Pax3/7 paired homeodomain transcription factors are important markers of muscle stem cells. Pax3 is required upstream of myod for lateral dermomyotomal cells in the amniote somite to form particular muscle cells. Later Pax3/7-dependent cells generate satellite cells and most body muscle. Here we analyse early myogenesis from, and regulation of, a population of Pax3-expressing dermomyotome-like cells in the zebrafish. Zebrafish pax3 is widely expressed in the lateral somite and, along with pax7, becomes restricted anteriorly and then to the external cells on the lateral somite surface. Midline-derived Hedgehog signals appear to act directly on lateral somite cells to repress Pax3/7. Both Hedgehog and Fgf8, signals that induce muscle formation within the somite, suppress Pax3/7 and promote expression of myogenic regulatory factors (MRFs) myf5 and myod in specific muscle precursor cell populations. Loss of MRF function leads to loss of myogenesis by specific populations of muscle fibres, with parallel up-regulation of Pax3/7. Myod is required for lateral fast muscle differentiation from pax3-expressing cells. In contrast, either Myf5 or Myod is sufficient to promote slow muscle formation from adaxial cells. Thus, myogenic signals act to drive somite cells to a myogenic fate through up-regulation of distinct combinations of MRFs. Our data show that the relationship between Pax3/7 genes and myogenesis is evolutionarily ancient, but that changes in the MRF targets for particular signals contribute to myogenic differences between species.

Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness. T. brucei multiplies extracellularly in the bloodstream, relying on antigenic variation of a dense variant surface glycoprotein (VSG) coat to escape antibody-mediated lysis. We investigated the role of VSG in proliferation and pathogenicity by using inducible RNA interference to ablate VSG transcript down to 1-2% normal levels. Inhibiting VSG synthesis in vitro triggers a rapid and specific cell cycle checkpoint blocking cell division. Parasites arrest at a discrete precytokinesis stage with two fulllength flagella and opposing flagellar pockets, without undergoing additional rounds of S phase and mitosis. A subset (<10%) of the stalled cells have internal flagella, indicating that the progenitors of these cells were already committed to cytokinesis when VSG restriction was sensed. Although there was no obvious VSG depletion in vitro after 24-h induction of VSG RNA interference, there was rapid clearance of these cells in vivo. We propose that a stringent block in VSG synthesis produces stalled trypanosomes with a minimally compromised VSG coat, which can be targeted by the immune system. Our data indicate that VSG protein or transcript is monitored during cell cycle progression in bloodstreamform T. brucei and describes precise precytokinesis cell cycle arrest. This checkpoint before cell division provides a link between the protective VSG coat and cell cycle progression and could function as a novel parasite safety mechanism, preventing extensive dilution of the protective VSG coat in the absence of VSG synthesis. antigenic variation ͉ Trypanosoma bruceiA frican trypanosomes, including Trypanosoma brucei, are unicellular parasites transmitted by tsetse flies in subSaharan Africa, which are responsible for debilitating disease in humans and livestock. African sleeping sickness is resurgent, with an estimated 300,000-500,000 cases a year, and it is invariably fatal if untreated (1). T. brucei multiplies extracellularly in the bloodstream, exposing it to immune attack. Each bloodstream-form cell is coated with Ϸ10 7 variant surface glycoprotein (VSG) molecules of a given antigenic type, making up Ϸ10% of total cellular protein (2, 3). Eventually the host mounts an antibody response against a given VSG variant. However, as parasites can switch between hundreds of antigenically diverse VSG coats during the course of a chronic infection, trypanosomes expressing a new VSG variant can escape the host antibody response against the old one (4, 5). This highly sophisticated strategy of antigenic variation makes African trypanosomes a paradigm for antigenic variation in general.Although a great deal is known about mechanisms mediating VSG switching, we know relatively little about the role of VSG itself in other aspects of immune evasion and pathogenicity. To investigate this, we inhibited VSG synthesis by performing inducible RNA interference (RNAi) both in vitro and in vivo. Inhibition of VSG synthesis in vitro triggers a rapid and specifi...

The Pax3/7 gene family has a fundamental and conserved role during neural crest formation. In people, PAX3 mutation causes Waardenburg syndrome, and murine Pax3 is essential for pigment formation. However, it is unclear exactly how Pax3 functions within the neural crest. Here we show that pax3 is expressed before other pax3/7 members, including duplicated pax3b, pax7 and pax7b genes, early in zebrafish neural crest development. Knockdown of Pax3 protein by antisense morpholino oligonucleotides results in defective fate specification of xanthophores, with complete ablation in the trunk. Other pigment lineages are specified and differentiate. As a consequence of xanthophore loss, expression of pax7, a marker of the xanthophore lineage, is reduced in neural crest. Morpholino knockdown of Pax7 protein shows that Pax7 itself is dispensable for xanthophore fate specification, although yellow pigmentation is reduced. Loss of xanthophores after reduction of Pax3 correlates with a delay in melanoblast differentiation followed by significant increase in melanophores, suggestive of a Pax3-driven fate switch within a chromatophore precursor or stem cell. Analysis of other neural crest derivatives reveals that, in the absence of Pax3, the enteric nervous system is ablated from its inception. Therefore, Pax3 in zebrafish is required for specification of two specific lineages of neural crest, xanthophores and enteric neurons.

The zebrafish model system offers significant utility for in vivo imaging of adipose tissue (AT) dynamics and for screening to identify chemical and genetic modifiers of adiposity. In particular, AT can be quantified accurately in live zebrafish using fluorescent lipophilic dyes. Although this methodology offers considerable promise, the comprehensive identification and classification of zebrafish ATs has not been performed. Here, we use fluorescent lipophilic dyes and in vivo imaging systematically to identify, classify and quantify the zebrafish AT pool. We identify 34 regionally distinct zebrafish ATs, including five visceral ATs and 22 subcutaneous ATs. For each of these ATs, we describe detailed morphological characteristics to aid their identification in future studies. Furthermore, we quantify the areas for each AT and construct regression models to allow prediction of expected AT size and variation across a range of developmental stages. Finally, we demonstrate the utility of this resource for identifying effects of strain variation and high-fat diet on AT growth. Altogether, this resource provides foundational information on the identity, dynamics and expected quantities of zebrafish ATs for use as a reference for future studies.

The forelimbs of higher vertebrates are composed of two portions: the appendicular region (stylopod, zeugopod and autopod) and the less prominent proximal girdle elements (scapula and clavicle) that brace the limb to the main trunk axis. We show that the formation of the muscles of the proximal limb occurs through two distinct mechanisms. The more superficial girdle muscles (pectoral and latissimus dorsi) develop by the "In-Out" mechanism whereby migration of myogenic cells from the somites into the limb bud is followed by their extension from the proximal limb bud out onto the thorax. In contrast, the deeper girdle muscles (e.g. rhomboideus profundus and serratus anterior) are induced by the forelimb field which promotes myotomal extension directly from the somites. Tbx5 inactivation demonstrated its requirement for the development of all forelimb elements which include the skeletal elements, proximal and distal muscles as well as the sternum in mammals and the cleithrum of fish. Intriguingly, the formation of the diaphragm musculature is also dependent on the Tbx5 programme. These observations challenge our classical views of the boundary between limb and trunk tissues. We suggest that significant structures located in the body should be considered as components of the forelimb.

SummaryHypothalamic melanocortin neurons play a pivotal role in weight regulation. Here, we examined the contribution of Semaphorin 3 (SEMA3) signaling to the development of these circuits. In genetic studies, we found 40 rare variants in SEMA3A-G and their receptors (PLXNA1-4; NRP1-2) in 573 severely obese individuals; variants disrupted secretion and/or signaling through multiple molecular mechanisms. Rare variants in this set of genes were significantly enriched in 982 severely obese cases compared to 4,449 controls. In a zebrafish mutagenesis screen, deletion of 7 genes in this pathway led to increased somatic growth and/or adiposity demonstrating that disruption of Semaphorin 3 signaling perturbs energy homeostasis. In mice, deletion of the Neuropilin-2 receptor in Pro-opiomelanocortin neurons disrupted their projections from the arcuate to the paraventricular nucleus, reduced energy expenditure, and caused weight gain. Cumulatively, these studies demonstrate that SEMA3-mediated signaling drives the development of hypothalamic melanocortin circuits involved in energy homeostasis.

Somatic growth and adipogenesis are closely associated with the development of obesity in humans. In this study, we identify a zebrafish mutant, vizzini, that exhibits both a severe defect in somatic growth and increased accumulation of adipose tissue. Positional cloning of vizzini revealed a premature stop codon in gh1. Although the effects of GH are largely through igfs in mammals, we found no decrease in the expression of igf transcripts in gh1 mutants during larval development. As development progressed, however, we found overall growth to be progressively retarded and the attainment of specific developmental stages to occur at abnormally small body sizes relative to wild type. Moreover, both subcutaneous (sc) and visceral adipose tissues underwent precocious development in vizzini mutants, and at maturity, the sizes of different fat deposits were greatly expanded relative to wild type. In vivo confocal imaging of sc adipose tissue (SAT) expansion revealed that vizzini mutants exhibit extreme enlargement of adipocyte lipid droplets without a corresponding increase in lipid droplet number. These findings suggest that GH1 signaling restricts SAT hypertrophy in zebrafish. Finally, nutrient deprivation of vizzini mutants revealed that SAT mobilization was greatly diminished during caloric restriction, further implicating GH1 signaling in adipose tissue homeostasis. Overall, the zebrafish gh1 mutant, vizzini, exhibits decreased somatic growth, increased adipose tissue accumulation, and disrupted adipose plasticity after nutrient deprivation and represents a novel model to investigate the in vivo dynamics of vertebrate obesity.

Genome-wide association studies have implicated PLEXIN D1 (PLXND1) in body fat distribution and type 2 diabetes. However, a role for PLXND1 in regional adiposity and insulin resistance is unknown. Here we use in vivo imaging and genetic analysis in zebrafish to show that Plxnd1 regulates body fat distribution and insulin sensitivity. Plxnd1 deficiency in zebrafish induced hyperplastic morphology in visceral adipose tissue (VAT) and reduced lipid storage. In contrast, subcutaneous adipose tissue (SAT) growth and morphology were unaffected, resulting in altered body fat distribution and a reduced VAT:SAT ratio in zebrafish. A VAT-specific role for Plxnd1 appeared conserved in humans, as PLXND1 mRNA was positively associated with hypertrophic morphology in VAT, but not SAT. In zebrafish plxnd1 mutants, the effect on VAT morphology and body fat distribution was dependent on induction of the extracellular matrix protein collagen type V alpha 1 (col5a1). Furthermore, after high-fat feeding, zebrafish plxnd1 mutant VAT was resistant to expansion, and excess lipid was disproportionately deposited in SAT, leading to an even greater exacerbation of altered body fat distribution. Plxnd1-deficient zebrafish were protected from high-fat-diet-induced insulin resistance, and human VAT PLXND1 mRNA was positively associated with type 2 diabetes, suggesting a conserved role for PLXND1 in insulin sensitivity. Together, our findings identify Plxnd1 as a novel regulator of VAT growth, body fat distribution, and insulin sensitivity in both zebrafish and humans.zebrafish | body fat distribution | adipose development | insulin resistance | extracellular matrix T he regional distribution and morphology of adipose tissue (AT) are strong predictors of metabolic disease (1-3). Excess lipid deposition in visceral AT (VAT; adipose associated with visceral organs) is associated with increased susceptibility to insulin resistance and type 2 diabetes (4), whereas expansion of subcutaneous AT (SAT; adipose between muscle and skin) is associated with reduced risk for metabolic disease and is even protective against hyperglycemia and dyslipidemia (4-7). In turn, hypertrophic AT morphology (few large adipocytes) is associated with insulin resistance and AT dysfunction, whereas hyperplastic AT morphology (many small adipocytes) is associated with improved metabolic parameters (4, 7-9). Therefore, the identification of factors that regulate regional distribution and AT morphology could lead to new therapies to treat metabolic disease.Genome-wide association studies have implicated the PLEXIN D1 (PLXND1) locus in waist:hip ratio (a measurement of regional AT distribution) and type 2 diabetes (10). However, a role for Plxnd1 in AT morphology, distribution, and metabolism is unknown. Plxnd1 is a transmembrane receptor that controls the migration, proliferation, and survival of diverse cell types (11).Mutation of Plxnd1 in mouse and zebrafish leads to hypervascularization in many tissues (12, 13), and vascular endothelial cell Plxnd1 modulates extracellu...