A spatiotemporal barrier formed by Follistatin is required for left-right patterning.

How left-right (LR) asymmetry emerges in a patterning field along the anterior-posterior axis remains an unresolved problem in developmental biology. Left-biased Nodal emanating from the LR organizer propagates from posterior to anterior (PA) and establishes the LR pattern of the whole embryo. However, little is known about the regulatory mechanism of the PA spread of Nodal and its asymmetric activation in the forebrain. Here, we identify bilaterally expressed Follistatin (Fst) as a regulator blocking the propagation of the zebrafish Nodal ortholog Southpaw (Spaw) in the right lateral plate mesoderm (LPM), and restricting Spaw transmission in the left LPM to facilitate the establishment of a robust LR asymmetric Nodal patterning. In addition, Fst inhibits the Activin-Nodal signaling pathway in the forebrain thus preventing Nodal activation prior to the arrival, at a later time, of Spaw emanating from the left LPM. This contributes to the orderly propagation of asymmetric Nodal activation along the PA axis. The LR regulation function of Fst is further confirmed in chick and frog embryos. Overall, our results suggest that a robust LR patterning emerges by counteracting a Fst barrier formed along the PA axis.

Mycn regulates intestinal development through ribosomal biogenesis in a zebrafish model of Feingold syndrome 1.

Feingold syndrome type 1, caused by loss-of-function of MYCN, is characterized by varied phenotypes including esophageal and duodenal atresia. However, no adequate model exists for studying the syndrome's pathological or molecular mechanisms, nor is there a treatment strategy. Here, we developed a zebrafish Feingold syndrome type 1 model with nonfunctional mycn, which had severe intestinal atresia. Single-cell RNA-seq identified a subcluster of intestinal cells that were highly sensitive to Mycn, and impaired cell proliferation decreased the overall number of intestinal cells in the mycn mutant fish. Bulk RNA-seq and metabolomic analysis showed that expression of ribosomal genes was down-regulated and that amino acid metabolism was abnormal. Northern blot and ribosomal profiling analysis showed abnormal rRNA processing and decreases in free 40S, 60S, and 80S ribosome particles, which led to impaired translation in the mutant. Besides, both Ribo-seq and western blot analysis showed that mTOR pathway was impaired in mycn mutant, and blocking mTOR pathway by rapamycin treatment can mimic the intestinal defect, and both L-leucine and Rheb, which can elevate translation via activating TOR pathway, could rescue the intestinal phenotype of mycn mutant. In summary, by this zebrafish Feingold syndrome type 1 model, we found that disturbance of ribosomal biogenesis and blockage of protein synthesis during development are primary causes of the intestinal defect in Feingold syndrome type 1. Importantly, our work suggests that leucine supplementation may be a feasible and easy treatment option for this disease.

Mutational analysis of dishevelled genes in zebrafish reveals distinct functions in embryonic patterning and gastrulation cell movements.

Wnt signaling plays critical roles in dorsoventral fate specification and anteroposterior patterning, as well as in morphogenetic cell movements. Dishevelled proteins, or Dvls, mediate the activation of Wnt/ß-catenin and Wnt/planar cell polarity pathways. There are at least three highly conserved Dvl proteins in vertebrates, but the implication of each Dvl in key early developmental processes remains poorly understood. In this study, we use genome-editing approach to generate different combinations of maternal and zygotic dvl mutants in zebrafish, and examine their functions during early development. Maternal transcripts for dvl2 and dvl3a are most abundantly expressed, whereas the transcript levels of other dvl genes are negligible. Phenotypic and molecular analyses show that early dorsal fate specification is not affected in maternal and zygotic dvl2 and dvl3a double mutants, suggesting that the two proteins may be dispensable for the activation of maternal Wnt/ß-catenin signaling. Interestingly, convergence and extension movements and anteroposterior patterning require both maternal and the zygotic functions of Dvl2 and Dvl3a, but these processes are more sensitive to Dvl2 dosage. Zygotic dvl2 and dvl3a double mutants display mild axis extension defect with correct anteroposterior patterning. However, maternal and zygotic double mutants exhibit most strongly impaired convergence and extension movements, severe trunk and posterior deficiencies, and frequent occurrence of cyclopia and craniofacial defects. Our results suggest that Dvl2 and Dvl3a products are required for the activation of zygotic Wnt/ß-catenin signaling and Wnt/planar cell polarity pathway, and regulate zygotic developmental processes in a dosage-dependent manner. This work provides insight into the mechanisms of Dvl-mediated Wnt signaling pathways during early vertebrate development.

Protocol for generation and assessment of head-like structure in zebrafish.

In vitro embryonic analogue models, such as gastruloids, trunk-like structures and embryoids, have been developed to understand principles of early development and morphogenesis. However, models that can fully mimic head formation are still missing. Here, we present a protocol for generating the head-like structure (HLS) in zebrafish embryonic explants. We describe steps for dissection and constructing cell and patterning landscapes. We then detail assessment of this structure through axis induction. For complete details on the use and execution of this protocol, please refer to Cheng et al. (2023).1.

Nodal coordinates the anterior-posterior patterning of germ layers and induces head formation in zebrafish explants.

Much progress has been made toward generating analogs of early embryos, such as gastruloids and embryoids, in vitro. However, methods for how to fully mimic the cell movements of gastrulation and coordinate germ-layer patterning to induce head formation are still lacking. Here, we show that a regional Nodal gradient applied to zebrafish animal pole explant can generate a structure that recapitulates the key cell movements of gastrulation. Using single-cell transcriptome and in situ hybridization analysis, we assess the dynamics of the cell fates and patterning of this structure. The mesendoderm differentiates into the anterior endoderm, prechordal plate, notochord, and tailbud-like cells along an anterior-posterior axis, and an anterior-posterior-patterned head-like structure (HLS) progressively forms during late gastrulation. Among 105 immediate Nodal targets, 14 genes contain axis-induction ability, and 5 of them induce a complete or partial head structure when overexpressed in the ventral side of zebrafish embryos.

[Determination of doping in human urine by gas chromatography-high resolution mass spectrometry].

A method was evaluated for determination of twenty-one doping (including nandrolone, boldenone and methandienone) in human urine by gas chromatography-high resolution mass spectrometry. Samples were prepared by liquid-liquid extraction, concentrated, TMS derivatization and limit of detection at ng x mL(-1) by MID/GC/HRMS. According to the code of the World Anti-Doping Agency (WADA), precision and recoveries of the procedure were evaluated by replicate analysis (n = 6), the recoveries in the range of 66%-103%, with the RSD below 10.0%. The precision within the day of the method with three different concentrations was also determined RSD were less than 9.5%, 10.0% and 9.7%.

[Determination methods for erythropoietin receptor activator in human urine].

In the present study, isoelectronic focusing with different pH gradients (pH 3-5, 2-6) or migrating distances (8.5, 12 and 17 cm) and SDS-PAGE was used to separate continuous erythropoietin receptor activator (CERA), recombinant human erythropoietin (rhEPO), darbepoetin and endogenous EPO spiked in human urine with 37 degrees C overnight incubation. Double blotting and chemiluminescent visualization were used to detect the IEF and SDS-PAGE profiles. The bands of CERA profile were detected and well separated from the endogenous EPO and the other two EPO preparations with both SDS-PAGE and the IEF method using a gradient pH 3-5 and a migrating distance of 17 cm, and a significant particular band of CERA profile was found in the IEF result. These preliminary results indicated that the methods were reliable and reproducible for detecting CERA, and could be used as a routine procedure for anti-doping analysis.