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Lead (Pb) is a heavy metal associated with a range of toxic effects. Relatively few studies attempt to understand the impact of lead on development from a mechanistic perspective. Danio rerio (zebrafish) embryos are a model organism for studying the developmental consequences of exposure to chemical agents. This study examined the metabolome of developing zebrafish embryos exposed to 5 ppb, 15 ppb, 150 ppb, and 1500 ppb Pb concentrations during the first 24 h post fertilization, followed by 24 h of unexposed development and harvest at 48 h. Untargeted metabolomics and multivariate analysis revealed that various Pb exposures differentially affected the embryonic metabolome. Pathway analyses showed the dysregulation of biopterin, purine, alanine, and aspartate metabolism. Inductively coupled plasma mass spectrometry demonstrated Pb accumulation in embryos. Additionally, decreases in oxidation-reduction ratios were observed in 5-150 ppb groups but not in the 1500 ppb exposure group. This finding, along with several metabolite abundances, suggests a hormetic effect of Pb concentrations on the developing zebrafish metabolome. Together, these data reveal persistent global changes in the embryonic metabolome, pin-point biomarkers for Pb exposure, unveil dose-dependent relationships, and reflect Pb-induced changes in cellular energy. This work highlights aberrant processes and persistent changes underlying low-dose heavy metal exposure during early development.

Aquaporins and aquaglyceroporins are a large family of membrane channel proteins that allow rapid movement of water and small, uncharged solutes into and out of cells along concentration gradients. Recently, aquaporins have been gaining recognition for more complex biological roles than the regulation of cellular osmotic homeostasis. We have identified a specific expression pattern for Xenopus aqp3b (also called aqp3.L) during gastrulation, where it is localized to the sensorial (deep) layer of the blastocoel roof and dorsal margin. Interference with aqp3b expression resulted in loss of fibrillar fibronectin matrix in Brachet's cleft at the dorsal marginal zone, but not on the free surface of the blastocoel. Detailed observation showed that the absence of fibronectin matrix correlated with compromised border integrities between involuted mesendoderm and noninvoluted ectoderm in the marginal zone. Knockdown of aqp3b also led to delayed closure of the blastopore, suggesting defects in gastrulation movements. Radial intercalation was not affected in aqp3b morphants, while the data presented are consistent with impeded convergent extension movements of the dorsal mesoderm in response to loss of aqp3b. Our emerging model suggests that aqp3b is part of a mechanism that promotes proper interaction between cells and the extracellular matrix, thereby playing a critical role in gastrulation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Human ZIC1 (zinc finger protein of cerebellum 1), one of five homologs of the Drosophila pair-rule gene odd-paired, encodes a transcription factor previously implicated in vertebrate brain development. Heterozygous deletions of ZIC1 and its nearby paralog ZIC4 on chromosome 3q25.1 are associated with Dandy-Walker malformation of the cerebellum, and loss of the orthologous Zic1 gene in the mouse causes cerebellar hypoplasia and vertebral defects. We describe individuals from five families with heterozygous mutations located in the final (third) exon of ZIC1 (encoding four nonsense and one missense change) who have a distinct phenotype in which severe craniosynostosis, specifically involving the coronal sutures, and variable learning disability are the most characteristic features. The location of the nonsense mutations predicts escape of mutant ZIC1 transcripts from nonsense-mediated decay, which was confirmed in a cell line from an affected individual. Both nonsense and missense mutations are associated with altered and/or enhanced expression of a target gene, engrailed-2, in a Xenopus embryo assay. Analysis of mouse embryos revealed a localized domain of Zic1 expression at embryonic days 11.5-12.5 in a region overlapping the supraorbital regulatory center, which patterns the coronal suture. We conclude that the human mutations uncover a previously unsuspected role for Zic1 in early cranial suture development, potentially by regulating engrailed 1, which was previously shown to be critical for positioning of the murine coronal suture. The diagnosis of a ZIC1 mutation has significant implications for prognosis and we recommend genetic testing when common causes of coronal synostosis have been excluded., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

A particularly critical event in avian evolution was the transition from long- to short-tailed birds. Primitive bird tails underwent significant alteration, most notably reduction of the number of caudal vertebrae and fusion of the distal caudal vertebrae into an ossified pygostyle. These changes, among others, occurred over a very short evolutionary interval, which brings into focus the underlying mechanisms behind those changes. Despite the wealth of studies delving into avian evolution, virtually nothing is understood about the genetic and developmental events responsible for the emergence of short, fused tails. In this review, we summarize the current understanding of the signaling pathways and morphological events that contribute to tail extension and termination and examine how mutations affecting the genes that control these pathways might influence the evolution of the avian tail. To generate a list of candidate genes that may have been modulated in the transition to short-tailed birds, we analyzed a comprehensive set of mouse mutants. Interestingly, a prevalent pleiotropic effect of mutations that cause fused caudal vertebral bodies (as in the pygostyles of birds) is tail truncation. We identified 23 mutations in this class, and these were primarily restricted to genes involved in axial extension. At least half of the mutations that cause short, fused tails lie in the Notch/Wnt pathway of somite boundary formation or differentiation, leading to changes in somite number or size. Several of the mutations also cause additional bone fusions in the trunk skeleton, reminiscent of those observed in primitive and modern birds. All of our findings were correlated to the fossil record. An open question is whether the relatively sudden appearance of short-tailed birds in the fossil record could be accounted for, at least in part, by the pleiotropic effects generated by a relatively small number of mutational events.

Background: The zic genes encode a family of transcription factors with important roles during early development. Since little is known about zic gene expression in chick embryos, we have characterized the expression patterns of the zic1, zic2, zic3, and zic4 (zic1-4) genes during neurulation and somitogenesis., Findings: We used in situ hybridization to analyze the expression patterns of the zic1-4 genes during early chick development (HH stages 7-19). The zic1-3 genes showed both overlapping and gene-specific expression patterns along the length of the dorsal neural tube and in the dorsal parts of the somites. In addition, unique expression domains of zic genes included: zic2 in the neural plate, periotic mesoderm and limb buds; zic3 in the paraxial mesoderm surrounding the neural plate, in presomitic mesoderm and in the most recently formed epithelial somites; zic2 and zic3 in developing eyes. zic4 expression was limited to dorsal fore- and midbrain regions and, unlike the expression of the zic1-3 genes, zic4 expression was not detected in the hindbrain and trunk. This was in contrast to more extensive zic4 expression in other vertebrates., Conclusions: The zic1-3 genes were expressed in both overlapping and unique domains within the neural tube, somites and other ectoderm and mesoderm-derived structures in the future head and trunk. zic4 expression, however, was limited to dorso-anterior regions of the future brain. This is the first comprehensive study of zic1-4 gene expression in chick embryos during neurulation and somitogenesis.

The Zic1 transcription factor plays multiple roles during early development, for example, in patterning the early neural plate and formation of the neural crest, somites, and cerebellum. To identify direct downstream target genes of Zic1, a microarray screen was conducted in Xenopus laevis that identified 85 genes upregulated twofold or more. These include transcription factors, receptors, enzymes, proteins involved in retinoic acid signaling, and an aquaglyceroporin (aqp-3b), but surprisingly no genes known to be involved in cell proliferation. We show that both aqp-3 and aqp-3b were expressed in adult tissues, while during early embryonic development, only aqp-3b was transcribed. During neurula stages, aqp-3b was expressed specifically in the neural folds. This pattern of aqp-3b expression closely resembled that of NF-protocadherin (NFPC), which is involved in cell adhesion and neural tube closure. Aqp-3b may also be involved in neural tube closure, since mammalian Aqp-3 promotes cell migration and proliferation.

Members of the Zic family of zinc finger transcription factors play critical roles in a variety of developmental processes. They are involved in development of neural tissues and the neural crest, in left-right axis patterning, in somite development, and in formation of the cerebellum. In addition to their roles in cell-fate specification, zic genes also promote cell proliferation. Further, they are expressed in postmitotic cells of the cerebellum and in retinal ganglion cells. Efforts to determine the role of individual zic genes within an array of developmental and cellular processes are complicated by overlapping patterns of zic gene expression and strong sequence conservation within this gene family. Nevertheless, substantial progress has been made. This review summarizes our knowledge of the molecular events that govern the activities of zic family members, including emerging relationships between upstream signaling pathways and zic genes. In addition, advancements in our understanding of the molecular events downstream of Zic transcription factors are reviewed. Despite significant progress, however, much remains to be learned regarding the mechanisms through which zic genes exert their function in a variety of different contexts.

The transcription factor Zic1 plays important roles in patterning the neural plate in early vertebrate development. However, few genes that are regulated by Zic1 are known. We have identified a new direct downstream target gene of Zic1 that we have named Xfeb. Xfeb is a member of the pathogenesis-related (PR) protein superfamily and contains five tandem SCP domains. The sequence of Xfeb suggests that it may possess serine protease activity. Xfeb is expressed in the presumptive hindbrain region during neurula stages and in somite tissues later in development. Xfeb represses the hindbrain gene hoxB1 and the anterior neural gene otx2, suggesting that Xfeb is involved in regionalizing the neural plate, possibly by ensuring a posterior expression limit for otx2., ((c) 2006 Wiley-Liss, Inc.)

Zic transcription factors regulate the expression of neural and neural crest-specific genes and are expressed in the cells of the dorsal neural tube and the premigratory neural crest. Here we characterize zic1 expression in the chick embryo during somite formation and neural crest migration. zic1 is expressed in the dorsomedial portion of epithelial somites and subsequently in the dorsomedial lip of the dermomyotome. Although zic1 is expressed in cells of the nascent myotome, it is absent from differentiated myotome cells that express myosin. As the dorsal root ganglia form, zic1 is expressed at high levels in the dorsal sclerotome and zic1 expression is more pronounced in the caudal regions of the somites. Double-label experiments showed that cells expressing zic1 are not labeled by the HNK-1 antibody specific for migratory neural crest cells. Thus, migrating neural crest cells do not express zic1.

The zic1 gene plays an important role in early patterning of the Xenopus neurectoderm. While Zic1 does not act as a neural inducer, it synergizes with the neural inducing factor Noggin to activate expression of posterior neural genes, including the midbrain/hindbrain boundary marker engrailed-2. Since the Drosophila homologue of zic1, odd-paired (opa), regulates expression of the wingless and engrailed genes and since Wnt proteins posteriorize neural tissue in Xenopus, we asked whether Xenopus Zic1 acted through the Wnt pathway. Using Wnt signaling inhibitors, we demonstrate that an active Wnt pathway is required for activation of en-2 expression by zic1. Consistent with this result, Zic1 induces expression of several wnt genes, including wnt1, wnt4 and wnt8b. wnt1 gene expression activates expression of engrailed in various organisms, including Xenopus, as demonstrated here. Together, our data suggest that zic1 is an upstream regulator of several wnt genes and that the regulatory relationships between opa, wingless and engrailed seen in Drosophila are also present in vertebrates.

Formation of the blastocoel in early Xenopus embryos was studied with a novel biotin-permeability assay and newly generated tight junction markers. The blastocoel forms at the first cleavage division since functional tight junctions which excluded biotin and established a segregated intraembryonic compartment were found at the 2-cell and all subsequent developmental stages. Unexpectedly, tight junctions before the 64-cell stage were not at their normal apical positions, but were found deep in the embryos, up to 200 micron from the apical surface. In these positions, the tight junctions left large areas of ion permeable lateral membranes exposed to the extraembryonic environment, explaining why electrophysiological experiments record a decrease in embryonic input resistances concomitant with early cleavage stages. Immunohistochemistry revealed that the recessed tight junctions did not influence the distribution of C-cadherin and Na+,K+ATPase. Both markers were present apical to recessed tight junctions, indicating that the maintenance of polarization of these basolateral markers does not require tight junctions. With further development, tight junctions assumed an increasingly apical location until, by the 2000-cell stage, they occupied their conventional positions between the blastomeres at the apical/lateral membrane boundaries., (Copyright 1998 Academic Press.)

The tight junction is the most apical member of the intercellular junctional complex. It functions as a permeability barrier between epithelial cells and maintains the integrity of the apical and basolateral membrane domains. In order to study tight junctions in Xenopus laevis, a polyclonal antibody was raised which recognized Xenopus ZO-1. Monoclonal antibody 19B1 (mAb 19B1) was generated in rats using a crude membrane preparation from Xenopus lung as antigen. mAb 19B1 gave immunofluorescent staining patterns identical to those seen with anti-ZO-1 on monolayers of Xenopus A6 kidney epithelial cells and on frozen sections of Xenopus kidney, liver, and embryos. Electron microscopy showed that the 19B1 antigen colocalized with ZO-1 at the tight junction. Western blotting and immunoprecipitation demonstrated that ZO-1 is an approximately 220 kDa protein in Xenopus, while mAb 19B1 identified an approximately 210 kDa antigen on immunoblots. Immunoprecipitates of ZO-1 were not recognized by mAb 19B1 by western analysis. The solubility properties of the 19B1 antigen suggested that it is a peripheral membrane protein. Thus, the antigen recognized by the new monoclonal antibody 19B1 is not ZO-1 and represents a different Xenopus tight junction associated protein.