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FUS (Fused in sarcoma) is a ubiquitously expressed DNA/RNA binding protein. Mutations in FUS cause aggressive juvenile forms of amyotrophic lateral sclerosis (ALS), as in the case with the FUSDelta14 mutation. While most studies have focused on the role of FUS in motor neuron degeneration, little is known about the effect ofFUSmutations in the whole body, and the impact ofFUSmutations in the correct development of the nervous system. We studied pleiotropic phenotypes in a physiological knock-in mouse model carrying the FUSDelta14 mutation in homozygosity. RNA sequencing was conducting in six different tissues (frontal cortex, spinal cord, tibialis anterior muscle, white and brown adipose tissue and liver) to identify the genes and pathways altered by the FUSDelta14 mutant protein in the systemic transcriptome. Additionally, brain structural magnetic resonance imaging (MRI) and histological characterisation was conducted in young mice to study the role of FUS mutation in the brain development. FUS mutant protein was upregulated and mislocalised in the cytoplasm in most cells of the tissues analysed. We identified few genes commonly altered in all tissues by this mutation, although most genes and pathways affected were generally tissue-specific. Phenotypic assessment of mice revealed systemic metabolic alterations related to the pathway changes identified. MRI brain scans revealed that homozygous FUSDelta14 brains were smaller and displayed significant morphological alterations including a thinner cortex, reduced neuronal number and increased gliosis, which correlated with early cognitive impairment and fatal seizures. We demonstrated that the disease aetiology of FUS mutations can include neurodevelopmental and systemic alterations, which should be taken into consideration in the clinic.

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common human prion disease, which occurs when the cellular prion protein (PrPC) spontaneously misfolds into disease-associated forms, culminating in fatal neurodegeneration. In a genome-wide association study of sCJD we recently identified risk variants in and around the geneSTX6, with evidence to suggest a causal increase ofSTX6expression in disease-relevant brain regions.STX6encodes syntaxin-6, a SNARE protein primarily involved in early endosome totrans-Golgi network retrograde transport. Here we investigated a causal role ofStx6expression in mouse prion disease through a classical prion transmission study assessing the impact of homozygous and heterozygous syntaxin-6 knockout on disease incubation time and prion-related neuropathology. Homozygous (Stx6-/-) and heterozygous (Stx6+/-) knockout ofStx6expression extended survival by 12 days following inoculation with RML prions relative to wildtype controls. Similarly, inStx6-/-mice, disease incubation time following inoculation with ME7 prions was extended by 12 days. Histopathological analysis revealed a modest increase in astrogliosis in ME7-inoculatedStx6-/-animals and a variable effect ofStx6expression on microglia activation, however no differences in neuronal loss, spongiform change or PrP deposition were observed at endpoint. Importantly,Stx6-/-mice are viable and fertile with no gross impairments on a range of neurological, biochemical, histological and skeletal structure tests. Our results provide confirmatory evidence for a pathological role ofStx6expression in prion disease and support further exploration of syntaxin-6 lowering as a potential therapeutic strategy.Significance StatementSporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is an invariably fatal disease with no disease-modifying treatments. The identification ofSTX6as a proposed risk gene for sCJD motivated the generation of a new mouse knockout model, in which we found no grossly deleterious phenotypes. A transmission study inStx6-/-,Stx6+/-andStx6+/+mice showed a statistically significant prolongation in survival with two prion strains, providing evidence for a causal link ofStx6expression to prion disease pathogenesis. Syntaxin-6 appears to have pleiotropic risk effects across multiple neurodegenerative diseases including progressive supranuclear palsy and Alzheimer’s disease. Thus, this work supports further exploration ofSTX6as a potential therapeutic target across multiple neurodegenerative diseases.

This themed collection of articles was prompted by a collaboration between Neuronal Signaling and the British Neuroscience Association. The Biochemical Society and Portland Press organised a symposium at the BNA Festival of Neuroscience in 2021, focused on the development and use of experimental models of human neuronal disorders. One aspect dealt with how new technologies are being (or could be) used both as a substitute for, or to complement, research that uses whole animal models. Another aspect discussed factors that need to be considered when appraising the validity of animal models of complex, multifactorial neuronal disorders. Given its relevance to the scope of Neuronal Signaling, the journal’s Editorial Board developed a themed collection of content around this symposium entitled Emerging technologies for research models of human neuronal disorders in vivo and in vitro. We were delighted that speakers from the symposium and other experts working in this field agreed to submit reviews for the collection, which offers an invaluable resource both for researchers who are already experts in this field and those who need merely to learn about its scope and potential.

Graphical abstract, Highlights • Large GGGGCC repeat expansions within BAC vectors are highly unstable. • CRISPR-Cas9 screening of BAC vector clones to determine repeat length. • CRISPR-Cas9 cloning of GGGGCC repeat expansion regions into the linear pJazz vector. • pJazz dramatically stabilizes GGGGCC repeat expansions over 4 kb in length., Aberrant microsatellite repeat-expansions at specific loci within the human genome cause several distinct, heritable, and predominantly neurological, disorders. Creating models for these diseases poses a challenge, due to the instability of such repeats in bacterial vectors, especially with large repeat expansions. Designing constructs for more precise genome engineering projects, such as engineering knock-in mice, proves a greater challenge still, since these unstable repeats require numerous cloning steps in order to introduce homology arms or selection cassettes. Here, we report our efforts to clone a large hexanucleotide repeat in the C9orf72 gene, originating from within a BAC construct, derived from a C9orf72-ALS patient. We provide detailed methods for efficient repeat sizing and growth conditions in bacteria to facilitate repeat retention during growth and sub-culturing. We report that sub-cloning into a linear vector dramatically improves stability, but is dependent on the relative orientation of DNA replication through the repeat, consistent with previous studies. We envisage the findings presented here provide a relatively straightforward route to maintaining large-range microsatellite repeat-expansions, for efficient cloning into vectors.

The small EDRK-rich factor 2 (SERF2) is a highly conserved protein that modifies amyloid fibre assembly in vitro and promotes protein misfolding. However, the role of SERF2 in regulating age-related proteotoxicity remains largely unexplored due to a lack of in vivo models. Here, we report the generation of Serf2 knockout mice using an ES cell targeting approach, with Serf2 knockout alleles being bred onto different defined genetic backgrounds. We highlight phenotyping data from heterozygous Serf2+/− mice, including unexpected male-specific phenotypes in startle response and pre-pulse inhibition. We report embryonic lethality in Serf2−/− null animals when bred onto a C57BL/6 N background. However, homozygous null animals were viable on a mixed genetic background and, remarkably, developed without obvious abnormalities. The Serf2 knockout mice provide a powerful tool to further investigate the role of SERF2 protein in previously unexplored pathophysiological pathways in the context of a whole organism.

Mutations in the RNA-binding protein (RBP) FUS have been genetically associated with the motoneuron disease amyotrophic lateral sclerosis (ALS). Using both human induced pluripotent stem cells and mouse models, we found that FUS-ALS causative mutations affect the activity of two relevant RBPs with important roles in neuronal RNA metabolism: HuD/ELAVL4 and FMRP. Mechanistically, mutant FUS leads to upregulation of HuD protein levels through competition with FMRP for HuD mRNA 3’UTR binding. In turn, increased HuD levels overly stabilize the transcript levels of its targets, NRN1 and GAP43. As a consequence, mutant FUS motoneurons show increased axon branching and growth upon injury, which could be rescued by dampening NRN1 levels. Since similar phenotypes have been previously described in SOD1 and TDP-43 mutant models, increased axonal growth and branching might represent broad early events in the pathogenesis of ALS., Maria Giovanna Garone et al. use iPSC and mouse models to evaluate a mechanistic link between aberrant axonal phenotypes in ALS and the alteration of a cross-regulatory circuitry involving three RNA binding proteins: FUS, HuD and FMRP. Their results suggest NRN1 as a potential therapeutic target for ALS and provide further insight into the pathogenesis of this critical disorder.

SUMMARYAmyotrophic lateral sclerosis - frontotemporal dementia spectrum disorder (ALS/FTD) is a complex neurodegenerative disease; up to 10% of cases are familial, usually arising from single dominant mutations in >30 causative genes. Transgenic mouse models that overexpress human ALS/FTD causative genes have been the preferred organism for in vivo modelling. However, while conferring human protein biochemistry, these overexpression models are not ideal for dosage-sensitive proteins such as TDP-43 or FUS.We have created three next-generation genomically humanised knock-in mouse models for ALS/FTD research, by replacing the entire mouse coding region of Sod1, Tardbp (TDP-43) and Fus, with their human orthologues to preserve human protein biochemistry, with exons and introns intact to enable future modelling of coding or non-coding mutations and variants and to preserve human splice variants. In generating these mice, we have established a new-standard of quality control: we demonstrate the utility of indirect capture for enrichment of a region of interest followed by Oxford Nanopore sequencing for robustly characterising large knock-in alleles. This approach confirmed that targeting occurred at the correct locus and to map homologous recombination events. Furthermore, extensive expression data from the three lines shows that homozygous humanised animals only express human protein, at endogenous levels. Characterisation of humanised FUS animals showed that they are phenotypically normal compared to wildtype littermates throughout their lifespan.These humanised mouse strains are critically needed for preclinical assessment of interventions, such as antisense oligonucleotides (ASOs), to modulate expression levels in patients, and will serve as templates for the addition of human ALS/FTD mutations to dissect disease pathomechanisms.

The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor neuron axon terminal and a muscle fibre. NMJs are thought to be the primary site of peripheral pathology in many neuromuscular diseases, but innervation/denervation status is often assessed qualitatively with poor systematic criteria across studies, and separately from 3D morphological structure. Here, we describe the development of ‘NMJ-Analyser’, to comprehensively screen the morphology of NMJs and their corresponding innervation status automatically. NMJ-Analyser generates 29 biologically relevant features to quantitatively define healthy and aberrant neuromuscular synapses and applies machine learning to diagnose NMJ degeneration. We validated this framework in longitudinal analyses of wildtype mice, as well as in four different neuromuscular disease models: three for amyotrophic lateral sclerosis (ALS) and one for peripheral neuropathy. We showed that structural changes at the NMJ initially occur in the nerve terminal of mutant TDP43 and FUS ALS models. Using a machine learning algorithm, healthy and aberrant neuromuscular synapses are identified with 95% accuracy, with 88% sensitivity and 97% specificity. Our results validate NMJ-Analyser as a robust platform for systematic and structural screening of NMJs, and pave the way for transferrable, and cross-comparison and high-throughput studies in neuromuscular diseases.

Neurodegenerative disease encompasses a wide range of disorders afflicting the central and peripheral nervous systems and is a major unmet biomedical need of our time. There are very limited treatments, and no cures, for most of these diseases, including Alzheimer’s Disease, Parkinson's Disease, Huntington Disease, and Motor Neuron Diseases. Mouse and other animal models provide hope by analysing them to understand pathogenic mechanisms, to identify drug targets, and to develop gene therapies and stem cell therapies. However, despite many decades of research, virtually no new treatments have reached the clinic. Increasingly, it is apparent that human heterogeneity within clinically defined neurodegenerative disorders, and between patients with the same genetic mutations, significantly impacts disease presentation and, potentially, therapeutic efficacy. Therefore, stratifying patients according to genetics, lifestyle, disease presentation, ethnicity, and other parameters may hold the key to bringing effective therapies from the bench to the clinic. Here, we discuss genetic and cellular humanised mouse models, and how they help in defining the genetic and environmental parameters associated with neurodegenerative disease, and so help in developing effective precision medicine strategies for future healthcare.

Metabolic reprogramming is an active regulator of stem cell fate choices, and successful stem cell differentiation in different compartments requires the induction of oxidative phosphorylation. However, the mechanisms that promote mitochondrial respiration during stem cell differentiation are poorly understood. Here we demonstrate that Stat3 promotes muscle stem cell myogenic lineage progression by stimulating mitochondrial respiration in mice. We identify Fam3a, a cytokine-like protein, as a major Stat3 downstream effector in muscle stem cells. We demonstrate that Fam3a is required for muscle stem cell commitment and skeletal muscle development. We show that myogenic cells secrete Fam3a, and exposure of Stat3-ablated muscle stem cells to recombinant Fam3a in vitro and in vivo rescues their defects in mitochondrial respiration and myogenic commitment. Together, these findings indicate that Fam3a is a Stat3-regulated secreted factor that promotes muscle stem cell oxidative metabolism and differentiation, and suggests that Fam3a is a potential tool to modulate cell fate choices., Induction of mitochondrial oxidative respiration is required for stem cell differentiation, but the mechanisms underlying this process are poorly understood. Here, the authors report that Stat3 promotes muscle stem cell differentiation by stimulating mitochondrial respiration via Fam3a.

To better understand human health and disease, researchers create a wide variety of mouse models that carry human DNA. With recent advances in genome engineering, the targeted replacement of mouse genomic regions with orthologous human sequences has become increasingly viable, ranging from finely tuned humanisation of individual nucleotides and amino acids to the incorporation of many megabases of human DNA. Here, we examine emerging technologies for targeted genomic humanisation, we review the spectrum of existing genomically humanised mouse models and the insights such models have provided, and consider the lessons learned for designing such models in the future., Generation of transgenic mice has become routine in studying gene function and disease mechanisms, but often this is not enough to fully understand human biology. Here, the authors review the current state of the art of targeted genomic humanisation strategies and their advantages over classic approaches.

The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor neuron axon terminal and a muscle fibre. NMJs are thought to be the primary site of peripheral pathology in many neuromuscular diseases, but innervation/denervation status is often assessed qualitatively with poor systematic criteria across studies, and separately from 3D morphological structure. Here, we describe the development of ‘NMJ-Analyser’, to comprehensively screen the morphology of NMJs and their corresponding innervation status automatically. NMJ-Analyser generates 29 biologically relevant features to quantitatively define healthy and aberrant neuromuscular synapses and applies machine learning to diagnose NMJ degeneration. We validated this framework in longitudinal analyses of wildtype mice, as well as in four different neuromuscular disease models: three for amyotrophic lateral sclerosis (ALS) and one for peripheral neuropathy. We showed that structural changes at the NMJ initially occur in the nerve terminal of mutant TDP43 and FUS ALS models. Using a machine learning algorithm, healthy and aberrant neuromuscular synapses are identified with 95% accuracy, with 88% sensitivity and 97% specificity. Our results validate NMJ-Analyser as a robust platform for systematic and structural screening of NMJs, and pave the way for transferrable, and cross-comparison and high-throughput studies in neuromuscular diseases.

Mutations in the RNA-binding protein (RBPs) FUS have been genetically associated with the motoneuron disease amyotrophic lateral sclerosis (ALS). Using both human induced pluripotent stem cells and mouse models, we found that FUS-ALS causative mutations affect the activity of two relevant RBPs with important roles in neuronal RNA metabolism: HuD/ELAVL4 and FMRP. Mechanistically, mutant FUS leads to upregulation of HuD protein levels through competition with FMRP for HuD mRNA 3’UTR binding. In turn, increased HuD levels overly stabilize the transcript levels of its targets, NRN1 and GAP43. As a consequence, mutant FUS motoneurons show increased axon branching and growth upon injury, which could be rescued by dampening NRN1 levels. Since similar phenotypes have been previously described in SOD1 and TDP-43 mutant models, increased axonal growth and branching might represent broad early events in the pathogenesis of ALS.

Gene therapy in a mouse model for amyotrophic lateral sclerosis (ALS) illustrates the rapid deployment of base editing in therapeutic modeling of neurodegenerative disease.

SUMMARY A standard approach in the identification of transcriptional enhancers is the use of transgenic animals carrying DNA elements joined to reporter genes inserted randomly in the genome. We examined elements near Tbx5, a gene required for forelimb development in humans and other vertebrates. Previous transgenic studies reported a mammalian Tbx5 fore-limb enhancer located in intron 2 containing a putative retinoic acid response element and a zebrafish tbx5a forelimb (pectoral fin) enhancer located downstream that is conserved from fish to mammals. We used CRISPR/Cas9 gene editing to knockout the endogenous elements and unexpectedly found that deletion of the intron 2 and downstream elements, either singly or together in double knockouts, resulted in no effect on forelimb development. Our findings show that reporter transgenes may not identify endogenous enhancers and that in vivo genetic loss-of-function studies are required, such as CRISPR/Cas9, which is similar in effort to production of animals carrying reporter transgenes., In Brief Forelimb development requires Tbx5. Using CRISPR/Cas9 gene editing to create homozygous deletions, Cunningham et al. show that two Tbx5 forelimb enhancers identified with reporter transgenes are not required for Tbx5 activation or forelimb development. These observations demonstrate that knockout studies are required to identify endogenous enhancers necessary for biological processes.

Bipotent neuromesodermal progenitors (NMPs) residing in the caudal epiblast drive coordinated body axis extension by generating both posterior neuroectoderm and presomitic mesoderm. Retinoic acid (RA) is required for body axis extension, however the early molecular response to RA signaling is poorly defined, as is its relationship to NMP biology. As endogenous RA is first seen near the time when NMPs appear, we used WNT/FGF agonists to differentiate embryonic stem cells to NMPs which were then treated with a short 2-h pulse of 25 nM RA or 1 µM RA followed by RNA-seq transcriptome analysis. Differential expression analysis of this dataset indicated that treatment with 25 nM RA, but not 1 µM RA, provided physiologically relevant findings. The 25 nM RA dataset yielded a cohort of previously known caudal RA target genes including Fgf8 (repressed) and Sox2 (activated), plus novel early RA signaling targets with nearby conserved RA response elements. Importantly, validation of top-ranked genes in vivo using RA-deficient Raldh2−/− embryos identified novel examples of RA activation (Nkx1-2, Zfp503, Zfp703, Gbx2, Fgf15, Nt5e) or RA repression (Id1) of genes expressed in the NMP niche or progeny. These findings provide evidence for early instructive and permissive roles of RA in controlling differentiation of NMPs to neural and mesodermal lineages., Summary: The findings here demonstrate that the signaling molecule retinoic acid (RA) plays an early role in determining how embryonic progenitor cells decide to differentiate into either mesodermal or neural tissues.

Retinoic acid (RA) repression of Fgf8 is required for many different aspects of organogenesis, however relatively little is known about how endogenous RA controls gene repression as opposed to gene activation. Here, we show that nuclear receptor corepressors NCOR1 and NCOR2 (SMRT) redundantly mediate the ability of RA to repress Fgf8. Ncor1;Ncor2 double mutants generated by CRISPR/Cas9 gene editing exhibited a small somite and distended heart phenotype similar to that of RA-deficient Raldh2−/− embryos, associated with increased Fgf8 expression and FGF signaling in caudal progenitors and heart progenitors. Embryo chromatin immunoprecipitation studies revealed that NCOR1/2 but not coactivators are recruited to the Fgf8 RA response element (RARE) in an RA-dependent manner, whereas coactivators but not NCOR1/2 are recruited RA-dependently to a RARE near Rarb that is activated by RA. CRISPR/Cas9-mediated genomic deletion of the Fgf8 RARE in mouse embryos often resulted in a small somite defect with Fgf8 derepression caudally, but no defect was observed in heart development or heart Fgf8 expression. This suggests the existence of another DNA element whose function overlaps with the Fgf8 RARE to mediate Fgf8 repression by RA and NCOR1/2. Our studies support a model in which NCOR1/2 mediates direct RA-dependent repression of Fgf8 in caudal progenitors in order to control somitogenesis.

In mouse, retinoic acid (RA) is required for the early phase of body axis extension controlled by a population of neuromesodermal progenitors (NMPs) in the trunk called expanding-NMPs, but not for the later phase of body axis extension controlled by a population of NMPs in the tail called depleting-NMPs. Recent observations suggest that zebrafish utilize depleting-NMPs but not expanding-NMPs for body axis extension. In zebrafish, a role for RA in body axis extension was not supported by previous studies on aldh1a2 (raldh2) mutants lacking RA synthesis. Here, by treating zebrafish embryos with an RA synthesis inhibitor, we also found that body axis extension and somitogenesis was not perturbed, although loss of pectoral fin and cardiac edema were observed consistent with previous studies. The conclusion that zebrafish diverges from mouse in not requiring RA for body axis extension is consistent with zebrafish lacking early expanding-NMPs to generate the trunk. We suggest that RA control of body axis extension was added to higher vertebrates during evolution of expanding-NMPs.

Background: Vertebrate body axis extension occurs in a head-to-tail direction from a caudal progenitor zone that responds to interacting signals. Wnt/β-catenin signaling is critical for generation of paraxial mesoderm, somite formation, and maintenance of the axial stem cell pool. Body axis extension requires Wnt8a in lower vertebrates, but in mammals Wnt3a is required, although the anterior trunk develops in the absence of Wnt3a. Results: We examined mouse Wnt8a–/– and Wnt3a–/– single and double mutants to explore whether mammalian Wnt8a contributes to body axis extension and to determine whether a posterior growth function for Wnt8a is conserved throughout the vertebrate lineage. We find that caudal Wnt8a is expressed only during early somite stages and is required for normal development of the anterior trunk in the absence of Wnt3a. During this time, we show that Wnt8a and Wnt3a cooperate to maintain Fgf8 expression and prevent premature Sox2 up-regulation in the axial stem cell niche, critical for posterior growth. Similar to Fgf8, Wnt8a requires retinoic acid (RA) signaling to restrict its caudal expression boundary and possesses an upstream RA response element that binds RA receptors. Conclusions: These findings provide new insight into interaction of caudal Wnt-FGF-RA signals required for body axis extension. Developmental Dynamics 244:797–807, 2015. © 2015 Wiley Periodicals, Inc.

International audience; Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix–loop–helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation—Tcf3 and Foxa2—and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1–4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

Here, we generated a genome-scale shRNA library targeting long intergenic noncoding RNAs (lincRNAs) in the mouse. We performed an unbiased loss-of-function study in mouse embryonic stem cells (mESCs) and identified 20 lincRNAs involved in the maintenance of pluripotency. Among these, TUNA (Tcl1 Upstream Neuron-Associated lincRNA, or megamind) was required for pluripotency and formed a complex with three RNA-binding proteins (RBPs). The TUNA-RBP complex was detected at the promoters of Nanog, Sox2, and Fgf4, and knockdown of TUNA or the individual RBPs inhibited neural differentiation of mESCs. TUNA showed striking evolutionary conservation of both sequence- and CNS-restricted expression in vertebrates. Accordingly, knockdown of tuna in zebrafish caused impaired locomotor function, and TUNA expression in the brains of Huntington's disease patients was significantly associated with disease grade. Our results suggest that the lincRNA TUNA plays a vital role in pluripotency and neural differentiation of ESCs and is associated with neurological function of adult vertebrates.

Background: Retinoic acid (RA) signaling controls patterning and neuronal differentiation within the hindbrain, but forebrain RA function remains controversial. RA is produced from metabolism of retinol to retinaldehyde by retinol dehydrogenase (RDH), followed by metabolism of retinaldehyde to RA by retinaldehyde dehydrogenase (RALDH). Previous studies on Raldh2−/− and Raldh3−/− mice demonstrated an RA requirement for γ-aminobutyric acid (GABA)ergic and dopaminergic differentiation in forebrain basal ganglia, but no RA requirement was observed during early forebrain patterning or subsequent forebrain cortical expansion. However, other studies suggested that RA controls forebrain patterning, and analysis of ethylnitrosourea-induced Rdh10 mutants suggested that RA synthesized in the meninges stimulates forebrain cortical expansion. Results: We generated Rdh10−/− mouse embryos that lack RA activity early in the head and later in the meninges. We observed defects in hindbrain patterning and eye RA signaling, but early forebrain patterning was unaffected. Retinaldehyde treatment of Rdh10−/− embryos from E7–E9 rescues a cranial skeletal defect, resulting in E14.5 embryos lacking meningeal RA activity but maintaining normal forebrain shape and cortical expansion. Conclusions: Rdh10−/− embryos demonstrate that RA controls hindbrain but not early forebrain patterning, while studies on retinaldehyde-rescued Rdh10−/− embryos show that meningeal RA synthesis is unnecessary to stimulate forebrain cortical expansion. Developmental Dynamics 242:1056–1065, 2013. © 2013 Wiley Periodicals, Inc.

We have demonstrated a direct detection of 100-5000 eV electrons with a back-illuminated boron delta-doped hybrid silicon complementary metal-oxide-semiconductor imager operating in full depletion and a silicon electron-multiplying charge-coupled device (CCD) operating in partial depletion. The delta-doping molecular beam epitaxy increases sensitivity to low-energy electrons and improves low-energy electron detection threshold relative to conventional solid-state detectors. We compare the gain measured in these two delta-doped devices with gain measured from control delta-doped CCDs.

Retinoic acid (RA) signalling has a central role during vertebrate development. RA synthesized in specific locations regulates transcription by interacting with nuclear RA receptors (RARs) bound to RA response elements (RAREs) near target genes. RA was first implicated in signalling on the basis of its teratogenic effects on limb development. Genetic studies later revealed that endogenous RA promotes forelimb initiation by repressing fibroblast growth factor 8 (Fgf8). Insights into RA function in the limb serve as a paradigm for understanding how RA regulates other developmental processes. In vivo studies have identified RAREs that control repression of Fgf8 during body axis extension or activation of homeobox (Hox) genes and other key regulators during neuronal differentiation and organogenesis.

Bipotent axial stem cells residing in the caudal epiblast during late gastrulation generate neuroectodermal and presomitic mesodermal progeny that coordinate somitogenesis with neural tube formation, but the mechanism that controls these two fates is not fully understood. Retinoic acid (RA) restricts the anterior extent of caudal fibroblast growth factor 8 (Fgf8) expression in both mesoderm and neural plate to control somitogenesis and neurogenesis, however it remains unclear where RA acts to control the spatial expression of caudal Fgf8. Here, we found that mouse Raldh2-/- embryos, lacking RA synthesis and displaying a consistent small somite defect, exhibited abnormal expression of key markers of axial stem cell progeny, with decreased Sox2+ and Sox1+ neuroectodermal progeny and increased Tbx6+ presomitic mesodermal progeny. The Raldh2-/- small somite defect was rescued by treatment with an FGF receptor antagonist. Rdh10 mutants, with a less severe RA synthesis defect, were found to exhibit a small somite defect and anterior expansion of caudal Fgf8 expression only for somites 1-6, with normal somite size and Fgf8 expression thereafter. Rdh10 mutants were found to lack RA activity during the early phase when somites are small, but at the 6-somite stage RA activity was detected in neural plate although not in presomitic mesoderm. Expression of a dominant-negative RA receptor in mesoderm eliminated RA activity in presomitic mesoderm but did not affect somitogenesis. Thus, RA activity in the neural plate is sufficient to prevent anterior expansion of caudal Fgf8 expression associated with a small somite defect. Our studies provide evidence that RA restriction of Fgf8 expression in undifferentiated neural progenitors stimulates neurogenesis while also restricting the anterior extent of the mesodermal Fgf8 mRNA gradient that controls somite size, providing new insight into the mechanism that coordinates somitogenesis with neurogenesis.

Vertebrate body axis extension occurs in a head-to-tail direction from a caudal progenitor zone that responds to interacting signals. Wnt/β-catenin signaling is critical for generation of paraxial mesoderm, somite formation, and maintenance of the axial stem cell pool. Body axis extension requires Wnt8a in lower vertebrates, but in mammals Wnt3a is required, although the anterior trunk develops in the absence of Wnt3a.We examined mouse Wnt8a(-/-) and Wnt3a(-/-) single and double mutants to explore whether mammalian Wnt8a contributes to body axis extension and to determine whether a posterior growth function for Wnt8a is conserved throughout the vertebrate lineage. We find that caudal Wnt8a is expressed only during early somite stages and is required for normal development of the anterior trunk in the absence of Wnt3a. During this time, we show that Wnt8a and Wnt3a cooperate to maintain Fgf8 expression and prevent premature Sox2 up-regulation in the axial stem cell niche, critical for posterior growth. Similar to Fgf8, Wnt8a requires retinoic acid (RA) signaling to restrict its caudal expression boundary and possesses an upstream RA response element that binds RA receptors.These findings provide new insight into interaction of caudal Wnt-FGF-RA signals required for body axis extension.

A new front-end for photodiode-based CMOS imagers is presented. Degradation in imaging performance due to conventional hard- and soft-reset of pixels is analyzed. To overcome these limitations, the design and operation of a flushed-reset pixel is described. The flushed-reset pixel combines the best of hard- and soft-reset to simultaneously provide excellent radiometric accuracy, high linearity, no image lag, high saturation level, and reduced read-noise. The new front-end is implemented by changes to the column-circuitry only, leaving the pixel unchanged, preventing degradation of any unrelated imaging performance. It is compatible with large format imager implementation, has minimal impact on the frame-rate, and does not introduce any additional hot-carrier stress in the pixel. Data from a large format (512/sup 2/) imager demonstrates the efficacy of the flushed-reset pixel approach.

Background Laparoscopy is the most common procedure for minimally invasive intraabdominal surveys. Patients with pulmonary hypertension (PHTN) may need an alternate approach because the systemic absorption of carbon dioxide may lead to hypercapnia, acidemia, and increases in systemic and pulmonary pressures. We report a case of intraabdominal survey using a microcatheter trocar (Check-Flo Introducer) in a patient with a large adnexal mass and PHTN. Case An adolescent girl with severe PHTN and multiple cardiac anomalies presented with oligomenorrhea. During the course of workup, an ultrasound revealed an 8-cm simple cyst in the right adnexa, which required removal. The decision was made to proceed with cystectomy. The patient was not a candidate for laparoscopy, and an alternative method was used. The Check-Flo Introducer, a microcatheter trocar; crystalloid fluid infusion; and a 3-mm laparoscopic camera were used to survey the abdominal cavity, revealing a 10-cm simple paratubal cyst that could be safely drained and removed via a minilaparotomy incision. Conclusion The microcatheter trocar is a useful and novel alternative in patients who cannot tolerate increases in intraabdominal pressure.

Active pixel sensors (APS) are micro-fabricated CMOS amplifier arrays that are rapidly replacing CCD devices in many electronic imaging applications. Unlike the pixels of a CCD device, the sensing elements of the APS will respond to locally situated electrostatic charge, owing to the amplifier present in each pixel. We have built two small test arrays with microscopic aluminum electrodes integrated onto standard APS readout circuitry for the purpose of detecting low-energy gas-phase ions in mass spectrometers and other analytical instruments. The devices exhibit a near-linear dynamic range greater than four orders of magnitude, and a noise level of less than 100 electrons at room temperature. Data are presented for the response of the APS detectors to small ions in a miniature magnetic sector mass spectrometer and in an atmospheric pressure jet of helium. Data for individual highly-charged electrospray droplets are presented as well. Anticipated improvements suggest that in the near future APS ion detectors will posses noise levels approaching 10 electrons and will have a useful dynamic range over six orders of magnitude.

Although many regulatory networks involved in defining definitive endoderm have been identified, the mechanisms through which these networks interact to pattern the endoderm are less well understood. To explore the mechanisms involved in midgut patterning, we dissected the transcriptional regulatory elements of nephrocan (Nepn), the earliest known midgut specific gene in mice. We observed that Nepn expression is dramatically reduced in Sox17−/− and Raldh2−/− embryos compared with wild-type embryos. We further show that Nepn is directly regulated by Sox17 and the retinoic acid (RA) receptor via two enhancer elements located upstream of the gene. Moreover, Nepn expression is modulated by Activin signaling, with high levels inhibiting and low levels enhancing RA-dependent expression. In Foxh1−/− embryos in which Nodal signaling is reduced, the Nepn expression domain is expanded into the anterior gut region, confirming that Nodal signaling can modulate its expression in vivo. Together, Sox17 is required for Nepn expression in the definitive endoderm, while RA signaling restricts expression to the midgut region. A balance of Nodal/Activin signaling regulates the anterior boundary of the midgut expression domain.