Inbred Mouse Populations Exhibit Intergenerational Changes in Intestinal Microbiota Composition and Function Following Introduction to a Facility.

Inbred mice are used to investigate many aspects of human physiology, including susceptibility to disease and response to therapies. Despite increasing evidence that the composition and function of the murine intestinal microbiota can substantially influence a broad range of experimental outcomes, relatively little is known about microbiome dynamics within experimental mouse populations. We investigated changes in the intestinal microbiome between C57BL/6J mice spanning six generations (assessed at generations 1, 2, 3, and 6), following their introduction to a stringently controlled facility. Fecal microbiota composition and function were assessed by 16S rRNA gene amplicon sequencing and liquid chromatography mass spectrometry, respectively. Significant divergence of the intestinal microbiota between founder and second generation mice, as well as continuing inter-generational variance, was observed. Bacterial taxa whose relative abundance changed significantly through time included Akkermansia, Turicibacter, and Bifidobacterium (p < 0.05), all of which are recognized as having the potential to substantially influence host physiology. Shifts in microbiota composition were mirrored by corresponding differences in the fecal metabolome (r = 0.57, p = 0.0001), with notable differences in levels of tryptophan pathway metabolites and amino acids, including glutamine, glutamate and aspartate. We related the magnitude of changes in the intestinal microbiota and metabolome characteristics during acclimation to those observed between populations housed in separate facilities, which differed in regards to husbandry, barrier conditions and dietary intake. The microbiome variance reported here has implications for experimental reproducibility, and as a consequence, experimental design and the interpretation of research outcomes across wide range of contexts.

Genetic and functional analyses demonstrate a role for abnormal glycinergic signaling in autism.

Autism spectrum disorder (ASD) is a common neurodevelopmental condition characterized by marked genetic heterogeneity. Recent studies of rare structural and sequence variants have identified hundreds of loci involved in ASD, but our knowledge of the overall genetic architecture and the underlying pathophysiological mechanisms remains incomplete. Glycine receptors (GlyRs) are ligand-gated chloride channels that mediate inhibitory neurotransmission in the adult nervous system but exert an excitatory action in immature neurons. GlyRs containing the α2 subunit are highly expressed in the embryonic brain, where they promote cortical interneuron migration and the generation of excitatory projection neurons. We previously identified a rare microdeletion of the X-linked gene GLRA2, encoding the GlyR α2 subunit, in a boy with autism. The microdeletion removes the terminal exons of the gene (GLRA2(Δex8-9)). Here, we sequenced 400 males with ASD and identified one de novo missense mutation, p.R153Q, absent from controls. In vitro functional analysis demonstrated that the GLRA2(Δex8)(-)(9) protein failed to localize to the cell membrane, while the R153Q mutation impaired surface expression and markedly reduced sensitivity to glycine. Very recently, an additional de novo missense mutation (p.N136S) was reported in a boy with ASD, and we show that this mutation also reduced cell-surface expression and glycine sensitivity. Targeted glra2 knockdown in zebrafish induced severe axon-branching defects, rescued by injection of wild type but not GLRA2(Δex8-9) or R153Q transcripts, providing further evidence for their loss-of-function effect. Glra2 knockout mice exhibited deficits in object recognition memory and impaired long-term potentiation in the prefrontal cortex. Taken together, these results implicate GLRA2 in non-syndromic ASD, unveil a novel role for GLRA2 in synaptic plasticity and learning and memory, and link altered glycinergic signaling to social and cognitive impairments.

Identification and characterization of two novel calpain large subunit genes.

Calpains are a family of related proteins, some of which have been shown to function as calcium-dependent cysteine proteases. CAPN1 and CAPN2, the most well characterized calpains, consist of a large (80 kDa) and a small (30 kDa) subunit. In mammals, 11 different paralogous genes encoding calpain large subunits have been identified. We report the identification of two further genes, CAPN13 and CAPN14, potentially encoding calpain large subunits. Radiation hybrid mapping localized both genes within a region mapped to 2p21-2p22. The CAPN13 mRNA exhibits a restricted tissue distribution with low levels of expression detected only in human testis and lung while CAPN14 mRNA could not be detected in any of the 76 tissues examined. Examination of the human genome sequence in the public and private consortia databases did not detect any further members of this gene family. Thus, there would seem to be 13 large subunit calpain genes in the human genome. Phylogenetic analysis reveals that the putative calpain large subunit proteins can be divided into three major groups. The 13 human large subunit genes and the single small subunit gene are located in eight syntenic groups on chromosomes 1, 2, 3, 6, 11, 15, 19 and X.

Hox11 acts cell autonomously in spleen development and its absence results in altered cell fate of mesenchymal spleen precursors.

The genetic steps governing development of the spleen are largely unknown. Absence of Hox11 in mice results in asplenia, but it is unclear how Hox11 exerts its effect on spleen development. To more precisely define Hox11's role in spleen morphogenesis, we have examined the fate of the developing spleen in Hox11(-/-) mice. Perturbation of spleen development begins between dE13 and dE13.5. Cells of the spleen anlage persist past this developmental stage as an unorganized rudiment between the stomach and the pancreas. They fail to proliferate, and haematopoietic cells do not colonize the rudiment. At later stages of embryonic development, the cells can be observed in the mesenchyme of the pancreas, also an expression site of Hox11. In Hox11-/-<-->+/+ chimaeras, spleens were devoid of Hox11(-/-) cells, indicating that the genetic defect is cell autonomous and not due to failure of the organ anlage to attract and retain haematopoietic cells. In -/-<-->+/+ chimaeric embryos, Hox11(-/-) cells were initially present in the spleen anlage. However, at dE13, a reorganization of the spleen occurred in the chimaeras and Hox11(-/-) cells were subsequently excluded from the spleen, suggesting that a change in the affinity for one of the spleen cells had occurred. These observations demonstrate that spleen development consists of genetically separable steps and that absence of Hox11 arrests spleen development at an early stage. The formation of the spleen primordium before the entry of haematopoietic cells does not require the activity of Hox11. However, subsequent differentiation of spleen precursor cells is dependent on the Hox11 gene.

Gene structure, chromosomal localization, and expression pattern of Capn12, a new member of the calpain large subunit gene family.

We report the identification of mouse Capn12, a new member of the calpain large subunit gene family. It possesses potential protease and calcium-binding domains, features typical of the classical calpains. In situ hybridization and Northern blot analysis demonstrate that during the anagen phase of the hair cycle the cortex of the hair follicle is the major expression site of Capn12. The gene was sequenced in its entirety and consists of 21 exons spanning 13 kb with an exon-intron structure typical of the calpain gene family. The last exon of the mouse Actn4 gene overlaps the 3' end of Capn12 but in the opposite orientation. This overlap between the two genes is conserved in the human genome. Three versions of the Capn12 mRNA transcript were identified. They occur as a result of alternative splicing, and two of these encode a protein lacking the C-terminal calmodulin-like domain. Radiation hybrid mapping localized Capn12 to mouse chromosome 7, closely linked to a marker positioned at 10.4 cM. Refined mapping of Capn5, also previously localized to chromosome 7, indicated that it was not closely linked to Capn12, mapping tightly linked to a marker positioned at 48.5 cM.

Hox11 is required to maintain normal Wt1 mRNA levels in the developing spleen.

Mice deficient in Hox11 are asplenic. As Hoxll can function as a transcription factor, we examined the spatial and temporal mRNA expression patterns of Hox11 and a candidate target gene, the Wilm's tumor gene Wt1, in the developing spleen. Hox11 mRNA first appears at approximately dE10.5 in the dorsal mesogastrium while Wt1 mRNA is expressed from dE11.5, approximately 24 hours after Hox11 mRNA first appears. Wt1 mRNA was significantly reduced in the spleen anlage of Hox11-null mice suggesting that Wt1 acts downstream of Hox11 in a transcriptional cascade. Additionally, Hox11 protein is able to transactivate the WT1 promoter in a Hox11-null fibroblast cell line. As Wt1-null embryos have recently been reported to be asplenic, these findings suggest that Wt1 and Hox11 may be components common to a genetic hierarchy that is required for spleen development.

Diverse mRNA expression patterns of the mouse calpain genes Capn5, Capn6 and Capn11 during development.

Calpains are a family of related proteins, originally classified on the basis of their calcium dependence and protease activity. Here we report the mRNA expression patterns during mouse development of the recently identified Capn5, Capn6 and Capn11 genes. The major expression sites of Capn5 during embryogenesis are the developing thymus, sympathetic and dorsal root ganglia. Capn6 mRNA is exclusively expressed during embryogenesis predominantly in developing skeletal and heart muscle overlapping closely with Capn3 expression domains. Expression was also observed in specific cells of the lung, kidney and placenta and in various epithelial cell types where the Capn6 mRNA appeared to be localized within the cell to the basal and apical ends. Capn11 mRNA is restricted exclusively to spermatocytes and only during the later stages of meiosis.

Whn and mHa3 are components of the genetic hierarchy controlling hair follicle differentiation.

The molecular basis of the characteristic hair growth disorder in nude mice that carry a defective Whn transcription factor gene is unknown. A comparison of mRNA populations from wild-type and nude mice back skin by representational difference analysis revealed the absence of acidic hair keratin gene 3 (mHa3) mRNA in mutant mice. Whn and acidic hair keratin genes are co-expressed in hair follicles, nail forming regions and filiform papillae of the tongue: expression of the mHa3 gene is generally detectable about 1 day after Whn mRNA and rapidly ceases in its absence. Whn is strongly expressed during the anagen (growth) phase of the hair cycle in matrix, cortex and outer root sheath; its expression rapidly declines during catagen and is undetectable in telogen phases. In nude mice, low levels of mHa3 expression are maintained in nails and whisker follicles, whereas expression is completely absent in pelage hair follicles and filiform papillae. Thus, the nude phenotype represents the first example of an inherited skin disorder that is associated with the loss of expression rather than structural mutation of keratin genes. The distinct molecular difference between pelage and whisker follicles correlates with the improved mechanical stability of vibrissae in nude mice, implicating mHa3 as an important structural component of the hair shaft.

CAPN11: A calpain with high mRNA levels in testis and located on chromosome 6.

Calpains are a superfamily of related proteins, some of which have been shown to function as calcium-dependent cysteine proteases. In mammals, eight different calpains have been identified. We report the identification of a new mammalian calpain gene, CAPN11. The predicted protein possesses the features typical of calpains including potential protease and calcium-binding domains. The CAPN11 mRNA exhibits a highly restricted tissue distribution with highest levels present in testis. Radiation hybrid mapping localized the gene to human chromosome 6, within a region mapped to p12. Phylogenetic analysis suggests that, in mammals, the predicted CAPN11 protein is most closely related to CAPN1 and CAPN2. However, of the calpain sequences available, the predicted CAPN11 sequence exhibits greatest homology to the chicken micro/m calpain. Thus CAPN11 may be the human orthologue of micro/m calpain. The discovery of this new calpain emphasizes the complexity of the calpain family, with members being distinguished on the basis of protease activity, calcium dependence, and tissue expression.