Terminal differentiation of villus tip enterocytes is governed by distinct Tgfβ superfamily members

The protective and absorptive functions of the intestinal epithelium rely on differentiated enterocytes in the villi. The differentiation of enterocytes is orchestrated by sub-epithelial mesenchymal cells producing distinct ligands along the villus axis, in particular Bmps and Tgfβ. Here, we show that individual Bmp ligands and Tgfβ drive distinct enterocytic programs specific to villus zonation. Bmp4 is expressed from the centre to the upper part of the villus and activates preferentially genes connected to lipid uptake and metabolism. In contrast, Bmp2 is produced by villus tip mesenchymal cells and it influences the adhesive properties of villus tip epithelial cells and the expression of immunomodulators. Additionally, Tgfβ induces epithelial gene expression programs similar to those triggered by Bmp2. Bmp2-driven villus tip program is activated by a canonical Bmp receptor type I/Smad-dependent mechanism. Finally, we establish an organoid cultivation system that enriches villus tip enterocytes and thereby better mimics the cellular composition of the intestinal epithelium. Our data suggest that not only a Bmp gradient but also the activity of individual Bmp drives specific enterocytic programs

Interspecific variation of gastointestinal microbiota in passerine birds

in English Vertebrates host complex microbial communities in their intestinal system. This gut microbiome (GM) comprises billions of bacterial cells closely interacting with a host's physiology. Despite contemporary progress in this field, present knowledge of vertebrate GM is based on studies carried out only on a limited set of host species, including humans, captive rodents and few economically important mammals and birds. On the contrary, knowledge of GM in wild living, non-mammalian species is still rather insufficient. Thus, this thesis aims to analyze GM in wild passerine populations and to investigate potential patterns in GM composition. Main objective is to provide evidence that GM composition is dependent on host species, and of co-divergence between GM composition variability and host phylogeny. Additional focus is devoted to diversity analysis among individual bacterial genera regarding their phylogeny. Using the Illumina MySeq platform, we sequenced amplicons of bacterial 16s rRNA gene obtained from fecal samples of 486 individuals representing 57 Czech Republic passerine species and 107 individuals representing 38 Cameroon Republic species. A modified approach was applied during data processing. In the first step, analyzed dataset was sorted by classifying sequences to respective..

Research of vertebrate-microbiota relationship using germ-free organisms

Germ free (axenic) animals are individuals reared under specific conditions preventing their contact with surrounding microorganisms. Some of the features observed in these individuals vary from those observed in naturally colonized counterparts. These differences probably reflect the influence of presence of a complex intestinal microbial population in the intestine, which influences important physiological functions of the host body by various mechanisms. Thus, nature of these differences allows to study relationship of the host, vertebrate in this case and its microbiota, which evolved into a complicated system of interactions providing relatively stable coexistence. Germ free research of this relationship is focused on interactions between microbiota and host's immune system, metabolism, morphology of digestive tract and behavior. This thesis provides summary of research outcomes on previously mentioned topics. Powered by TCPDF (www.tcpdf.org

Retinitis pigmentosa-linked mutation in DHX38 modulates its splicing activity.

Retinitis pigmentosa (RP) is a hereditary disease affecting tens of thousands of people world-wide. Here we analyzed the effect of an amino acid substitution in the RNA helicase DHX38 (Prp16) causing RP. DHX38 has been proposed as the helicase important for the 2nd step of splicing. We showed that DHX38 associates with key splicing factors involved in both splicing steps but did not find any evidence that the RP mutations changes DHX38 interaction profile with the spliceosome. We further downregulated DHX38 and monitored changes in splicing. We observed only minor perturbations of general splicing but detected modulation of ~70 alternative splicing events. Next, we probed DHX38 function in splicing of retina specific genes and found that FSCN2 splicing is dependent on DHX38. In addition, RHO splicing was inhibited specifically by expression of DHX38 RP variant. Finally, we showed that overexpression of DHX38 promotes usage of canonical as well as cryptic 5' splice sites in HBB splicing reporter. Together, our data show that DHX38 is a splicing factor that promotes splicing of cryptic splice sites and regulate alternative splicing. We further provide evidence that the RP-linked substitution G332D modulates DHX38 splicing activity

Sodium Accumulation and Blood Capillary Rarefaction in the Skin Predispose Spontaneously Hypertensive Rats to Salt Sensitive Hypertension

Recent studies in humans and rats suggested that increased Na+ storage in the skin without parallel water retention may predispose to salt-sensitive hypertension. In the current studies, we compared tissue Na+ storage in salt sensitive spontaneously hypertensive rats (SHR) versus salt resistant normotensive Brown Norway (BN-Lx) rats. After salt loading (10 days drinking 1% NaCl solution), the SHR showed significant parallel increase in Na+-to-water as well as (Na++K+)-to-water ratios suggesting increased storage of osmotically inactive Na+ in the skin while no significant changes in skin electrolyte concentrations were observed in BN-Lx rats. SHR rats after salt treatment exhibited a nonsignificant decrease in skin blood capillary number (rarefaction) while BN-Lx rats showed significantly increased skin blood capillary density. Analysis of dermal gene expression profiles in BN-Lx rats after salt treatment showed significant up-regulation of genes involved in angiogenesis and proliferation of endothelial cells contrary to the SHR. Since the skin harbors most of the body’s resistance vessels it is possible that blood capillary rarefaction may lead to increased peripheral resistance and salt sensitivity in the SHR

Within‐community variation of interspecific divergence patterns in passerine gut microbiota

Abstract Gut microbiota (GM) often exhibit variation between different host species and co‐divergence with hosts' phylogeny. Identifying these patterns is a key for understanding the mechanisms that shaped symbiosis between GM and its hosts. Therefore, both GM‐host species specificity and GM‐host co‐divergence have been investigated by numerous studies. However, most of them neglected a possibility that different groups of bacteria within GM can vary in the tightness of their association with the host. Consequently, unlike most of these studies, we aimed to directly address how the strength of GM‐host species specificity and GM‐host co‐divergence vary across different GM clades. We decomposed GM communities of 52 passerine species (394 individuals), characterized by 16S rRNA amplicon sequence variant (ASV) profiles, into monophyletic Binned Taxonomic units (BTUs). Subsequently, we analyzed strength of host species specificity and correlation with host phylogeny separately for resulting BTUs. We found that most BTUs exhibited significant host‐species specificity in their composition. Notably, BTUs exhibiting high host‐species specificity comprised bacterial taxa known to impact host's physiology and immune system. However, BTUs rarely displayed significant co‐divergence with host phylogeny, suggesting that passerine GM evolution is not shaped primarily through a shared evolutionary history between the host and its gut microbes

DataSheet1_Two complementary approaches for efficient isolation of Sertoli cells for transcriptomic analysis.PDF

Sertoli cells (SCs) are the only somatic cells that reside in seminiferous tubules of testis. They directly interact with and support the development of germ cells, thus have an indispensable role in the process of spermatogenesis. SCs first appear in a proliferative state and then, with the initiation of the first wave of spermatogenesis, progress to a mature “nurturing” state which supports lifelong continuous sperm production. During this development, the SC transcriptome must adapt rapidly as obstacles in SC maturation often result in deficiencies in male fertility. Due to its importance in spermatogenesis, a reliable, rapid, and precise method for the isolation of high purity, viable and unadulterated SC has been largely missing. We have developed an improved method for the preparation of a testicular single cell suspension comprised of two alternative protocols to separate SCs from the rest of the testicular cells by FACS. The first sorting scheme is based on their co-expression of surface specific markers, FSHr and Occludin-1, while the second focuses on the co-staining of SCs with FSHr-specific antibody and Hoechst 33342, which discriminates DNA content of testicular cells. The entire procedure can be completed in less than 3 h which permits the analysis of the development-related transcriptional profile of these cells. Notably, our comparative study showed that this method resulted in a SC transcriptome that is largely comparable to SCs which were briskly isolated due to their cell-specific expression of fluorescent protein. Interestingly, we also show that SCs sorted as FSHr+Occludin+ cells contained a tangible portion of transcripts from all types of testicular germ cells. Sorting of SCs according to their 2C DNA content significantly reduced the presence of these transcripts, thus seems to be the most suitable approach for accurate determination of the SC transcriptome. We believe that these novel approaches for the isolation of SCs will assist researchers in the elucidation of their function as well as their role in spermatogenesis and disorders related to male infertility.</p

Data_Sheet_1_Gut microbiota variation between climatic zones and due to migration strategy in passerine birds.pdf

IntroductionDecreasing biotic diversity with increasing latitude is an almost universal macroecological pattern documented for a broad range of taxa, however, there have been few studies focused on changes in gut microbiota (GM) across climatic zones.MethodsUsing 16S rRNA amplicon profiling, we analyzed GM variation between temperate (Czechia) and tropical (Cameroon) populations of 99 passerine bird species and assessed GM similarity of temperate species migrating to tropical regions with that of residents/short-distance migrants and tropical residents. Our study also considered the possible influence of diet on GM.ResultsWe observed no consistent GM diversity differences between tropical and temperate species. In the tropics, GM composition varied substantially between dry and rainy seasons and only a few taxa exhibited consistent differential abundance between tropical and temperate zones, irrespective of migration behavior and seasonal GM changes. During the breeding season, trans-Saharan migrant GM diverged little from species not overwintering in the tropics and did not show higher similarity to tropical passerines than temperate residents/short-distance migrants. Interestingly, GM of two temperate-breeding trans-Saharan migrants sampled in the tropical zone matched that of tropical residents and converged with other temperate species during the breeding season. Diet had a slight effect on GM composition of tropical species, but no effect on GM of temperate hosts.DiscussionConsequently, our results demonstrate extensive passerine GM plasticity, the dominant role of environmental factors in its composition and limited effect of diet.</p

Data_Sheet_2_Gut microbiota variation between climatic zones and due to migration strategy in passerine birds.xlsx

IntroductionDecreasing biotic diversity with increasing latitude is an almost universal macroecological pattern documented for a broad range of taxa, however, there have been few studies focused on changes in gut microbiota (GM) across climatic zones.MethodsUsing 16S rRNA amplicon profiling, we analyzed GM variation between temperate (Czechia) and tropical (Cameroon) populations of 99 passerine bird species and assessed GM similarity of temperate species migrating to tropical regions with that of residents/short-distance migrants and tropical residents. Our study also considered the possible influence of diet on GM.ResultsWe observed no consistent GM diversity differences between tropical and temperate species. In the tropics, GM composition varied substantially between dry and rainy seasons and only a few taxa exhibited consistent differential abundance between tropical and temperate zones, irrespective of migration behavior and seasonal GM changes. During the breeding season, trans-Saharan migrant GM diverged little from species not overwintering in the tropics and did not show higher similarity to tropical passerines than temperate residents/short-distance migrants. Interestingly, GM of two temperate-breeding trans-Saharan migrants sampled in the tropical zone matched that of tropical residents and converged with other temperate species during the breeding season. Diet had a slight effect on GM composition of tropical species, but no effect on GM of temperate hosts.DiscussionConsequently, our results demonstrate extensive passerine GM plasticity, the dominant role of environmental factors in its composition and limited effect of diet.</p