Molecular Bases of Human Malformation Syndromes Involving the SHH Pathway: GLIA/R Balance and Cardinal Phenotypes

Human hereditary malformation syndromes are caused by mutations in the genes of the signal transduction molecules involved in fetal development. Among them, the Sonic hedgehog (SHH) signaling pathway is the most important, and many syndromes result from its disruption. In this review, we summarize the molecular mechanisms and role in embryonic morphogenesis of the SHH pathway, then classify the phenotype of each malformation syndrome associated with mutations of major molecules in the pathway. The output of the SHH pathway is shown as GLI activity, which is generated by SHH in a concentration-dependent manner, i.e., the sum of activating form of GLI (GLIA) and repressive form of GLI (GLIR). Which gene is mutated and whether the mutation is loss-of-function or gain-of-function determine in which concentration range of SHH the imbalance occurs. In human malformation syndromes, too much or too little GLI activity produces symmetric phenotypes affecting brain size, craniofacial (midface) dysmorphism, and orientation of polydactyly with respect to the axis of the limb. The symptoms of each syndrome can be explained by the GLIA/R balance model.

Stromal remodeling regulates dendritic cell abundance and activity in the tumor microenvironment

Stimulatory dendritic cells (SDC), enriched within Batf3-DC (cDC1), engage in productive interactions with CD8+ effectors along tumor-stroma boundaries. The paradoxical accumulation of poised cross-presenting Batf3-DC within stromal sheets, distal to tumoral nests, is unlikely to simply reflect passive exclusion away from immunosuppressive tumor cores. Drawing parallels with embryonic morphogenesis, we hypothesized that invasive margin stromal remodeling may generate developmentally conserved cell-fate cues that regulate Batf3-DC behavior. We find that CD8+ T-cells massively infiltrate tumor matrices undergoing proteoglycan versican (VCAN) proteolysis, an essential organ-sculpting modification in development and adult tissue-plane forging. VCAN proteolysis releases a bioactive fragment (matrikine), versikine, that is necessary and sufficient for Batf3-DC accumulation. Versikine does not influence tumor-seeding pre-DC differentiation; rather, it orchestrates a distinctive activation program conferring exquisite sensitivity to DNA-sensing, coupled with survival support from atypical innate lymphoid cells. Thus, homeostatic signals from stroma invasion regulate SDC survival and activity to promote T-cell inflammation.

TES-1/Tes protects junctional actin networks under tension from self-injury during epidermal morphogenesis in the C. elegans embryo

During embryonic morphogenesis, the integrity of epithelial tissues depends on the ability of cells in tissue sheets to undergo rapid changes in cell shape while preventing self-injury to junctional actin networks. LIM domain-containing repeat (LCR) proteins are recruited to sites of strained actin filaments in cultured cells, and are therefore promising candidates for mediating self-healing of actin networks, but whether they play similar roles in living organisms has not been determined. Here, we establish roles for Caenorhabditis elegans TES-1/Tes, an actin-binding LCR protein present at apical junctions, during epithelial morphogenesis. TES-1∷GFP is recruited to apical junctions during embryonic elongation, when junctions are under tension; in embryos in which stochastic failure of cell elongation occurs, TES-1 is only strongly recruited to junctions in cells that successfully elongate, and recruitment is severely compromised in genetic backgrounds in which cell shape changes do not successfully occur. tes-1 mutant embryos display junctional F-actin defects, and loss of TES-1 strongly enhances tension-dependent injury of junctional actin networks in hypomorphic mutant backgrounds for CCC components, suggesting that TES-1 helps to prevent self-injury of junctional actin networks during rapid cell shape change. Consistent with such role, a fragment of TES-1 containing its LIM domains localizes to stress fiber strain sites (SFSS) in cultured vertebrate cells. Together, these data establish TES-1 as a tension-sensitive stabilizer of the junctional actin cytoskeleton during embryonic morphogenesis.

Engineering Threshold-Based Selection Systems

Using model organisms to identify novel therapeutic targets is frequently constrained by pre-existing genetic toolkits. To expedite positive selection for identification of novel downstream effectors, we engineered conditional expression of activated CED-10/Rac to disrupt C. elegans embryonic morphogenesis, titrated to 100% lethality. The strategy of engineering thresholds for positive selection using experimental animals was validated with pharmacological and genetic suppression and is generalizable to diverse molecular processes and experimental systems.

Exposure to dexamethasone modifies transcriptomic responses of free-living stages of Strongyloides stercoralis

Hyperinfection and disseminated infection by the parasitic nematode Strongyloides stercoralis can be induced by iatrogenic administration of steroids and immunosuppression and lead to an elevated risk of mortality. Responses of free-living stages of S. stercoralis to the therapeutic corticosteroid dexamethasone (DXM) were investigated using RNA-seq transcriptomes of DXM-treated female and male worms. A total of 17,950 genes representing the transcriptome of these free-living adult stages were obtained, among which 199 and 263 were differentially expressed between DXM-treated females and DXM-treated males, respectively, compared with controls. According to Gene Ontology analysis, differentially expressed genes from DXM-treated females participate in developmental process, multicellular organismal process, cell differentiation, carbohydrate metabolic process and embryonic morphogenesis. Others are involved in signaling and signal transduction, including cAMP, cGMP-dependent protein kinase pathway, endocrine system, and thyroid hormone pathway, as based on Kyoto Encyclopedia of Genes and Genomes analysis. The novel findings warrant deeper investigation of the influence of DXM on growth and other pathways in this neglected tropical disease pathogen, particularly in a setting of autoimmune and/or allergic disease, which may require the clinical use of steroid-like hormones during latent or covert strongyloidiasis.

Tissue-specific regulation of epidermal contraction during C. elegans embryonic morphogenesis

Actin and myosin mediate the epidermal cell contractions that elongate the Caenorhabditis elegans embryo from an ovoid to a tubular-shaped worm. Contraction occurs mainly in the lateral epidermal cells, while the dorsoventral epidermis plays a more passive role. Two parallel pathways trigger actinomyosin contraction, one mediated by LET-502/Rho kinase and the other by PAK-1/p21 activated kinase. A number of genes mediating morphogenesis have been shown to be sufficient when expressed either laterally or dorsoventrally. Additional genes show either lateral or dorsoventral phenotypes. This led us to a model where contractile genes have discrete functions in one or the other cell type. We tested this by examining several genes for either lateral or dorsoventral sufficiency. LET-502 expression in the lateral cells was sufficient to drive elongation. MEL-11/Myosin phosphatase, which antagonizes contraction, and PAK-1 were expected to function dorsoventrally, but we could not detect tissue-specific sufficiency. Double mutants of lethal alleles predicted to decrease lateral contraction with those thought to increase dorsoventral force were previously shown to be viable. We hypothesized that these mutant combinations shifted the contractile force from the lateral to the dorsoventral cells and so the embryos would elongate with less lateral cell contraction. This was tested by examining ten single and double mutant strains. In most cases, elongation proceeded without a noticeable alteration in lateral contraction. We suggest that many embryonic elongation genes likely act in both lateral and dorsoventral cells, even though they may have their primary focus in one or the other cell type.

The initiation knot is a signaling center required for molar tooth development

ABSTRACT Signaling centers, or organizers, regulate many aspects of embryonic morphogenesis. In the mammalian molar tooth, reiterative signaling in specialized centers called enamel knots (EKs) determines tooth patterning. Preceding the primary EK, transient epithelial thickening appears, the significance of which remains debated. Using tissue confocal fluorescence imaging with laser ablation experiments, we show that this transient thickening is an earlier signaling center, the molar initiation knot (IK), that is required for the progression of tooth development. IK cell dynamics demonstrate the hallmarks of a signaling center: cell cycle exit, condensation and eventual silencing through apoptosis. IK initiation and maturation are defined by the juxtaposition of cells with high Wnt activity to Shh-expressing non-proliferating cells, the combination of which drives the growth of the tooth bud, leading to the formation of the primary EK as an independent cell cluster. Overall, the whole development of the tooth, from initiation to patterning, is driven by the iterative use of signaling centers.

A genetic screen for temperature-sensitive morphogenesis-defectiveCaenorhabditis elegansmutants

Morphogenesis involves coordinated cell migrations and cell shape changes that generate tissues and organs, and organize the body plan. Cell adhesion and the cytoskeleton are important for executing morphogenesis, but their regulation remains poorly understood. As genes required for embryonic morphogenesis may have earlier roles in development, temperature-sensitive embryonic-lethal mutations are useful tools for investigating this process. From a collection of ∼200 such Caenorhabditis elegans mutants, we have identified 17 that have highly penetrant embryonic morphogenesis defects after upshifts from the permissive to the restrictive temperature, just prior to the cell shape changes that mediate elongation of the ovoid embryo into a vermiform larva. Using whole genome sequencing, we identified the causal mutations in seven affected genes. These include three genes that have roles in producing the extracellular matrix, which is known to affect the morphogenesis of epithelial tissues in multicellular organisms: the rib-1 and rib-2 genes encode glycosyltransferases, and the emb-9 gene encodes a collagen subunit. We also used live imaging to characterize epidermal cell shape dynamics in one mutant, or1219ts, and observed cell elongation defects during dorsal intercalation and ventral enclosure that may be responsible for the body elongation defects. These results indicate that our screen has identified factors that influence morphogenesis and provides a platform for advancing our understanding of this fundamental biological process.