Transcriptomic analysis reveals that RasGEF1b deletion alters basal and LPS-induced expression of genes involved in chemotaxis and cytokine responses in macrophages.

Ras guanine nucleotide exchange factor member 1b (RasGEF1b) of the RasGEF/CDC25 domain-containing family is preferentially expressed by macrophages. However, information is lacking about its role in macrophage function. In this study, we generated mice with ubiquitous deletion of Rasgef1b and used RNA-seq-based transcriptomics to compare the global gene expression in wild-type and knock-out primary bone-marrow-derived macrophages under basal conditions and after lipopolysaccharide (LPS) treatment. Transcriptional filtering identified several genes with significantly different transcript levels between wild-type and knock-out macrophages. In total, 49 and 37 differentially expressed genes were identified at baseline and in LPS-activated macrophages, respectively. Distinct biological processes were significantly linked to down-regulated genes at the basal condition only, and largely included chemotaxis, response to cytokines, and positive regulation of GTPase activity. Importantly, validation by RT-qPCR revealed that the expression of genes identified as down-regulated after LPS stimulation was also decreased in the knock-out cells under basal conditions. We used a luciferase-based reporter assay to showcase the capability of RasGEF1b in activating the Serpinb2 promoter. Notably, knockdown of RasGEF1b in RAW264.7 macrophages resulted in impaired transcriptional activation of the Serpinb2 promoter, both in constitutive and LPS-stimulated conditions. This study provides a small collection of genes that shows relative expression changes effected by the absence of RasGEF1b in macrophages. Thus, we present the first evidence that RasGEF1b mediates the regulation of both steady-state and signal-dependent expression of genes and propose that this GEF plays a role in the maintenance of the basal transcriptional level in macrophages.

Morphological And Molecular Analyses of an Anatomical Novelty: The Pelvic Fin Filaments of the South American Lungfish.

The pelvic fins of male South American lungfish, Lepidosiren paradoxa, are adorned with a distinctive array of filaments, which grow and become highly vascularized during the breeding season. The resemblance between these pelvic fin filaments (PFFs) and external gills of other vertebrates suggested that this gill-like structure was used for physiological gas exchange. It has been proposed that the unique pelvic fin of male L. paradoxa is used for release of oxygen from its blood into the environment in order to aerate its nesting brood, or, conversely, as an auxiliary respiratory organ by absorbing oxygen from the environment into its bloodstream. Here, we employed histology, scanning electron microscopy (SEM) and quantitative PCR (qPCR) to assess whether the morphology and molecular profile of PFFs are compatible with a role in gas exchange. First, we closely examined its external morphology and showed that PFFs develop from short papillae during the rainy season, but remain covered by a thick nonvascularized epithelium. Histological examination confirmed that capillaries within the filaments are separated from the exterior by a basement membrane and a stratified epithelium composed of four to five cell layers. In addition, SEM analysis revealed significant differences between the fin filament epithelium and typical gill epithelium. Finally, our qPCR results showed that five genes commonly expressed in gills were downregulated in PFFs relative to their expression in regular pectoral fin epidermis. Collectively, our results do not directly support a role for PFFs, commonly referred to as "limb gills", in oxygen release or uptake.