Prioritizing Women's Health in Germline Editing Research.

Although women are inextricably involved in the study of germline editing, their interests have not been significantly represented in debates about the evolution of genome editing technology. Discussions have taken place about effects of germline editing on women as parents and members of families, but key discussions about women's health and well-being as patients and subjects are lacking. This neglect is due in part to restrictions on uterine transfer of modified human embryos, a boundary that has now been crossed. As a result, only scant discussion has taken place about safeguards needed to ensure that women who participate in germline modification research are not exposed to disproportionate risk in exchange for benefits they might expect for future offspring. This omission sets the stage for serious ethical implications for women and their families.

Developmental gene expression provides clues to relationships between sponge and eumetazoan body plans.

Elucidation of macroevolutionary transitions between diverse animal body plans remains a major challenge in evolutionary biology. We address the sponge-eumetazoan transition by analyzing expression of a broad range of eumetazoan developmental regulatory genes in Sycon ciliatum (Calcispongiae). Here we show that many members of surprisingly numerous Wnt and Tgfß gene families are expressed higher or uniquely in the adult apical end and the larval posterior end. Genes involved in formation of the eumetazoan endomesoderm, such as ß-catenin, Brachyury and Gata, as well as germline markers Vasa and Pl10, are expressed during formation and maintenance of choanoderm, the feeding epithelium of sponges. Similarity in developmental gene expression between sponges and eumetazoans, especially cnidarians, is consistent with Haeckel's view that body plans of sponges and cnidarians are homologous. These results provide a framework for further studies aimed at deciphering ancestral developmental regulatory networks and their modifications during animal body plans evolution.

A randomised controlled pilot study of standardised counselling and cost-free pharmacotherapy for smoking cessation among stroke and TIA patients.

Background Tobacco use is a major risk factor for recurrent stroke. The provision of cost-free quit smoking medications has been shown to be efficacious in increasing smoking abstinence in the general population. Objective The objective of this pilot study was to assess the feasibility and obtain preliminary data on the effectiveness of providing cost-free quit smoking pharmacotherapy and counselling to smokers identified in a stroke prevention clinic. Trial design Cluster randomised controlled trial. Methods All patients seen at the Ottawa Hospital Stroke Prevention Clinic who smoked more five or more cigarettes per day, were ready to quit smoking in the next 30 days, and were willing to use pharmacotherapy were invited to participate in the study. All participants were advised to quit smoking and treated using a standardised protocol including counselling and pharmacotherapy. Participants were randomly assigned to either a prescription only usual care group or an experimental group who received a 4-week supply of cost-free quit smoking medications and a prescription for medication renewal. All patients received follow-up counselling. The primary outcome was biochemically validated quit rates at 26 weeks. The research coordinator conducting outcome assessment was blind to group allocation. Results Of 219 smokers screened, 73 were eligible, 28 consented and were randomised, and 25 completed the 26-week follow-up assessment. All 28 patients randomised were included in the analysis. The biochemically validated 7-day point prevalence abstinence rate in the experimental group compared to the usual care group was 26.6% vs 15.4% (adjusted OR 2.00, 95% CI 0.33 to 13.26; p=0.20). Conclusions It would be feasible to definitively evaluate this intervention in a large multi-site trial. Trial registration number http://ClinicalTrials.gov # UOHI2010-1.

meis1 regulates cyclin D1 and c-myc expression, and controls the proliferation of the multipotent cells in the early developing zebrafish eye.

During eye development, retinal progenitors are drawn from a multipotent, proliferative cell population. In Drosophila the maintenance of this cell population requires the function of the TALE-homeodomain transcription factor Hth, although its mechanisms of action are still unknown. Here we investigate whether members of the Meis gene family, the vertebrate homologs of hth, are also involved in early stages of eye development in the zebrafish. We show that meis1 is initially expressed throughout the eye primordium. Later, meis1 becomes repressed as neurogenesis is initiated, and its expression is confined to the ciliary margin, where the retinal stem population resides. Knocking down meis1 function through morpholino injection causes a delay in the G1-to-S phase transition of the eye cells, and results in severely reduced eyes. This role in cell cycle control is mediated by meis1 regulating cyclin D1 and c-myc transcription. The forced maintenance of meis1 expression in cell clones is incompatible with the normal differentiation of the meis1-expressing cells, which in turn tend to reside in undifferentiated regions of the retinal neuroepithelium, such as the ciliary margin. Together, these results implicate meis1 as a positive cell cycle regulator in early retinal cells, and provide evidence of an evolutionary conserved function for Hth/Meis genes in the maintenance of the proliferative, multipotent cell state during early eye development.

Identification and real-time imaging of a myc-expressing neutrophil population involved in inflammation and mycobacterial granuloma formation in zebrafish.

By enhancer trap screening we identified a transgenic zebrafish line showing leukocyte-specific YFP expression during late embryo and early larval development. Its enhancer detection insertion was mapped near a novel member of the myc proto-oncogene family, encoding transcription factors known to be important for regulating human myelopoiesis. Characterization of the zebrafish myc family showed that only this particular myc gene is strongly expressed in leukocytes. To identify the myc/YFP-expressing cell type, we re-examined specificity of described myeloid markers by multiplex fluorescent in situ hybridization, showing that lcp1 can be considered as a general leukocyte marker, csf1r as a macrophage-specific marker, and mpx and lyz as neutrophil-specific markers. Subsequent colocalization analysis defined the YFP-positive cells as a subset of the neutrophil population. Using real-time confocal imaging we demonstrate that these cells migrate to sites of inflammation and are involved in innate immune responses towards infections, including Mycobacterium marinum-induced granuloma formation.

Genomic regulatory blocks encompass multiple neighboring genes and maintain conserved synteny in vertebrates.

We report evidence for a mechanism for the maintenance of long-range conserved synteny across vertebrate genomes. We found the largest mammal-teleost conserved chromosomal segments to be spanned by highly conserved noncoding elements (HCNEs), their developmental regulatory target genes, and phylogenetically and functionally unrelated "bystander" genes. Bystander genes are not specifically under the control of the regulatory elements that drive the target genes and are expressed in patterns that are different from those of the target genes. Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2, rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the expression patterns of these genes even if located inside or beyond bystander genes, suggesting that the regulatory domain of a developmental regulatory gene can extend into and beyond adjacent transcriptional units. We termed these chromosomal segments genomic regulatory blocks (GRBs). After whole genome duplication in teleosts, GRBs, including HCNEs and target genes, were often maintained in both copies, while bystander genes were typically lost from one GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy GRBs of higher vertebrates intact. We show that loss of bystander genes and other mutational events suffered by duplicated GRBs in teleost genomes permits target gene identification and HCNE/target gene assignment. These findings explain the absence of evolutionary breakpoints from large vertebrate chromosomal segments and will aid in the recognition of position effect mutations within human GRBs.

Completing the set of h/E(spl) cyclic genes in zebrafish: her12 and her15 reveal novel modes of expression and contribute to the segmentation clock.

Somitogenesis is the key developmental process that lays down the framework for a metameric body in vertebrates. Somites are generated from the un-segmented presomitic mesoderm (PSM) by a pre-patterning process driven by a molecular oscillator termed the segmentation clock. The Delta-Notch intercellular signaling pathway and genes belonging to the hairy (h) and Enhancer of split (E(spl))-related (h/E(spl)) family of transcriptional repressors are conserved components of this oscillator. A subset of these genes, called cyclic genes, is characterized by oscillating mRNA expression that sweeps anteriorly like a wave through the embryonic PSM. Periodic transcriptional repression by H/E(spl) proteins is thought to provide a critical part of a negative feedback loop in the oscillatory process, but it is an open question how many cyclic h/E(spl) genes are involved in the somitogenesis clock in any species, and what distinct roles they might play. From a genome-wide search for h/E(spl) genes in the zebrafish, we previously estimated a total of five cyclic members. Here we report that one of these, the mHes5 homologue her15 actually exists as a very recently duplicated gene pair. We investigate the expression of this gene pair and analyse its regulation and activity in comparison to the paralogous her12 gene, and the other cyclic h/E(spl) genes in the zebrafish. The her15 gene pair and her12 display novel and distinct expression features, including a caudally restricted oscillatory domain and dynamic stripes of expression in the rostral PSM that occur at the future segmental borders. her15 expression stripes demarcate a unique two-segment interval in the rostral PSM. Mutant, morpholino, and inhibitor studies show that her12 and her15 expression in the PSM is regulated by Delta-Notch signaling in a complex manner, and is dependent on her7, but not her1 function. Morpholino-mediated her12 knockdown disrupts cyclic gene expression, indicating that it is a non-redundant core component of the segmentation clock. Over-expression of her12, her15 or her7 disrupts cyclic gene expression and somite border formation, and structure function analysis of Her7 indicates that DNA binding, but not Groucho-recruitment seems to be important in this process. Thus, the zebrafish has five functional cyclic h/E(spl) genes, which are expressed in a distinct spatial configuration. We propose that this creates a segmentation oscillator that varies in biochemical composition depending on position in the PSM.

Enhancer detection in the zebrafish using pseudotyped murine retroviruses.

Vectors based on murine retroviruses are among the most efficient means to insert reporter constructs into the context of a vertebrate chromosome with the aim to visualize cis-regulatory information available to a basal promoter at the site of insertion. In combination with using the zebrafish embryo as a readout for the activity of regulatory elements, enhancer detection becomes a powerful technique for gene discovery and for the mapping of the extent of regulatory domains in a vertebrate genome. Our laboratory has performed the only large-scale enhancer detection screen to date in any vertebrate and we describe in this paper the methods we developed to generate viral particles, to insert reporter constructs into the zebrafish germ line, the screening of detection events in heterozygous F1 embryos, and the isolation of genomic sequence flanking the inserted vector for the purpose of genomic mapping. Given sufficient scale, the technology described here can be used to obtain cis-regulatory information across the entire zebrafish genome for any given basal promoter.

Segregation of telencephalic and eye-field identities inside the zebrafish forebrain territory is controlled by Rx3.

Anteroposterior patterning of the vertebrate forebrain during gastrulation involves graded Wnt signaling, which segregates anterior fields (telencephalon and eye) from the diencephalon. How the telencephalic and retinal primordia are subsequently subdivided remains largely unknown. We demonstrate that at late gastrulation the Paired-like homeodomain transcription factor Rx3 biases cell specification choices towards the retinal fate within a population of bipotential precursors of the anterior forebrain: direct cell tracing demonstrates that retinal precursors acquire a telencephalic fate in embryos homozygous for the rx3-null allele ckh(ne2611), characterized by an enlarged telencephalon and a lack of eyes. Chimera analyses further indicate that this function of Rx3 is cell autonomous. Transfating of the eye field in the absence of Rx3 function correlates with a substantial posterior expansion of expression of the Wnt antagonist Tlc and the winged-helix transcription factor Foxg1. These results suggest that the process segregating the telencephalic and eye fields is isolated from diencephalic patterning, and is mediated by Rx3.

Duplicate sfrp1 genes in zebrafish: sfrp1a is dynamically expressed in the developing central nervous system, gut and lateral line.

The secreted frizzled-related proteins (Sfrp) are a family of soluble proteins with diverse biological functions having the capacity to bind Wnt ligands, to modulate Wnt signalling, and to signal directly via the Wnt receptor, Frizzled. In an enhancer trap screen for embryonic expression in zebrafish we identified an sfrp1 gene. Previous studies suggest an important role for sfrp1 in eye development, however, no data have been reported using the zebrafish model. In this paper, we describe duplicate sfrp1 genes in zebrafish and present a detailed analysis of the expression profile of both genes. Whole mount in situ hybridisation analyses of sfrp1a during embryonic and larval development revealed a dynamic expression profile, including: the central nervous system, where sfrp1a was regionally expressed throughout the brain and developing eye; the posterior gut, from the time of endodermal cell condensation; the lateral line, where sfrp1a was expressed in the migrating primordia and interneuromast cells that give rise to the sensory organs. Other sites included the blastoderm, segmenting mesoderm, olfactory placode, developing ear, pronephros and fin-bud. We have also analysed sfrp1b expression during embryonic development. Surprisingly this gene exhibited a divergent expression profile being limited to the yolk syncytium under the elongating tail-bud, which later covered the distal yolk extension, and transiently in the tail-bud mesenchyme. Overall, our studies provide a basis for future analyses of these developmentally important factors using the zebrafish model.

Large-scale enhancer detection in the zebrafish genome.

Murine retroviral vectors carrying an enhancer detection cassette were used to generate 95 transgenic lines of fish in which reporter expression is observed in distinct patterns during embryonic development. We mapped 65 insertion sites to the as yet unfinished zebrafish genome sequence. Many integrations map close to previously known developmental genes, including transcription factors of the Pax, Hox, Sox, Pou, Otx, Emx, zinc-finger and bHLH gene families. In most cases, the activated provirus is located in, or within a 15 kb interval around, the corresponding transcriptional unit. The exceptions include four insertions into a gene desert on chromosome 20 upstream of sox11b, and an insertion upstream of otx1. In these cases, the activated insertions are found at a distance of between 32 kb and 132 kb from the coding region. These as well as seven other insertions described here identify genes that have recently been associated with ultra conserved non-coding elements found in all vertebrate genomes.