the 3C techniques are grounded in basic molecular biology methods that are applicable to a broad range of studies. By addressing tightly focused questions of chromatin organization, this cutting-edge technique can be used to enhance the undergraduate research and teaching lab experience. This paper presents a 3C protocol and provides adaptations and points of emphasis for implementation at primarily undergraduate institutions in undergraduate research and teaching experiences.

A novel ex vivo method for measuring whole brain metabolism in model systems.

BACKGROUND: Many neuronal and glial diseases have been associated with changes in metabolism. Therefore, metabolic reprogramming has become an important area of research to better understand disease at the cellular level, as well as to identify targets for treatment. Model systems are ideal for interrogating metabolic questions in a tissue dependent context. However, while new tools have been developed to study metabolism in cultured cells there has been less progress towards studies in vivo and ex vivo. NEW METHOD: We have developed a method using newly designed tissue restraints to adapt the Agilent XFe96 metabolic analyzer for whole brain analysis. These restraints create a chamber for Drosophila brains and other small model system tissues to reside undisrupted, while still remaining in the zone for measurements by sensor probes. RESULTS: This method generates reproducible oxygen consumption and extracellular acidification rate data for Drosophila larval and adult brains. Single brains are effectively treated with inhibitors and expected metabolic readings are observed. Measuring metabolic changes, such as glycolytic rate, in transgenic larval brains demonstrates the potential for studying how genotype affects metabolism. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: Current methodology either utilizes whole animal chambers to measure respiration, not allowing for targeted tissue analysis, or uses technically challenging MRI technology for in vivo analysis that is not suitable for smaller model systems. This new method allows for novel metabolic investigation of intact brains and other tissues ex vivo in a quick, and simplistic way with the potential for large-scale studies.

Dynamic nucleosome organization at hox promoters during zebrafish embryogenesis.

Nucleosome organization at promoter regions plays an important role in regulating gene activity. Genome-wide studies in yeast, flies, worms, mammalian embryonic stem cells and transformed cell lines have found well-positioned nucleosomes flanking a nucleosome depleted region (NDR) at transcription start sites. This nucleosome arrangement depends on DNA sequence (cis-elements) as well as DNA binding factors and ATP-dependent chromatin modifiers (trans-factors). However, little is understood about how the nascent embryonic genome positions nucleosomes during development. This is particularly intriguing since the embryonic genome must undergo a broad reprogramming event upon fusion of sperm and oocyte. Using four stages of early embryonic zebrafish development, we map nucleosome positions at the promoter region of 37 zebrafish hox genes. We find that nucleosome arrangement at the hox promoters is a progressive process that takes place over several stages. At stages immediately after fertilization, nucleosomes appear to be largely disordered at hox promoter regions. At stages after activation of the embryonic genome, nucleosomes are detectable at hox promoters, with positions becoming more uniform and more highly occupied. Since the genomic sequence is invariant during embryogenesis, this progressive change in nucleosome arrangement suggests that trans-factors play an important role in organizing nucleosomes during embryogenesis. Separating hox genes into expressed and non-expressed groups shows that expressed promoters have better positioned and occupied nucleosomes, as well as distinct NDRs, than non-expressed promoters. Finally, by blocking the retinoic acid-signaling pathway, we disrupt early hox gene transcription, but observe no effect on nucleosome positions, suggesting that active hox transcription is not a driving force behind the arrangement of nucleosomes at the promoters of hox genes during early development.