Comparative analysis of the sea urchin genome has broad implications for the primitive state of deuterostome host defense and the genetic underpinnings of immunity in vertebrates. The sea urchin has an unprecedented complexity of innate immune recognition receptors relative to other animal species yet characterized. These receptor genes include a vast repertoire of 222 Toll-like receptors, a superfamily of more than 200 NACHT domain-leucine-rich repeat proteins (similar to nucleotide-binding and oligomerization domain (NOD) and NALP proteins of vertebrates), and a large family of scavenger receptor cysteine-rich proteins. More typical numbers of genes encode other immune recognition factors. Homologs of important immune and hematopoietic regulators, many of which have previously been identified only from chordates, as well as genes that are critical in adaptive immunity of jawed vertebrates, also are present. The findings serve to underscore the dynamic utilization of receptors and the complexity of immune recognition that may be basal for deuterostomes and predicts features of the ancestral bilaterian form.
In metazoans, the epithelial-mesenchymal transition (EMT) is a crucial process for placing the mesoderm beneath the ectoderm. Primary mesenchyme cells (PMCs) at the vegetal pole of the sea urchin embryo ingress into the floor of the blastocoele from the blastula epithelium and later become the skeletogenic mesenchyme. This ingression movement is a classic EMT during which the PMCs penetrate the basal lamina, lose adherens junctions and migrate into the blastocoele. Later, secondary mesenchyme cells (SMCs) also enter the blastocoele via an EMT, but they accompany the invagination of the archenteron initially, in much the same way vertebrate mesenchyme enters the embryo along with endoderm. Here we identify a sea urchin ortholog of the Snail transcription factor, and focus on its roles regulating EMT during PMC ingression. Functional knockdown analyses of Snail in whole embryos and chimeras demonstrate that Snail is required in micromeres for PMC ingression. Snail represses the transcription of cadherin, a repression that appears evolutionarily conserved throughout the animal kingdom. Furthermore, Snail expression is required for endocytosis of cadherin, a cellular activity that accompanies PMC ingression. Perturbation studies position Snail in the sea urchin micromere-PMC gene regulatory network (GRN), downstream of Pmar1 and Alx1, and upstream of several PMCexpressed proteins. Taken together, our findings indicate that Snail plays an essential role in PMCs to control the EMT process, in part through its repression of cadherin expression during PMC ingression, and in part through its role in the endocytosis that helps convert an epithelial cell to a mesenchyme cell.
SUMMARYIn the sea urchin, entry of -catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of -catenin, of a dominant-negative variant of GSK-3 or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network.
αA- and αB-crystallins are small heat shock proteins that bind thermodynamically destabilized proteins thereby inhibiting their aggregation. Highly expressed in the mammalian lens, the α-crystallins have been postulated to play a critical role in the maintenance of lens optical properties by sequestering age-damaged proteins prone to aggregation as well as through a multitude of roles in lens epithelial cells. Here, we have examined the role of α-crystallins in the development of the vertebrate zebrafish lens. For this purpose, we have carried out morpholino-mediated knockdown of αA-, αBa- and αBb-crystallin and characterized the gross morphology of the lens. We observed lens abnormalities, including increased reflectance intensity, as a consequence of the interference with expression of these proteins. These abnormalities were less frequent in transgenic zebrafish embryos expressing rat αA-crystallin suggesting a specific role of α-crystallins in embryonic lens development. To extend and confirm these findings, we generated an αA-crystallin knockout zebrafish line. A more consistent and severe lens phenotype was evident in maternal/zygotic αA-crystallin mutants compared to those observed by morpholino knockdown. The penetrance of the lens phenotype was reduced by transgenic expression of rat αA-crystallin and its severity was attenuated by maternal αA-crystallin expression. These findings demonstrate that the role of α-crystallins in lens development is conserved from mammals to zebrafish and set the stage for using the embryonic lens as a model system to test mechanistic aspects of α-crystallin chaperone activity and to develop strategies to fine-tune protein-protein interactions in aging and cataracts.
We identify a novel mechanism of β-catenin inhibition involving, chemokine-GPCR signaling, which limits axis formation during zebrafish embryogenesis.
Recent work on the sea urchin endomesoderm gene regulatory network (GRN) offers many opportunities to study the specification and differentiation of each cell type during early development at a mechanistic level. The mesoderm lineages consist of two cell populations, primary and secondary mesenchyme cells (PMCs and SMCs). The micromere-PMC GRN governs the development of the larval skeleton, which is the exclusive fate of PMCs, and SMCs diverge into four lineages, each with its own GRN state. Here we identify a sea urchin ortholog of the Twist transcription factor, and show that it plays an essential role in the PMC GRN and later is involved in SMC formation. Perturbations of Twist either by morpholino knockdown or by overexpression result in defects in progressive phases of PMC development, including specification, ingression/EMT, differentiation and skeletogenesis. Evidence is presented that Twist expression is required for the maintenance of the PMC specification state, and a reciprocal regulation between Alx1 and Twist offers stability for the subsequent processes, such as PMC differentiation and skeletogenesis. These data illustrate the significance of regulatory state maintenance and continuous progression during cell specification, and the dynamics of the sequential events that depend on those earlier regulatory states.
Epithelial-mesenchyme transitions (EMTs) are familiar to all scholars of development. Each animal system utilizes an EMT to produce mesenchyme cells. In vertebrates, for example, there are a number of EMTs that shape the embryo. Early, entry of epiblast cells into the primitive streak is followed by the emergence of mesoderm via an EMT process. The departure of neural crest cells from the margin of the neural folds is an EMT process, and the delamination of cells from the endomesoderm to form the supporting mesenchyme of the lung, liver, and pancreas are EMTs. EMTs are observed in Drosophila following invagination of the ventral furrow, and even in Cnidarians, which have only two germ layers, yet mesoglial and stem cells delaminate from the epithelia and occupy the matrix between the ectoderm and endoderm. This review will focus on a classic example of an EMT, which occurs in the sea urchin embryo. The primary mesenchyme cells (PMCs) ingress from the vegetal plate of this embryo precociously and in advance of archenteron invagination. Because ingression is precisely timed, the PMC lineage precisely known, and the embryo easily observed and manipulated, much has been learned about how the ingression of PMCs works in the sea urchin. Though the focus of this review is the sea urchin PMCs, there is evidence that all EMTs share many common features at both cellular and molecular levels, and many of these mechanisms are also shown to be involved in tumor progression, especially metastasizing carcinomas.
Genetic mutations in the human small heat shock protein αB-crystallin have been implicated in autosomal cataracts and skeletal myopathies, including heart muscle diseases (cardiomyopathy). Although these mutations lead to modulation of their chaperone activity , the functions of αB-crystallin in the maintenance of both lens transparency and muscle integrity remain unclear. This lack of information has hindered a mechanistic understanding of these diseases. To better define the functional roles of αB-crystallin, we generated loss-of-function zebrafish mutant lines by utilizing the CRISPR/Cas9 system to specifically disrupt the two αB-crystallin genes, α and α We observed lens abnormalities in the mutant lines of both genes, and the penetrance of the lens phenotype was higher in α than α mutants. This finding is in contrast with the lack of a phenotype previously reported in αB-crystallin knock-out mice and suggests that the elevated chaperone activity of the two zebrafish orthologs is critical for lens development. Besides its key role in the lens, we uncovered another critical role for αB-crystallin in providing stress tolerance to the heart. The αB-crystallin mutants exhibited hypersusceptibility to develop pericardial edema when challenged by crowding stress or exposed to elevated cortisol stress, both of which activate glucocorticoid receptor signaling. Our work illuminates the involvement of αB-crystallin in stress tolerance of the heart presumably through the proteostasis network and reinforces the critical role of the chaperone activity of αB-crystallin in the maintenance of lens transparency.
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