Cells sense their environment and adapt to it by fine-tuning their transcriptome. Wired into this network of gene expression control are mechanisms to compensate for gene dosage. The increasing use of reverse genetics in zebrafish, and other model systems, has revealed profound differences between the phenotypes caused by genetic mutations and those caused by gene knockdowns at many loci, an observation previously reported in mouse and Arabidopsis. To identify the reasons underlying the phenotypic differences between mutants and knockdowns, we generated mutations in zebrafish egfl7, an endothelial extracellular matrix gene of therapeutic interest, as well as in vegfaa. Here we show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl7 morpholino (morphants) exhibit severe vascular defects. We further observe that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose. Comparing egfl7 mutant and morphant proteomes and transcriptomes, we identify a set of proteins and genes that are upregulated in mutants but not in morphants. Among them are extracellular matrix genes that can rescue egfl7 morphants, indicating that they could be compensating for the loss of Egfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPR interference, which obstructs transcript elongation and causes severe vascular defects, does not cause the upregulation of these genes. Similarly, vegfaa mutants but not morphants show an upregulation of vegfab. Taken together, these data reveal the activation of a compensatory network to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown.

Genetic compensation induced by deleterious mutations but not gene knockdowns

Signaling and Gene Regulatory Networks in Mammalian Lens Development

Although successful in identifying new cataract-linked genes, the previous version of the database iSyTE (integrated Systems Tool for Eye gene discovery) was based on expression information on just three mouse lens stages and was functionally limited to visualization by only UCSC-Genome Browser tracks. To increase its efficacy, here we provide an enhanced iSyTE version 2.0 (URL: http://research.bioinformatics.udel.edu/iSyTE) based on well-curated, comprehensive genome-level lens expression data as a one-stop portal for the effective visualization and analysis of candidate genes in lens development and disease. iSyTE 2.0 includes all publicly available lens Affymetrix and Illumina microarray datasets representing a broad range of embryonic and postnatal stages from wild-type and specific gene-perturbation mouse mutants with eye defects. Further, we developed a new user-friendly web interface for direct access and cogent visualization of the curated expression data, which supports convenient searches and a range of downstream analyses. The utility of these new iSyTE 2.0 features is illustrated through examples of established genes associated with lens development and pathobiology, which serve as tutorials for its application by the end-user. iSyTE 2.0 will facilitate the prioritization of eye development and disease-linked candidate genes in studies involving transcriptomics or next-generation sequencing data, linkage analysis and GWAS approaches.

/pdf/isyte-2-0-a-database-for-expression-based-gene-discovery-in-2kxgscfa4q.pdf

iSyTE 2.0: a database for expression-based gene discovery in the eye.

Oral squamous cell carcinoma is a common and lethal malignancy affecting the head and neck region. CCAT2 (colon cancer-associated transcript 2) gene is affiliated with long non-coding RNAs, which are often found to have important regulatory roles in cancers. This study aims to assess the expression and clinical significance of CCAT2 gene, identify its malignant biological behaviors, and explore the possible mechanisms in oral squamous cell carcinoma. CCAT2 expression was detected by quantitative real-time polymerase chain reaction, and its relationship with clinical factors was assayed using the Kaplan-Meier survival curve. The biological behaviors of CCAT2 and its potential mechanisms in oral squamous cell carcinoma were explored by the combined use of CCAT2 knockdown technology and the Wnt/β-catenin pathway agonist lithium chloride (LiCl). Our results showed that CCAT2 functioning as a potential oncogene was upregulated in oral squamous cell carcinoma. CCAT2 with high expression level was correlated with poor differentiation, higher T stage, and clinical stage, which made CCAT2 to be a prognostic biomarker in oral squamous cell carcinoma. LiCl-activated Wnt/β-catenin signaling pathway could partly restore the CCAT2-mediated malignant biological behaviors of oral squamous cell carcinoma cells by suppressing β-catenin, CCND1, and MYC and activating glycogen synthase kinase 3 beta expression. These findings might assist in the discovery of novel potential diagnostic and therapeutic target for oral squamous cell carcinoma, thereby improve the effects of clinical treatment in patients.

https://journals.sagepub.com/doi/pdf/10.1177/1010428317717670

Silencing of long non-coding RNA CCAT2 depressed malignancy of oral squamous cell carcinoma via Wnt/β-catenin pathway:

Isolated or syndromic congenital cataracts are heterogeneous developmental defects, making the identification of the associated genes challenging. In the past, mouse lens expression microarrays have been successfully applied in bioinformatics tools (e.g., iSyTE) to facilitate human cataract-associated gene discovery. To develop a new resource for geneticists, we report high-throughput RNA sequencing (RNA-seq) profiles of mouse lens at key embryonic stages (E)10.5 (lens pit), E12.5 (primary fiber cell differentiation), E14.5 and E16.5 (secondary fiber cell differentiation). These stages capture important events as the lens develops from an invaginating placode into a transparent tissue. Previously, in silico whole-embryo body (WB)-subtraction-based “lens-enriched” expression has been effective in prioritizing cataract-linked genes. To apply an analogous approach, we generated new mouse WB RNA-seq datasets and show that in silico WB subtraction of lens RNA-seq datasets successfully identifies key genes based on lens-enriched expression. At ≥2 counts-per-million expression, ≥1.5 log2 fold-enrichment (p < 0.05) cutoff, E10.5 lens exhibits 1401 enriched genes (17% lens-expressed genes), E12.5 lens exhibits 1937 enriched genes (22% lens-expressed genes), E14.5 lens exhibits 2514 enriched genes (31% lens-expressed genes), and E16.5 lens exhibits 2745 enriched genes (34% lens-expressed genes). Biological pathway analysis identified genes associated with lens development, transcription regulation and signaling pathways, among other functional groups. Furthermore, these new RNA-seq data confirmed high expression of established cataract-linked genes and identified new potential regulators in the lens. Finally, we developed new lens stage-specific UCSC Genome Brower annotation tracks and made these publicly accessible through iSyTE (
 https://research.bioinformatics.udel.edu/iSyTE/
 ) for user-friendly visualization of lens gene expression/enrichment to prioritize genes from high-throughput data from cataract cases.

/pdf/rna-sequencing-based-transcriptomic-profiles-of-embryonic-1pm1zrnorb.pdf

RNA sequencing-based transcriptomic profiles of embryonic lens development for cataract gene discovery

The signal-induced proliferation-associated family of proteins comprises four members, SIPA1 and SIPA1L1-3. Mutations of the human SIPA1L3 gene result in congenital cataracts. In Xenopus, loss of Sipa1l3 function led to a severe eye phenotype that was distinguished by smaller eyes and lenses including lens fiber cell maturation defects. We found a direct interaction between Sipa1l3 and Epha4, building a functional platform for proper ocular development. Epha4 deficiency phenocopied loss of Sipa1l3 and rescue experiments demonstrated that Epha4 acts upstream of Sipa1l3 during eye development, with both Sipa1l3 and Epha4 required for early eye specification. The ocular phenotype, upon loss of either Epha4 or Sipa1l3, was partially mediated by rax. We demonstrate that canonical Wnt signaling is inhibited downstream of Epha4 and Sipa1l3 during normal eye development. Depletion of either Sipa1l3 or Epha4 resulted in an upregulation of axin2 expression, a direct Wnt/β-catenin target gene. In line with this, Sipa1l3 or Epha4 depletion could be rescued by blocking Wnt/β-catenin or activating non-canonical Wnt signaling. We therefore conclude that this pathomechanism prevents proper eye development and maturation of lens fiber cells, resulting in congenital cataracts.

/pdf/an-epha4-sipa1l3-wnt-pathway-regulates-eye-development-and-4wf3l03553.pdf

An Epha4/Sipa1l3/Wnt pathway regulates eye development and lens maturation

The human cytomegalovirus (HCMV) infects fibroblasts via an interaction of its envelope glycoprotein gO with the cellular platelet-derived growth factor receptor alpha (PDGFRα), and soluble derivatives of this receptor can inhibit viral entry. We aimed to select mutants with resistance against PDGFRα-Fc and the PDGFRα-derived peptides GT40 and IK40 to gain insight into the underlying mechanisms and determine the genetic barrier to resistance. An error-prone variant of strain AD169 was propagated in the presence of inhibitors, cell cultures were monitored weekly for signs of increased viral growth, and selected viruses were tested regarding their sensitivity to the inhibitor. Resistant virus was analyzed by DNA sequencing, candidate mutations were transferred into AD169 clone pHB5 by seamless mutagenesis, and reconstituted virus was again tested for loss of sensitivity by dose-response analyses. An S48Y mutation in gO was identified that conferred a three-fold loss of sensitivity against PDGFRα-Fc, a combination of mutations in gO, gH, gB and gN reduced sensitivity to GT40 by factor 4, and no loss of sensitivity occurred with IK40. The resistance-conferring mutations support the notion that PDGFRα-Fc and GT40 perturb the interaction of gO with its receptor, but the relatively weak effect indicates a high genetic barrier to resistance.

https://www.mdpi.com/1999-4915/13/6/1094/pdf

Rebecca Engels

Papers

An Epha4/Sipa1l3/Wnt pathway regulates eye development and lens maturation

Selection of Human Cytomegalovirus Mutants with Resistance against PDGFRα-Derived Entry Inhibitors.

LFA1 and ICAM1 are critical for fusion and spread of murine leukemia virus in vivo.