Transgenerational transmission and modification of pathological traits induced by prenatal immune activation.

Prenatal exposure to infectious or inflammatory insults is increasingly recognized to contribute to the etiology of psychiatric disorders with neurodevelopmental components, including schizophrenia, autism and bipolar disorder. It remains unknown, however, if such immune-mediated brain anomalies can be transmitted to subsequent generations. Using an established mouse model of prenatal immune activation by the viral mimetic poly(I:C), we show that reduced sociability and increased cued fear expression are similarly present in the first- and second-generation offspring of immune-challenged ancestors. We further demonstrate that sensorimotor gating impairments are confined to the direct descendants of infected mothers, whereas increased behavioral despair emerges as a novel phenotype in the second generation. These transgenerational effects are mediated via the paternal lineage and are stable until the third generation, demonstrating transgenerational non-genetic inheritance of pathological traits following in-utero immune activation. Next-generation sequencing further demonstrated unique and overlapping genome-wide transcriptional changes in first- and second-generation offspring of immune-challenged ancestors. These transcriptional effects mirror the transgenerational effects on behavior, showing that prenatal immune activation leads to a transgenerational transmission (presence of similar phenotypes across generations) and modification (presence of distinct phenotypes across generations) of pathological traits. Together, our study demonstrates for, we believe, the first time that prenatal immune activation can negatively affect brain and behavioral functions in multiple generations. These findings thus highlight a novel pathological aspect of this early-life adversity in shaping disease risk across generations.

Hypervulnerability of the adolescent prefrontal cortex to nutritional stress via reelin deficiency.

Overconsumption of high-fat diets (HFDs) can critically affect synaptic and cognitive functions within telencephalic structures such as the medial prefrontal cortex (mPFC). The underlying mechanisms, however, remain largely unknown. Here we show that adolescence is a sensitive period for the emergence of prefrontal cognitive deficits in response to HFD. We establish that the synaptic modulator reelin (RELN) is a critical mediator of this vulnerability because (1) periadolescent HFD (pHFD) selectively downregulates prefrontal RELN+ cells and (2) augmenting mPFC RELN levels using transgenesis or prefrontal pharmacology prevents the pHFD-induced prefrontal cognitive deficits. We further identify N-methyl-d-aspartate-dependent long-term depression (NMDA-LTD) at prefrontal excitatory synapses as a synaptic signature of this association because pHFD abolishes NMDA-LTD, a function that is restored by RELN overexpression. We believe this study provides the first mechanistic insight into the vulnerability of the adolescent mPFC towards nutritional stress, such as HFDs. Our findings have primary relevance to obese individuals who are at an increased risk of developing neurological cognitive comorbidities, and may extend to multiple neuropsychiatric and neurological disorders in which RELN deficiency is a common feature.

Abnormal context-reward associations in an immune-mediated neurodevelopmental mouse model with relevance to schizophrenia.

Impairments in central reward processing constitute an important aspect of the negative symptoms of schizophrenia. Despite its clinical relevance, the etiology of deficient reward processing in schizophrenia remains largely unknown. Here, we used an epidemiologically informed mouse model of schizophrenia to explore the effects of prenatal immune activation on reward-related functions. The model is based on maternal administration of the viral mimic PolyI:C and has been developed in relation to the epidemiological evidence demonstrating enhanced risk of schizophrenia and related disorders following prenatal maternal infection. We show that prenatal immune activation induces selective deficits in the expression (but not acquisition) of conditioned place preference for a natural reward (sucrose) without changing hedonic or neophobic responses to the reward. On the other hand, prenatal immune activation led to enhanced place preference for the psychostimulant drug cocaine, while it attenuated the locomotor reaction to the drug. The prenatal exposure did not alter negative reinforcement learning as assessed using a contextual fear conditioning paradigm. Our findings suggest that the nature of reward-related abnormalities following prenatal immune challenge depends on the specificity of the reward (natural reward vs drug of abuse) as well as on the valence domain (positive vs negative reinforcement learning). Moreover, our data indicate that reward abnormalities emerging in prenatally immune-challenged offspring may, at least in part, stem from an inability to retrieve previously established context-reward associations and to integrate such information for appropriate goal-directed behavior.

Vagal afferents mediate early satiation and prevent flavour avoidance learning in response to intraperitoneally infused exendin-4.

Glucagon-like peptide-1 receptor (GLP-1R) agonists such as exendin-4 (Ex-4) affect eating and metabolism and are potential candidates for treating obesity and type II diabetes. In the present study, we tested whether vagal afferents mediate the eating-inhibitory and avoidance-inducing effects of Ex-4. Subdiaphragmatic vagal deafferentation (SDA) blunted the short-term (< 1 h) but not long-term eating-inhibitory effect of i.p.-infused Ex-4 (0.1 µg/kg) in rats. A dose of 1 µg/kg Ex-4 reduced 0.5, 1, 2 and 4 h cumulative food intake in SDA and sham-operated rats to a similar extent. Paradoxically, SDA but not sham rats developed a conditioned flavour avoidance (CFA) after i.p. Ex-4 (0.1 µg/kg). SDA completely blunted the induction of c-Fos expression by Ex-4 in the hypothalamic paraventricular nucleus. Ex-4, however, increased the number of c-Fos expressing cells, independent of intact vagal afferents, in the nucleus accumbens and in the central nucleus of the amygdala, the lateral external parabrachial nucleus, the caudal ventrolateral medulla and the dorsal vagal complex. These data suggest that intact vagal afferents are only necessary for the full expression of the early satiating effect of Ex-4 but not for later eating-inhibitory actions, when circulating Ex-4 might reach the brain via the circulation. Our data also dissociate the satiating and avoidance-inducing effects of the low Ex-4 dose tested under our conditions and suggest that vagal afferent signalling may protect against the development of CFA. Taken together, these findings reveal a complex role of vagal afferents in mediating the effects of GLP-1R activation on ingestive behaviour.

Continuum model of epithelial morphogenesis during Caenorhabditis elegans embryonic elongation.

The purpose of this work is to provide a biomechanical model to investigate the interplay between cellular structures and the mechanical force distribution during the elongation process of Caenorhabditis elegans embryos. Epithelial morphogenesis drives the elongation process of an ovoid embryo to become a worm-shaped embryo about four times longer and three times thinner. The overall anatomy of the embryo is modelled in the continuum mechanics framework from the structural organization of the subcellular filaments within epithelial cells. The constitutive relationships consider embryonic cells as homogeneous materials with an active behaviour, determined by the non-muscle myosin II molecular motor, and a passive viscoelastic response, related to the directional properties of the filament network inside cells. The axisymmetric elastic solution at equilibrium is derived by means of the incompressibility conditions, the continuity conditions for the overall embryo deformation and the balance principles for the embryonic cells. A particular analytical solution is proposed from a simplified geometry, demonstrating the mechanical role of the microtubule network within epithelial cells in redistributing the stress from a differential contraction of circumferentially oriented actin filaments. The theoretical predictions of the biomechanical model are discussed within the biological scenario proposed through genetic analysis and pharmacological experiments.

Tissue integrity: hemidesmosomes and resistance to stress.

How do animal tissues resist the shearing forces to which they are exposed during locomotion or harsh encounters with the environment? Genetic analysis in Caenorhabditis elegans is furthering our understanding of the nature and function of the attachments that preserve tissue integrity.

Epithelial biology: lessons from Caenorhabditis elegans.

Epithelial cells are essential and abundant in all multicellular animals where their dynamic cell shape changes orchestrate morphogenesis of the embryo and individual organs. Genetic analysis in the simple nematode Caenorhabditis elegans provides some clues to the mechanisms that are involved in specifying epithelial cell fates and in controlling specific epithelial processes such as junction assembly, trafficking or cell fusion and cell adhesion. Here we review recent findings concerning C. elegans epithelial cells, focusing in particular on epithelial polarity, and transcriptional control.

Assembly of C. elegans apical junctions involves positioning and compaction by LET-413 and protein aggregation by the MAGUK protein DLG-1.

Specialised subapical junctions play a critical role in maintaining epithelial cell polarity and tissue integrity, and provide a platform for intracellular signalling. Here we analyse the roles of C. elegans genes let-413 and dlg-1, a homologue of Drosophila lethal discs large, in the assembly of the C. elegans apical junction (CeAJ), and provide the first characterisation of this structure. We have identified dlg-1 as an essential gene in an RNA interference screen against C. elegans homologues of genes encoding proteins involved in tight or septate junction formation. We show that DLG-1 colocalises with the junctional protein JAM-1 at CeAJs in a unit distinct from HMP-1/alpha-catenin, and apical to the laterally localised LET-413. Loss of dlg-1 activity leads to JAM-1 mislocalisation and the disappearance of the electron-dense component of the CeAJs, but only mild adhesion and polarity defects. In contrast, loss of let-413 activity leads to the formation of basally extended discontinuous CeAJs and strong adhesion and polarity defects. Interestingly, in LET-413-deficient embryos, CeAJ markers are localised along the lateral membrane in a manner resembling that observed in wild-type embryos at the onset of epithelial differentiation. We conclude that the primary function of LET-413 is to correctly position CeAJ components at a discrete subapical position. Furthermore, we propose that DLG-1 is required to aggregate JAM-1 and other proteins forming the electron-dense CeAJ structure. Our data suggest that epithelial adhesion is maintained by several redundant systems in C. elegans.

Human medulloblastoma cell line DEV is a potent tool to screen for factors influencing differentiation of neural stem cells.

The aim of our study was to investigate whether a human neural cell line could be used as a reliable screening tool to examine the functional conservation, in humans, of transcription factors involved in neuronal or glial specification in other species. Gain-of-function experiments were performed on DEV cells, a cell line derived from a human medulloblastoma. Genes encoding nine different transcription factors were tested for their influence on the process of specification of human DEV cells towards a neuronal or glial fate. In a first series of experiments, DEV cells were transfected with murine genes encoding transcription factors known to be involved in the neuronal differentiation cascade. Neurogenins-1, -2, and -3; Mash-1; and NeuroD increased the differentiation of DEV cells towards a neuronal phenotype by a factor of 2-3.5. In a second series of experiments, we tested transcription factors involved in invertebrate glial specification. In the embryonic Drosophila CNS, the development of most glial cells depends on the master regulatory gene glial cell missing (gcm). Expression of gcm in DEV cells induced a twofold increase of astrocytic and a sixfold increase of oligodendroglial cell types. Interestingly, expression of tramtrack69, which is required in all Drosophila glial cells, resulted in a fourfold increase of only the oligodendrocyte phenotype. Expression of the related tramtrack88 protein, which is not expressed in the fly glia, or the C. elegans lin26 protein showed no effect. These results show that the Drosophila transcription factor genes tested can conserve their function upon transfection into the human DEV cells, qualifying this cell line as a screening tool to analyze the mechanisms of neuronal and glial specification.

The Caenorhabditis elegans gene lin-26 can trigger epithelial differentiation without conferring tissue specificity.

How epithelial cell fates become specified is poorly understood. We have previously shown that the putative C2H2 zinc-finger transcription factor LIN-26 is required for the differentiation of ectodermal and mesodermal epithelial cells in Caenorhabditis elegans. Here, we report that ectopic LIN-26 expression during early gastrulation transforms most blastomeres into epithelial-like cells. Specifically, LIN-26 induced the expression of three epithelial markers: the adherens junction protein JAM-1; DLG-1, which is essential for the assembly of JAM-1 at junctions; and CHE-14, which is involved in apical trafficking. Furthermore, ultrastructural studies revealed that ectopic LIN-26 expression induced the formation of adherens-like junctions. However, ectopic lin-26 expression did not confer any tissue-specific cell fate, such as the epidermal cell fate, as evidenced from the observation that several epidermal-specific genes were not induced. Conversely, we show that epidermal cells displayed some polarity defects in lin-26 mutants. We conclude that lin-26 can induce epithelial differentiation and that epitheliogenesis is not a default pathway in C. elegans.

TBP-like factor is required for embryonic RNA polymerase II transcription in C. elegans.

Recently, a novel family of TATA binding protein (TBP)-like factors (TLFs) have been described in metazoan organisms; however, their function has not yet been elucidated. Using Caenorhabditis elegans (Ce) as a model, we demonstrate that CeTLF is required in vivo for zygotic transcription during embryogenesis. Elimination of CeTLF expression by RNA interference caused embryonic lethality either due to the lack of expression of early patterning genes or to their ectopic expression. Moreover, the absence of CeTLF in vivo prevented the correct soma-specific phosphorylation of RNA polymerase II (Pol II). Thus, CeTLF may positively or negatively regulate Pol II transcription, depending on the developmental stage of the embryo.

CHE-14, a protein with a sterol-sensing domain, is required for apical sorting in C. elegans ectodermal epithelial cells.

BACKGROUND: Polarised trafficking of proteins is critical for normal expression of the epithelial phenotype, but its genetic control is not understood. The regulatory gene lin-26 is essential for normal epithelial differentiation in the nematode Caenorhabditis elegans. To identify potential effectors of lin-26, we characterised mutations that result in lin-26-like phenotypes. Here, we report the phenotypic and molecular analysis of one such mutant line, che-14. RESULTS: Mutations in che-14 resulted in several partially penetrant phenotypes affecting the function of most epithelial or epithelial-like cells of the ectoderm, including the hypodermis, excretory canal, vulva, rectum and several support cells. The defects were generally linked to the accumulation of vesicles or amorphous material near the apical surface, suggesting that secretion was defective. The CHE-14 protein showed similarity to proteins containing sterol-sensing domains, including Dispatched, Patched and NPC1. A fusion protein between full-length CHE-14 and the green fluorescent protein became localised to the apical surface of epithelial cells that require che-14 function. Deletions that removed the predicted transmembrane domains or extracellular loops of CHE-14 abolished apical localisation and function of the protein. CONCLUSIONS: We propose that CHE-14 is involved in a novel secretory pathway dedicated to the exocytosis of lipid-modified proteins at the apical surface of certain epithelial cells. Our data raise the possibility that the primordial function of proteins containing a sterol-sensing domain is to control vesicle trafficking: CHE-14 and Dispatched in exocytosis, Patched and NPC1 in endocytosis.

LET-413 is a basolateral protein required for the assembly of adherens junctions in Caenorhabditis elegans.

Epithelial cells are polarized, with apical and basal compartments demarcated by tight and adherens junctions. Proper establishment of these subapical junctions is critical for normal development and histogenesis. We report the characterization of the gene let-413 which has a critical role in assembling adherens junctions in Caenorhabditis elegans. In let-413 mutants, adherens junctions are abnormal and mislocalized to more basolateral positions, epithelial cell polarity is affected and the actin cytoskeleton is disorganized. The LET-413 protein contains one PDZ domain and 16 leucine-rich repeats with high homology to proteins known to interact with small GTPases. Strikingly, LET-413 localizes to the basolateral membrane. We suggest that LET-413 acts as an adaptor protein involved in polarizing protein trafficking in epithelial cells.

RNA interference: genetic wand and genetic watchdog.

In many species, introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi. The apparently widespread nature of RNAi in eukaryotes, ranging from trypanosome to mouse, has sparked great interest from both applied and fundamental standpoints. Here we review the technical improvements being made to increase the experimental potential of this technique. We also discuss recent advances in uncovering the proteins that act during the RNAi process, discoveries that have revealed enticing links between transposition, transgene silencing and RNAi.

RNA interference can target pre-mRNA: consequences for gene expression in a Caenorhabditis elegans operon.

In nematodes, flies, trypanosomes, and planarians, introduction of double-stranded RNA results in sequence-specific inactivation of gene function, a process termed RNA interference (RNAi). We demonstrate that RNAi against the Caenorhabditis elegans gene lir-1, which is part of the lir-1/lin-26 operon, induced phenotypes very different from a newly isolated lir-1 null mutation. Specifically, lir-1(RNAi) induced embryonic lethality reminiscent of moderately strong lin-26 alleles, whereas the lir-1 null mutant was viable. We show that the lir-1(RNAi) phenotypes resulted from a severe loss of lin-26 gene expression. In addition, we found that RNAi directed against lir-1 or lin-26 introns induced similar phenotypes, so we conclude that lir-1(RNAi) targets the lir-1/lin-26 pre-mRNA. This provides direct evidence that RNA interference can prevent gene expression by targeting nuclear transcripts. Our results highlight that caution may be necessary when interpreting RNA interference without the benefit of mutant alleles.

Patterning the C. elegans embryo: moving beyond the cell lineage.

The Caenorhabditis elegans embryo undergoes a series of stereotyped cell cleavages that generates the organs and tissues necessary for an animal to survive. Here we review two models of embryonic patterning, one that is lineage-based, and one that focuses on domains of organ and tissue precursors. Our evolving view of C. elegans embryogenesis suggests that this animal develops by mechanisms that are qualitatively similar to those used by other animals.

lir-2, lir-1 and lin-26 encode a new class of zinc-finger proteins and are organized in two overlapping operons both in Caenorhabditis elegans and in Caenorhabditis briggsae.

lin-26, which encodes a unique Zn-finger protein, is required for differentiation of nonneuronal ectodermal cells in Caenorhabditis elegans. Here, we show that the two genes located immediately upstream of lin-26 encode LIN-26-like Zn-finger proteins; hence their names are lir-1 and lir-2 (lin-26 related). lir-2, lir-1, and lin-26 generate several isoforms by alternative splicing and/or trans-splicing at different positions. On the basis of their trans-splicing pattern, their intergenic distances, and their expression, we suggest that lir-2, lir-1, and lin-26 form two overlapping transcriptional operons. The first operon, which is expressed in virtually all cells, includes lir-2 and long lir-1 isoforms. The second operon, which is expressed in the nonneuronal ectoderm, includes short lir-1 isoforms, starting at exon 2 and lin-26. This unusual genomic organization has been conserved in C. briggsae, as shown by cloning the C. briggsae lir-2, lir-1, and lin-26 homologs. Particularly striking is the sequence conservation throughout the first lir-1 intron, which is very long in both species. Structural conservation is functionally meaningful as C. briggsae lin-26 is also expressed in the nonneuronal ectoderm and can complement a C. elegans lin-26 null mutation.

pha-4, an HNF-3 homolog, specifies pharyngeal organ identity in Caenorhabditis elegans.

To build complex organs, embryos have evolved mechanisms that integrate the development of cells unrelated to one another by cell type or ancestry. Here we show that the pha-4 locus establishes organ identity for the Caenorhabditis elegans pharynx. In pha-4 mutants, pharyngeal cells are transformed into ectoderm. Conversely, ectopic pha-4 expression produces excess pharyngeal cells. pha-4 encodes an HNF-3 homolog selectively expressed in the nascent digestive tract, including all pharynx precursors at the time they are restricted to a pharyngeal fate. We suggest that pha-4 is a key component of a transcription-based mechanism to endow cells with pharyngeal organ identity.

A tissue-specific knock-out strategy reveals that lin-26 is required for the formation of the somatic gonad epithelium in Caenorhabditis elegans.

The Caenorhabditis elegans LIN-26 protein is required to specify and/or maintain the fates of all non-neuronal ectodermal cells. Here we show that lin-26 is expressed until the somatic gonad primordium stage in all cells of the somatic gonad, except in distal tip cells, and later in all uterine cells. To determine if lin-26 functions in the somatic gonad, we have generated gonad-specific lin-26 alleles obtained by integration of lin-26 promoter deletion derivatives into a lin-26 null mutant background. In this way, we rescued the lethal phenotype imparted by lin-26 null mutations and uncovered a highly penetrant sterile phenotype. Specifically, the strongest of these new alleles was characterized by the absence of lin-26 expression in the somatic gonad, the presence of endomitotic oocytes, decreased germline proliferation, a protruding vulva and a less penetrant absence of gonad arms. Lineage analysis of mutant somatic gonads and examination of several markers expressed in the spermatheca, sheath cells, distal tip cells and the uterus, suggest that LIN-26 is required in sheath, spermatheca and uterine precursors, and in uterine cells. We conclude that lin-26 performs a similar function in the non-neuronal ectoderm and the somatic gonad, a mesoderm derivative, and we speculate that lin-26 is required to express epithelial characteristics.