Wnt-Dependent Epithelial Transitions Drive Pharyngeal Pouch Formation

The pharyngeal pouches, which form by budding of the foregut endoderm, are essential for segmentation of the vertebrate face. To date, the cellular mechanism and segmental nature of such budding have remained elusive. Here, we find that Wnt11r and Wnt4a from the head mesoderm and ectoderm, respectively, play distinct roles in the segmental formation of pouches in zebrafish. Time-lapse microscopy, combined with mutant and tissue-specific transgenic experiments, reveal requirements of Wnt signaling in two phases of endodermal epithelial transitions. Initially, Wnt11r and Rac1 destabilize the endodermal epithelium to promote the lateral movement of pouch-forming cells. Next, Wnt4a and Cdc42 signaling induce the rearrangement of maturing pouch cells into bilayers through junctional localization of the Alcama immunoglobulin-domain protein, which functions to restabilize adherens junctions. We propose that this dynamic control of epithelial morphology by Wnt signaling may be a common theme for the budding of organ anlagen from the endoderm

sox9b Is a Key Regulator of Pancreaticobiliary Ductal System Development

The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9bfh313, to dissect its function in the morphogenesis of this structure. Strikingly, sox9bfh313 homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9bfh313 mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9bfh313 mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9bfh313 mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies

Structure Tensor-Based Infrared Small Target Detection Method for a Double Linear Array Detector

The paper focuses on the mathematical modeling of a new double linear array detector. The special feature of the detector is that image pairs can be generated at short intervals in one scan. After registration and removal of dynamic cloud edges in each image, the image differentiation-based change detection method in the temporal domain is proposed to combine with the structure tensor edge suppression method in the spatial domain. Finally, experiments are conducted, and our results are compared with theoretic analyses. It is found that a high signal-to-clutter ratio (SCR) of camera input is required to obtain an acceptable detection rate and false alarm rate in real scenes. Experimental results also show that the proposed cloud edge removal solution can be used to successfully detect targets with a very low false alarm rate and an acceptable detection rate

Structure Tensor-Based Infrared Small Target Detection Method for a Double Linear Array Detector

The paper focuses on the mathematical modeling of a new double linear array detector. The special feature of the detector is that image pairs can be generated at short intervals in one scan. After registration and removal of dynamic cloud edges in each image, the image differentiation-based change detection method in the temporal domain is proposed to combine with the structure tensor edge suppression method in the spatial domain. Finally, experiments are conducted, and our results are compared with theoretic analyses. It is found that a high signal-to-clutter ratio (SCR) of camera input is required to obtain an acceptable detection rate and false alarm rate in real scenes. Experimental results also show that the proposed cloud edge removal solution can be used to successfully detect targets with a very low false alarm rate and an acceptable detection rate

Zebrafish Mef2ca and Mef2cb are essential for both first and second heart field cardiomyocyte differentiation

SummaryMef2 transcription factors have been strongly linked with early heart development. D-mef2 is required for heart formation in Drosophila, but whether Mef2 is essential for vertebrate cardiomyocyte (CM) differentiation is unclear. In mice, although Mef2c is expressed in all CMs, targeted deletion of Mef2c causes lethal loss of second heart field (SHF) derivatives and failure of cardiac looping, but first heart field CMs can differentiate. Here we examine Mef2 function in early heart development in zebrafish. Two Mef2c genes exist in zebrafish, mef2ca and mef2cb. Both are expressed similarly in the bilateral heart fields but mef2cb is strongly expressed in the heart poles at the primitive heart tube stage. By using fish mutants for mef2ca and mef2cb and antisense morpholinos to knock down either or both Mef2cs, we show that Mef2ca and Mef2cb have essential but redundant roles in myocardial differentiation. Loss of both Mef2ca and Mef2cb function does not interfere with early cardiogenic markers such as nkx2.5, gata4 and hand2 but results in a dramatic loss of expression of sarcomeric genes and myocardial markers such as bmp4, nppa, smyd1b and late nkx2.5 mRNA. Rare residual CMs observed in mef2ca;mef2cb double mutants are ablated by a morpholino capable of knocking down other Mef2s. Mef2cb over-expression activates bmp4 within the cardiogenic region, but no ectopic CMs are formed. Surprisingly, anterior mesoderm and other tissues become skeletal muscle. Mef2ca single mutants have delayed heart development, but form an apparently normal heart. Mef2cb single mutants have a functional heart and are viable adults. Our results show that the key role of Mef2c in myocardial differentiation is conserved throughout the vertebrate heart

Rapid identification and recovery of ENU-induced mutations with next-generation sequencing and Paired-End Low-Error analysis

AbstractBackgroundTargeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population. Classical TILLING processes, based on enzymatic detection of mutations in heteroduplex PCR amplicons, are slow and labor intensive.ResultsHere we describe a new TILLING strategy in zebrafish using direct next generation sequencing (NGS) of 250bp amplicons followed by Paired-End Low-Error (PELE) sequence analysis. By pooling a genomic DNA library made from over 9,000 N-ethyl-N-nitrosourea (ENU) mutagenized F1 fish into 32 equal pools of 288 fish, each with a unique Illumina barcode, we reduce the complexity of the template to a level at which we can detect mutations that occur in a single heterozygous fish in the entire library. MiSeq sequencing generates 250 base-pair overlapping paired-end reads, and PELE analysis aligns the overlapping sequences to each other and filters out any imperfect matches, thereby eliminating variants introduced during the sequencing process. We find that this filtering step reduces the number of false positive calls 50-fold without loss of true variant calls. After PELE we were able to validate 61.5% of the mutant calls that occurred at a frequency between 1 mutant call:100 wildtype calls and 1 mutant call:1000 wildtype calls in a pool of 288 fish. We then use high-resolution melt analysis to identify the single heterozygous mutation carrier in the 288-fish pool in which the mutation was identified.ConclusionsUsing this NGS-TILLING protocol we validated 28 nonsense or splice site mutations in 20 genes, at a two-fold higher efficiency than using traditional Cel1 screening. We conclude that this approach significantly increases screening efficiency and accuracy at reduced cost and can be applied in a wide range of organisms.</p

A study of the gas-water characteristics and their implications for the coalbed methane accumulation modes in the Southern Junggar Basin, China

Gas and water samples were collected from coalbed methane (CBM) wells, rivers, and springs in the southern Junggar Basin (SJB). These samples were analyzed for gas composition, stable isotopes, 16s ribosomal ribonucleic acid sequence, chemical compositions, and radioisotopes. The objective of this study was to understand CBM genesis in the Junggar Basin, the reason for abnormal CO2 accumulation, the development of microbial communities, the source of coalbed water, and the timing of methanogenesis. The CBM genesis is complex in the SJB, but it is closely related to microbial activities. The stagnant zone, which experiences limited groundwater recharge, may represent a relatively closed system where CO2 is easily trapped and the residual CO2 becomes progressively enriched in C-13. Only two families of methanogens (i. e., Methanobacteriaceae and Methanospirillaceae) are present in the coalbed waters, indicating that CO2 reduction is the main pathway for generating microbial gas. The coalbed water samples from the Houxia and Manasi-Hutubi regions plot around the local meteoric water line (LMWL), indicating recharge by modern meteoric water and rivers. However, the samples from the Miquan and Fukang regions plot below the LMWL, reflecting older snowmelt water recharge. Isotopic dating indicates that the age of coalbed water in the Miquan and Fukang regions is 43.5-2000 ka. Early coalification and later hydrological events collectively determined the regional variations in CBM genesis and gas composition in the SJB

Distinct Notch signaling outputs pattern the developing arterial system

Differentiation of arteries and veins is essential for the development of a functional circulatory system. In vertebrate embryos, genetic manipulation of Notch signaling has demonstrated the importance of this pathway in driving artery endothelial cell differentiation. However, when and where Notch activation occurs to affect endothelial cell fate is less clear. Using transgenic zebrafish bearing a Notch-responsive reporter, we demonstrate that Notch is activated in endothelial progenitors during vasculogenesis prior to blood vessel morphogenesis and is maintained in arterial endothelial cells throughout larval stages. Furthermore, we find that endothelial progenitors in which Notch is activated are committed to a dorsal aorta fate. Interestingly, some arterial endothelial cells subsequently downregulate Notch signaling and then contribute to veins during vascular remodeling. Lineage analysis, together with perturbation of both Notch receptor and ligand function, further suggests several distinct developmental windows in which Notch signaling acts to promote artery commitment and maintenance. Together, these findings demonstrate that Notch acts in distinct contexts to initiate and maintain artery identity during embryogenesis

Role of mef2ca in developmental buffering of the zebrafish larval hyoid dermal skeleton

AbstractPhenotypic robustness requires a process of developmental buffering that is largely not understood, but which can be disrupted by mutations. Here we show that in mef2cab1086 loss of function mutant embryos and early larvae, development of craniofacial hyoid bones, the opercle (Op) and branchiostegal ray (BR), becomes remarkably unstable; the large magnitude of the instability serves as a positive attribute to learn about features of this developmental buffering. The OpBR mutant phenotype variably includes bone expansion and fusion, Op duplication, and BR homeosis. Formation of a novel bone strut, or a bone bridge connecting the Op and BR together occurs frequently. We find no evidence that the phenotypic stability in the wild type is provided by redundancy between mef2ca and its co-ortholog mef2cb, or that it is related to the selector (homeotic) gene function of mef2ca. Changes in dorsal–ventral patterning of the hyoid arch also might not contribute to phenotypic instability in mutants. However, subsequent development of the bone lineage itself, including osteoblast differentiation and morphogenetic outgrowth, shows marked variation. Hence, steps along the developmental trajectory appear differentially sensitive to the loss of buffering, providing focus for the future study

Microarray Analysis Reveals a Potential Role of lncRNA Expression in 3,4-Benzopyrene/Angiotensin II-Activated Macrophage in Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is a fatal disease, and exposure to 3,4-benzopyrene (Bap) is closely related to the development of AAA. We have found that Bap could impair the biological function of endothelial progenitor cells (EPCs), which are associated with the occurrence of AAA. We have also demonstrated that macrophage activation plays a key role in Bap-induced AAA, but the mechanism is unknown. Here, we used a mouse lncRNA array to investigate the expression signatures of lncRNAs and mRNAs in Bap-activated macrophage. A total of 457 lncRNAs and 219 mRNAs were found to be differentially expressed. The function of differential mRNAs was determined by pathway and Gene Ontology analysis. Eight pathways associated with inflammation were upregulated, and seven pathways including cell apoptosis were downregulated. It was worth noting that AGE-RAGE pathway, which was involved in Bap-induced EPC dysfunction, was significantly upregulated in Bap-activated macrophage and may contribute to AAA formation. Thus, lncRNAs may exert a key role in activated macrophages and intervene the core lncRNAs and may inhibit the occurrence of a series of cascade reactions in the macrophages, which may provide potential targets for AAA caused by smoking