Publications by authors named "Benjamin Feldman"

29 Publications

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The X-linked acrogigantism-associated gene gpr101 is a regulator of early embryonic development and growth in zebrafish.

Mol Cell Endocrinol 2021 01 26;520:111091. Epub 2020 Nov 26.

Section on Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD, USA.

We recently described X-linked acrogigantism (X-LAG), a condition of early childhood-onset pituitary gigantism associated with microduplications of the GPR101 receptor. The expression of GPR101 in hyperplastic pituitary regions and tumors in X-LAG patients, and GPR101's normally transient pituitary expression during fetal development, suggest a role in the regulation of growth. Nevertheless, little is still known about GPR101's physiological functions, especially during development. By using zebrafish models, we investigated the role of gpr101 during embryonic development and somatic growth. Transient ectopic gpr101 expression perturbed the embryonic body plan but did not affect growth. Loss of gpr101 led to a significant reduction in body size that was even more pronounced in the absence of maternal transcripts, as well as subfertility. These changes were accompanied by gastrulation and hypothalamic defects. In conclusion, both gpr101 loss- and gain-of-function affect, in different ways, fertility, embryonic patterning, growth and brain development.
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http://dx.doi.org/10.1016/j.mce.2020.111091DOI Listing
January 2021

Squeezing strong correlations from graphene.

Science 2019 03;363(6431):1035-1036

Department of Physics and Geballe Laboratory for Advanced Materials, Stanford University, Stanford, CA, USA.

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http://dx.doi.org/10.1126/science.aaw4642DOI Listing
March 2019

Accessing analytes in biofluids for peripheral biochemical monitoring.

Nat Biotechnol 2019 04 25;37(4):407-419. Epub 2019 Feb 25.

Eccrine Systems Inc, Cincinnati, Ohio, USA.

Peripheral biochemical monitoring involves the use of wearable devices for minimally invasive or noninvasive measurement of analytes in biofluids such as interstitial fluid, saliva, tears and sweat. The goal in most cases is to obtain measurements that serve as surrogates for circulating analyte concentrations in blood. Key technological developments to date include continuous glucose monitors, which use an indwelling sensor needle to measure glucose in interstitial fluid, and device-integrated sweat stimulation for continuous access to analytes in sweat. Further development of continuous sensing technologies through new electrochemical sensing modalities will be a major focus of future research. While there has been much investment in wearable technologies to sense analytes, less effort has been directed to understanding the physiology of biofluid secretion. Elucidating the underlying biology is crucial for accelerating technological progress, as the biofluid itself often presents the greatest challenge in terms of sample volumes, secretion rates, filtration, active analyte channels, variable pH and salinity, analyte breakdown and other confounding factors.
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http://dx.doi.org/10.1038/s41587-019-0040-3DOI Listing
April 2019

Interacting multi-channel topological boundary modes in a quantum Hall valley system.

Nature 2019 02 6;566(7744):363-367. Epub 2019 Feb 6.

Joseph Henry Laboratories and Department of Physics, Princeton University, Princeton, NJ, USA.

Symmetry and topology are central to understanding quantum Hall ferromagnets (QHFMs), two-dimensional electronic phases with spontaneously broken spin or pseudospin symmetry whose wavefunctions also have topological properties. Domain walls between distinct broken-symmetry QHFM phases are predicted to host gapless one-dimensional modes-that is, quantum channels that emerge because of a topological change in the underlying electronic wavefunctions at such interfaces. Although various QHFMs have been identified in different materials, interacting electronic modes at these domain walls have not been probed. Here we use a scanning tunnelling microscope to directly visualize the spontaneous formation of boundary modes at domain walls between QHFM phases with different valley polarization (that is, the occupation of equal-energy but quantum mechanically distinct valleys in the electronic structure) on the surface of bismuth. Spectroscopy shows that these modes occur within a topological energy gap, which closes and reopens as the valley polarization switches across the domain wall. By changing the valley flavour and the number of modes at the domain wall, we can realize different regimes in which the valley-polarized channels are either metallic or develop a spectroscopic gap. This behaviour is a consequence of Coulomb interactions constrained by the valley flavour, which determines whether electrons in the topological modes can backscatter, making these channels a unique class of interacting one-dimensional quantum wires. QHFM domain walls can be realized in different classes of two-dimensional materials, providing the opportunity to explore a rich phase space of interactions in these quantum wires.
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http://dx.doi.org/10.1038/s41586-019-0913-0DOI Listing
February 2019

Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for screening of candidate therapeutic compounds.

Dis Model Mech 2018 08 15;11(9). Epub 2018 Aug 15.

Section on Molecular Dysmorphology, Division of Translational Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA

Niemann-Pick disease type C1 (NPC1) is a rare autosomal recessive lysosomal storage disease primarily caused by mutations in NPC1 is characterized by abnormal accumulation of unesterified cholesterol and glycolipids in late endosomes and lysosomes. Common signs include neonatal jaundice, hepatosplenomegaly, cerebellar ataxia, seizures and cognitive decline. Both mouse and feline models of NPC1 mimic the disease progression in humans and have been used in preclinical studies of 2-hydroxypropyl-β-cyclodextrin (2HPβCD; VTS-270), a drug that appeared to slow neurological progression in a Phase 1/2 clinical trial. However, there remains a need to identify additional therapeutic agents. High-throughput drug screens have been useful in identifying potential therapeutic compounds; however, current preclinical testing is time and labor intensive. Thus, development of a high-capacity platform suitable for screening candidate drugs/compounds would be valuable for compound optimization and prioritizing subsequent testing. Here, we generated and characterize two zebrafish -null mutants using CRISPR/Cas9-mediated gene targeting. The mutants model both the early liver and later neurological disease phenotypes of NPC1. LysoTracker staining of mutant larvae was notable for intense staining of lateral line neuromasts, thus providing a robust screen for lysosomal storage. As a proof of principle, we were able to show that treatment of the mutant larvae with 2HPβCD significantly reduced neuromast LysoTracker staining. These data demonstrate the potential value of using this zebrafish NPC1 model for efficient and rapid optimization and screening of potential therapeutic compounds.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.034165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176986PMC
August 2018

Visualizing heavy fermion confinement and Pauli-limited superconductivity in layered CeCoIn.

Nat Commun 2018 02 7;9(1):549. Epub 2018 Feb 7.

Joseph Henry Laboratories of Physics, Department of Physics, Princeton University, Princeton, NJ, 08544, USA.

Layered material structures play a key role in enhancing electron-electron interactions to create correlated metallic phases that can transform into unconventional superconducting states. The quasi-two-dimensional electronic properties of such compounds are often inferred indirectly through examination of bulk properties. Here we use scanning tunneling microscopy to directly probe in cross-section the quasi-two-dimensional electronic states of the heavy fermion superconductor CeCoIn. Our measurements reveal the strong confined nature of quasiparticles, anisotropy of tunneling characteristics, and layer-by-layer modulated behavior of the precursor pseudogap gap phase. In the interlayer coupled superconducting state, the orientation of line defects relative to the d-wave order parameter determines whether in-gap states form due to scattering. Spectroscopic imaging of the anisotropic magnetic vortex cores directly characterizes the short interlayer superconducting coherence length and shows an electronic phase separation near the upper critical in-plane magnetic field, consistent with a Pauli-limited first-order phase transition into a pseudogap phase.
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http://dx.doi.org/10.1038/s41467-018-02841-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803268PMC
February 2018

Observation of a nematic quantum Hall liquid on the surface of bismuth.

Science 2016 10;354(6310):316-321

Joseph Henry Laboratories and Department of Physics, Princeton University, Princeton, NJ 08544, USA.

Nematic quantum fluids with wave functions that break the underlying crystalline symmetry can form in interacting electronic systems. We examined the quantum Hall states that arise in high magnetic fields from anisotropic hole pockets on the Bi(111) surface. Spectroscopy performed with a scanning tunneling microscope showed that a combination of single-particle effects and many-body Coulomb interactions lift the six-fold Landau level (LL) degeneracy to form three valley-polarized quantum Hall states. We imaged the resulting anisotropic LL wave functions and found that they have a different orientation for each broken-symmetry state. The wave functions correspond to those expected from pairs of hole valleys and provide a direct spatial signature of a nematic electronic phase.
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http://dx.doi.org/10.1126/science.aag1715DOI Listing
October 2016

Characterization of GPR101 transcript structure and expression patterns.

J Mol Endocrinol 2016 08 9;57(2):97-111. Epub 2016 Jun 9.

Section on Endocrinology and GeneticsEunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, Maryland, USA

We recently showed that Xq26.3 microduplications cause X-linked acrogigantism (X-LAG). X-LAG patients mainly present with growth hormone and prolactin-secreting adenomas and share a minimal duplicated region containing at least four genes. GPR101 was the only gene highly expressed in their pituitary lesions, but little is known about its expression patterns. In this work, GPR101 transcripts were characterized in human tissues by 5'-Rapid Amplification of cDNA Ends (RACE) and RNAseq, while the putative promoter was bioinformatically predicted. We investigated GPR101 mRNA and protein expression by RT-quantitative PCR (qPCR), whole-mount in situ hybridization, and immunostaining, in human, rhesus monkey, rat and zebrafish. We identified four GPR101 isoforms characterized by different 5'-untranslated regions (UTRs) and a common 6.1kb long 3'UTR. GPR101 expression was very low or absent in almost all adult human tissues examined, except for specific brain regions. Strong GPR101 staining was observed in human fetal pituitary and during adolescence, whereas very weak/absent expression was detected during childhood and adult life. In contrast to humans, adult monkey and rat pituitaries expressed GPR101, but in different cell types. Gpr101 is expressed in the brain and pituitary during rat and zebrafish development; in rat pituitary, Gpr101 is expressed only after birth and shows sexual dimorphism. This study shows that different GPR101 transcripts exist and that the brain is the major site of GPR101 expression across different species, although divergent species- and temporal-specific expression patterns are evident. These findings suggest an important role for GPR101 in brain and pituitary development and likely reflect the very different growth, development and maturation patterns among species.
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http://dx.doi.org/10.1530/JME-16-0045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959428PMC
August 2016

Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

Nucleic Acids Res 2015 Apr 27;43(7):e48. Epub 2015 Jan 27.

Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA

Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.
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http://dx.doi.org/10.1093/nar/gkv035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4402511PMC
April 2015

Landau quantization and quasiparticle interference in the three-dimensional Dirac semimetal Cd₃As₂.

Nat Mater 2014 Sep 29;13(9):851-6. Epub 2014 Jun 29.

Joseph Henry Laboratories and Department of Physics, Princeton University, Princeton, New Jersey 08544, USA.

Condensed-matter systems provide a rich setting to realize Dirac and Majorana fermionic excitations as well as the possibility to manipulate them for potential applications. It has recently been proposed that chiral, massless particles known as Weyl fermions can emerge in certain bulk materials or in topological insulator multilayers and give rise to unusual transport properties, such as charge pumping driven by a chiral anomaly. A pair of Weyl fermions protected by crystalline symmetry effectively forming a massless Dirac fermion has been predicted to appear as low-energy excitations in a number of materials termed three-dimensional Dirac semimetals. Here we report scanning tunnelling microscopy measurements at sub-kelvin temperatures and high magnetic fields on the II-V semiconductor Cd3As2. We probe this system down to atomic length scales, and show that defects mostly influence the valence band, consistent with the observation of ultrahigh-mobility carriers in the conduction band. By combining Landau level spectroscopy and quasiparticle interference, we distinguish a large spin-splitting of the conduction band in a magnetic field and its extended Dirac-like dispersion above the expected regime. A model band structure consistent with our experimental findings suggests that for a magnetic field applied along the axis of the Dirac points, Weyl fermions are the low-energy excitations in Cd3As2.
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http://dx.doi.org/10.1038/nmat4023DOI Listing
September 2014

Fractional quantum Hall phase transitions and four-flux states in graphene.

Phys Rev Lett 2013 Aug 16;111(7):076802. Epub 2013 Aug 16.

Department of Physics, Harvard University, Cambridge, Massachusetts 02138, USA.

Graphene and its multilayers have attracted considerable interest because their fourfold spin and valley degeneracy enables a rich variety of broken-symmetry states arising from electron-electron interactions, and raises the prospect of controlled phase transitions among them. Here we report local electronic compressibility measurements of ultraclean suspended graphene that reveal a multitude of fractional quantum Hall states surrounding filling factors ν=-1/2 and -1/4. Several of these states exhibit phase transitions that indicate abrupt changes in the underlying order, and we observe many additional oscillations in compressibility as ν approaches -1/2, suggesting further changes in spin and/or valley polarization. We use a simple model based on crossing Landau levels of composite fermions with different internal degrees of freedom to explain many qualitative features of the experimental data. Our results add to the diverse array of many-body states observed in graphene and demonstrate substantial control over their order parameters.
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http://dx.doi.org/10.1103/PhysRevLett.111.076802DOI Listing
August 2013

Unconventional sequence of fractional quantum Hall states in suspended graphene.

Science 2012 Sep;337(6099):1196-9

Department of Physics, Harvard University, Cambridge, MA 02138, USA.

Graphene provides a rich platform to study many-body effects, owing to its massless chiral charge carriers and the fourfold degeneracy arising from their spin and valley degrees of freedom. We use a scanning single-electron transistor to measure the local electronic compressibility of suspended graphene, and we observed an unusual pattern of incompressible fractional quantum Hall states that follows the standard composite fermion sequence between filling factors ν = 0 and 1 but involves only even-numerator fractions between ν = 1 and 2. We further investigated this surprising hierarchy by extracting the corresponding energy gaps as a function of the magnetic field. The sequence and relative strengths of the fractional quantum Hall states provide insight into the interplay between electronic correlations and the inherent symmetries of graphene.
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http://dx.doi.org/10.1126/science.1224784DOI Listing
September 2012

Unique alterations of an ultraconserved non-coding element in the 3'UTR of ZIC2 in holoprosencephaly.

PLoS One 2012 31;7(7):e39026. Epub 2012 Jul 31.

Medical Genetics Branch, National Human Genome Research Institute (NHGRI), National Institutes of Health, Bethesda, Maryland, United States of America.

Coding region alterations of ZIC2 are the second most common type of mutation in holoprosencephaly (HPE). Here we use several complementary bioinformatic approaches to identify ultraconserved cis-regulatory sequences potentially driving the expression of human ZIC2. We demonstrate that an 804 bp element in the 3' untranslated region (3'UTR) is highly conserved across the evolutionary history of vertebrates from fish to humans. Furthermore, we show that while genetic variation of this element is unexpectedly common among holoprosencephaly subjects (6/528 or >1%), it is not present in control individuals. Two of six proband-unique variants are de novo, supporting their pathogenic involvement in HPE outcomes. These findings support a general recommendation that the identification and analysis of key ultraconserved elements should be incorporated into the genetic risk assessment of holoprosencephaly cases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039026PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409191PMC
April 2013

Social responsiveness and competence in Prader-Willi syndrome: direct comparison to autism spectrum disorder.

J Autism Dev Disord 2013 Jan;43(1):103-13

Department of Psychological Sciences, Case Western Reserve University, 11220 Bellflower Road, Cleveland, OH 44106-7123, USA.

Prader-Willi syndrome (PWS), a neurodevelopmental disorder primarily characterized by hyperphagia and food preoccupations, is caused by the absence of expression of the paternally active genes in the proximal arm of chromosome 15. Although maladaptive behavior and the cognitive profile in PWS have been well characterized, social functioning has only more recently been systematically examined. Findings to date indicate the social impairment exhibited may reflect specific difficulty interpreting and using social information effectively. In addition, evidence suggests that there is an increased risk of social deficits in people with the maternally-derived uniparental disomy (mUPD) subtype of PWS in comparison to those with 15q11-13 paternal deletion (DEL). Using the Social Responsiveness Scale (SRS) and the Social Competence Inventory, our goal was to compare social functioning in PWS to individuals with autism spectrum disorder (ASD). Participants with mUPD scored similarly to the ASD group across most SRS domains. All groups had difficulty with social competence, although the DEL group scored highest on prosocial behavior. Findings suggest further characterization of social behavior in PWS is necessary to aid in advancing the understanding of the contributions of genes in the 15q11-13 critical region to ASD susceptibility, particularly with respect to the overexpression of maternally expressed genes in this region, as well as aiding in awareness and development/implementation of interventions.
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http://dx.doi.org/10.1007/s10803-012-1547-3DOI Listing
January 2013

A psychodermatology clinic: the concept, the format, and our observations from Israel.

Am J Clin Dermatol 2012 Apr;13(2):97-101

Dermatology Unit, Kaplan Medical Center, Rehovot, Israel.

The connection between the brain and the skin is more than a physiologic fact. Skin conditions can impose great effects on every field in the patients' lives. Reciprocally, skin diseases can be evoked by psychological problems. A psychodermatology clinic is the format that enables dermatology patients to receive a comprehensive approach to their skin condition as well as to the difficulties it imposes on their lives, and vice versa. Although the notion that skin and mind are connected and have bilateral influences that should be addressed is now appreciated, it is surprising that such clinics are not more prevalent. In the literature there are only a few descriptions of such clinics, and even fewer descriptions of their format and their working formulations with their pluses and minuses. In this article, we describe in detail the working formulation of a new psychodermatology clinic in Israel, as well as our therapeutic methods, data, conclusions, and observations after the first 3 years. During a 3-year period, 124 patients were seen in our psychodermatology clinic, presenting with a vast array of dermatologic complaints. One of our major observations was that lack of proper patient-doctor communication resulted in the development of misconceptions about the disease, low compliance, and even long-lasting psychological difficulties. Another important observation was that there is a true need for such a clinic, among patients as well as among their doctors.
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http://dx.doi.org/10.2165/11630950-000000000-00000DOI Listing
April 2012

Embryonic mesoderm and endoderm induction requires the actions of non-embryonic Nodal-related ligands and Mxtx2.

Development 2011 Feb;138(4):787-95

Medical Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA.

Vertebrate mesoderm and endoderm formation requires signaling by Nodal-related ligands from the TGFβ superfamily. The factors that initiate Nodal-related gene transcription are unknown in most species and the relative contributions of Nodal-related ligands from embryonic, extraembryonic and maternal sources remain uncertain. In zebrafish, signals from the yolk syncytial layer (YSL), an extraembryonic domain, are required for mesoderm and endoderm induction, and YSL expression of nodal-related 1 (ndr1) and ndr2 accounts for a portion of this activity. A variable requirement of maternally derived Ndr1 for dorsal and anterior axis formation has also been documented. Here we show that Mxtx2 directly activates expression of ndr2 via binding to its first intron and is required for ndr2 expression in the YSL. Mxtx2 is also required for the Nodal signaling-independent expression component of the no tail a (ntla) gene, which is required for posterior (tail) mesoderm formation. Therefore, Mxtx2 defines a new pathway upstream of Nodal signaling and posterior mesoderm formation. We further show that the co-disruption of extraembryonic Ndr2, extraembryonic Ndr1 and maternal Ndr1 eliminates endoderm and anterior (head and trunk) mesoderm, recapitulating the loss of Nodal signaling phenotype. Therefore, non-embryonic sources of Nodal-related ligands account for the complete spectrum of early Nodal signaling requirements. In summary, the induction of mesoderm and endoderm depends upon the combined actions of Mxtx2 and Nodal-related ligands from non-embryonic sources.
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http://dx.doi.org/10.1242/dev.058974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3026419PMC
February 2011

A model of Costeff Syndrome reveals metabolic and protective functions of mitochondrial OPA3.

Development 2010 Aug;137(15):2587-96

Medical Genetics Branch, National Human Genome Research Institute, Bethesda, MD 20892, USA.

Costeff Syndrome, which is caused by mutations in the OPTIC ATROPHY 3 (OPA3) gene, is an early-onset syndrome characterized by urinary excretion of 3-methylglutaconic acid (MGC), optic atrophy and movement disorders, including ataxia and extrapyramidal dysfunction. The OPA3 protein is enriched in the inner mitochondrial membrane and has mitochondrial targeting signals, but a requirement for mitochondrial localization has not been demonstrated. We find zebrafish opa3 mRNA to be expressed in the optic nerve and retinal layers, the counterparts of which in humans have high mitochondrial activity. Transcripts of zebrafish opa3 are also expressed in the embryonic brain, inner ear, heart, liver, intestine and swim bladder. We isolated a zebrafish opa3 null allele for which homozygous mutants display increased MGC levels, optic nerve deficits, ataxia and an extrapyramidal movement disorder. This correspondence of metabolic, ophthalmologic and movement abnormalities between humans and zebrafish demonstrates a phylogenetic conservation of OPA3 function. We also find that delivery of exogenous Opa3 can reduce increased MGC levels in opa3 mutants, and this reduction requires the mitochondrial localization signals of Opa3. By manipulating MGC precursor availability, we infer that elevated MGC in opa3 mutants derives from extra-mitochondrial HMG-CoA through a non-canonical pathway. The opa3 mutants have normal mitochondrial oxidative phosphorylation profiles, but are nonetheless sensitive to inhibitors of the electron transport chain, which supports clinical recommendations that individuals with Costeff Syndrome avoid mitochondria-damaging agents. In summary, this paper introduces a faithful Costeff Syndrome model and demonstrates a requirement for mitochondrial OPA3 to limit HMG-CoA-derived MGC and protect the electron transport chain against inhibitory compounds.
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http://dx.doi.org/10.1242/dev.043745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2927703PMC
August 2010

Pre-gastrula expression of zebrafish extraembryonic genes.

BMC Dev Biol 2010 Apr 27;10:42. Epub 2010 Apr 27.

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Background: Many species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL).

Results: We hypothesize that key components of the yolk syncytial layer's mesoderm and endoderm inducing activity are expressed as mRNAs in the E-YSL. To identify genes expressed in the E-YSL, we used microarrays to compare the transcription profiles of intact pre-gastrula embryos with pre-gastrula embryonic cells that we had separated from the yolk and yolk syncytial layer. This identified a cohort of genes with enriched expression in intact embryos. Here we describe our whole mount in situ hybridization analysis of sixty-eight of them. This includes ten genes with E-YSL expression (camsap1l1, gata3, znf503, hnf1ba, slc26a1, slc40a1, gata6, gpr137bb, otop1 and cebpa), four genes with expression in the enveloping layer (EVL), a superficial epithelium that protects the embryo (zgc:136817, zgc:152778, slc14a2 and elovl6l), three EVL genes whose expression is transiently confined to the animal pole (elovl6l, zgc:136359 and clica), and six genes with transient maternal expression (mtf1, wu:fj59f04, mospd2, rftn2, arrdc1a and pho). We also assessed the requirement of Nodal signaling for the expression of selected genes in the E-YSL, EVL and margin. Margin expression was Nodal dependent for all genes we tested, including the concentrated margin expression of an EVL gene: zgc:110712. All other instances of EVL and E-YSL expression that we tested were Nodal independent.

Conclusion: We have devised an effective strategy for enriching and identifying genes expressed in the E-YSL of pre-gastrula embryos. To our surprise, maternal genes and genes expressed in the EVL were also enriched by this strategy. A number of these genes are promising candidates for future functional studies on early embryonic patterning.
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http://dx.doi.org/10.1186/1471-213X-10-42DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2873407PMC
April 2010

OPA3, mutated in 3-methylglutaconic aciduria type III, encodes two transcripts targeted primarily to mitochondria.

Mol Genet Metab 2010 Jun 16;100(2):149-54. Epub 2010 Mar 16.

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

3-Methylglutaconic aciduria type III (3-MGCA type III), caused by recessive mutations in the 2-exon gene OPA3, is characterized by early-onset bilateral optic atrophy, later-onset extrapyramidal dysfunction, and increased urinary excretion of 3-methylglutaconic acid and 3-methylglutaric acid. Here we report the identification of a novel third OPA3 coding exon, the apparent product of a segmental duplication event, resulting in two gene transcripts, OPA3A and OPA3B. OPA3A deficiency (as in optic atrophy type 3) causes up-regulation of OPA3B. OPA3 protein function remains unknown, but it contains a putative mitochondrial leader sequence, mitochondrial sorting signal and a peroxisomal sorting signal. Our green fluorescent protein tagged OPA3 expression studies found its localization to be predominantly mitochondrial. These findings thus place the cellular metabolic defect of 3-MGCA type III in the mitochondrion rather than the peroxisome and implicate loss of OPA3A rather than gain of OPA3B in disease etiology.
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http://dx.doi.org/10.1016/j.ymgme.2010.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872056PMC
June 2010

Cumulative ligand activity of NODAL mutations and modifiers are linked to human heart defects and holoprosencephaly.

Mol Genet Metab 2009 Sep-Oct;98(1-2):225-34. Epub 2009 May 27.

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3717, USA.

The cyclopic and laterality phenotypes in model organisms linked to disturbances in the generation or propagation of Nodal-like signals are potential examples of similar impairments resulting in birth defects in humans. However, the types of gene mutation(s) and their pathogenetic combinations in humans are poorly understood. Here we describe a mutational analysis of the human NODAL gene in a large panel of patients with phenotypes compatible with diminished NODAL ligand function. Significant reductions in the biological activity of NODAL alleles are detected among patients with congenital heart defects (CHD), laterality anomalies (e.g. left-right mis-specification phenotypes), and only rarely holoprosencephaly (HPE). While many of these NODAL variants are typical for family-specific mutations, we also report the presence of alleles with significantly reduced activity among common population variants. We propose that some of these common variants act as modifiers and contribute to the ultimate phenotypic outcome in these patients; furthermore, we draw parallels with strain-specific modifiers in model organisms to bolster this interpretation.
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http://dx.doi.org/10.1016/j.ymgme.2009.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774839PMC
November 2009

Identification of common and unique modifiers of zebrafish midline bifurcation and cyclopia.

Dev Biol 2009 Feb 19;326(1):201-11. Epub 2008 Nov 19.

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, 9000 Rockville Pike, Building 35, Room 1B 205, Bethesda, MD 20892, USA.

Loss of the zebrafish Nodal-related protein Squint causes a spectrum of phenotypes including cyclopia and midline bifurcations (MB). Here we examine MBs and their relation to cyclopia in maternal-zygotic squint (MZsqt) mutants. There is a concordance of MB with cyclopia in MZsqt embryos. Heat treatment and depletion of Hsp90a are "common" risk factors, each of which increases the incidence of both phenotypes. Midline identity is specified on both sides of MBs, and deep-layer cells are initially lacking within bifurcations, whereas enveloping layer cells are intact. Bifurcations do not appear until the completion of gastrulation and are preceded by gaps in the expression of wnt5b, an essential regulator of dorsal convergence. The incidence of early MBs and wnt5b expression defects in heated MZsqt embryos is high, but there is also substantial recovery. Wnt5b depletion increases the incidence of MB, but not cyclopia, and as such Wnt5b is a "unique" risk factor for MB. Reciprocally, depletion of Wnt11 or Hsp90b increases cyclopia only. In summary, we find that MB arises after gastrulation in regions that fail to express wnt5b, and we show that two complex dysmorphologies - MB and cyclopia - can be promoted by either common or unique risk factors.
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http://dx.doi.org/10.1016/j.ydbio.2008.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2666472PMC
February 2009

Mutations in the human SIX3 gene in holoprosencephaly are loss of function.

Hum Mol Genet 2008 Dec 12;17(24):3919-28. Epub 2008 Sep 12.

1Medical Genetics Branch, National Human Genome Research Institute, Bethesda, MD 20892, USA.

Holoprosencephaly (HPE) is the most common developmental anomaly of the human forebrain; however, the genetics of this heterogeneous and etiologically complex malformation is incompletely understood. Heterozygous mutations in SIX3, a transcription factor gene expressed in the anterior forebrain and eyes during early vertebrate development, have been frequently detected in human HPE cases. However, only a few mutations have been investigated with limited functional studies that would confirm a role in HPE pathogenesis. Here, we report the development of a set of robust and sensitive assays of human SIX3 function in zebrafish and apply these to the analysis of a total of 46 distinct mutations (19 previously published and 27 novel) located throughout the entire SIX3 gene. We can now confirm that 89% of these putative deleterious mutations are significant loss-of-function alleles. Since disease-associated single point mutations in the Groucho-binding eh1-like motif decreases the function in all assays, we can also confirm that this interaction is essential for human SIX3 co-repressor activity; we infer, in turn, that this function is important in HPE causation. We also unexpectedly detected truncated versions with partial function, yet missing a SIX3-encoded homeodomain. Our data indicate that SIX3 is a frequent target in the pathogenesis of HPE and demonstrate how this can inform the genetic counseling of families.
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http://dx.doi.org/10.1093/hmg/ddn294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2733808PMC
December 2008

Transcriptional profiling of endogenous germ layer precursor cells identifies dusp4 as an essential gene in zebrafish endoderm specification.

Proc Natl Acad Sci U S A 2008 Aug 21;105(34):12337-42. Epub 2008 Aug 21.

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

A major goal for developmental biologists is to define the behaviors and molecular contents of differentiating cells. We have devised a strategy for isolating cells from diverse embryonic regions and stages in the zebrafish, using computer-guided laser photoconversion of injected Kaede protein and flow cytometry. This strategy enabled us to perform a genome-wide transcriptome comparison of germ layer precursor cells. Mesendoderm and ectoderm precursors cells isolated by this method differentiated appropriately in transplantation assays. Microarray analysis of these cells reidentified known genes at least as efficiently as previously reported strategies that relied on artificial mesendoderm activation or inhibition. We also identified a large set of uncharacterized mesendoderm-enriched genes as well as ectoderm-enriched genes. Loss-of-function studies revealed that one of these genes, the MAP kinase inhibitor dusp4, is essential for early development. Embryos injected with antisense morpholino oligonucleotides that targeted Dusp4 displayed necrosis of head tissues. Marker analysis during late gastrulation revealed a specific loss of sox17, but not of other endoderm markers, and analysis at later stages revealed a loss of foregut and pancreatic endoderm. This specific loss of sox17 establishes a new class of endoderm specification defect.
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http://dx.doi.org/10.1073/pnas.0805589105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2527912PMC
August 2008

Reduced NODAL signaling strength via mutation of several pathway members including FOXH1 is linked to human heart defects and holoprosencephaly.

Am J Hum Genet 2008 Jul 5;83(1):18-29. Epub 2008 Jun 5.

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Abnormalities of embryonic patterning are hypothesized to underlie many common congenital malformations in humans including congenital heart defects (CHDs), left-right disturbances (L-R) or laterality, and holoprosencephaly (HPE). Studies in model organisms suggest that Nodal-like factors provide instructions for key aspects of body axis and germ layer patterning; however, the complex genetics of pathogenic gene variant(s) in humans are poorly understood. Here we report our studies of FOXH1, CFC1, and SMAD2 and summarize our mutational analysis of three additional components in the human NODAL-signaling pathway: NODAL, GDF1, and TDGF1. We identify functionally abnormal gene products throughout the pathway that are clearly associated with CHD, laterality, and HPE. Abnormal gene products are most commonly detected in patients within a narrow spectrum of isolated conotruncal heart defects (minimum 5%-10% of subjects), and far less commonly in isolated laterality or HPE patients (approximately 1% for each). The difference in the mutation incidence between these groups is highly significant. We show that apparent gene dosage discrepancies between humans and model organisms can be reconciled by considering a broader combination of sequence variants. Our studies confirm that (1) the genetic vulnerabilities inferred from model organisms with defects in Nodal signaling are indeed analogous to humans; (2) the molecular analysis of an entire signaling pathway is more complete and robust than that of individual genes and presages future studies by whole-genome analysis; and (3) a functional genomics approach is essential to fully appreciate the complex genetic interactions necessary to produce these effects in humans.
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http://dx.doi.org/10.1016/j.ajhg.2008.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2443854PMC
July 2008

Dynamics of zebrafish somitogenesis.

Dev Dyn 2008 Mar;237(3):545-53

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

Vertebrate somitogenesis is a rhythmically repeated morphogenetic process. The dependence of somitogenesis dynamics on axial position and temperature has not been investigated systematically in any species. Here we use multiple embryo time-lapse imaging to precisely estimate somitogenesis period and somite length under various conditions in the zebrafish embryo. Somites form at a constant period along the trunk, but the period gradually increases in the tail. Somite length varies along the axis in a stereotypical manner, with tail somites decreasing in size. Therefore, our measurements prompt important modifications to the steady-state Clock and Wavefront model: somitogenesis period, somite length, and wavefront velocity all change with axial position. Finally, we show that somitogenesis period changes more than threefold across the standard developmental temperature range, whereas the axial somite length distribution is temperature invariant. This finding indicates that the temperature-induced change in somitogenesis period exactly compensates for altered axial growth.
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http://dx.doi.org/10.1002/dvdy.21458DOI Listing
March 2008

An early requirement for maternal FoxH1 during zebrafish gastrulation.

Dev Biol 2007 Oct 19;310(1):10-22. Epub 2007 Jul 19.

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, 9000 Rockville Pike, Building 35, Room 1B 205, Bethesda, MD 20892, USA.

The Forkhead Box H1 (FoxH1) protein is a co-transcription factor recruited by phosphorylated Smad2 downstream of several TGFbetas, including Nodal-related proteins. We have reassessed the function of zebrafish FoxH1 using antisense morpholino oligonucleotides (MOs). MOs targeting translation of foxH1 disrupt embryonic epiboly movements during gastrulation and cause death on the first day of development. The FoxH1 morphant phenotype is much more severe than that of zebrafish carrying foxh1/schmalspur (sur) DNA-binding domain mutations, FoxH1 splice-blocking morphants or other Nodal pathway mutants, and it cannot be altered by concomitant perturbations in Nodal signaling. Apart from disrupting epiboly, FoxH1 MO treatment disrupts convergence and internalization movements. Late gastrula-stage FoxH1 morphants exhibit delayed mesoderm and endoderm marker gene expression and failed patterning of the central nervous system. Probing FoxH1 morphant RNA by microarray, we identified a cohort of five keratin genes--cyt1, cyt2, krt4, krt8 and krt18--that are normally transcribed in the embryo's enveloping layer (EVL) and which have significantly reduced expression in FoxH1-depleted embryos. Simultaneously disrupting these keratins with a mixture of MOs reproduces the FoxH1 morphant phenotype. Our studies thus point to an essential role for maternal FoxH1 and downstream keratins during gastrulation that is epistatic to Nodal signaling.
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http://dx.doi.org/10.1016/j.ydbio.2007.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2121100PMC
October 2007

Environmental and genetic modifiers of squint penetrance during zebrafish embryogenesis.

Dev Biol 2007 Aug 25;308(2):368-78. Epub 2007 May 25.

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

The Nodal-related subgroup of the TGFbeta superfamily of secreted cytokines regulates the specification of the mesodermal and endodermal germ layers during gastrulation. Two Nodal-related proteins - Squint (Sqt) and Cyclops (Cyc) - are expressed during germ-layer specification in zebrafish. Genetic sqt mutant phenotypes have defined a variable requirement for zygotic Sqt, but not for maternal Sqt, in midline mesendoderm development. However a comparison of phenotypes arising from oocytes or zygotes injected with Sqt antisense morpholinos has suggested a novel requirement for maternal Sqt in dorsal specification. In this study we examined maternal-zygotic mutants for each of two sqt alleles and we also compared phenotypes of closely related zygotic and maternal-zygotic sqt mutants. Each of these approaches indicated there is no general requirement for maternal Sqt. To better understand the dispensability of maternal and zygotic Sqt, we sought out developmental contexts that more rigorously demand intact Sqt signalling. We found that sqt penetrance is influenced by genetic modifiers, by environmental temperature, by levels of residual Activin-like activity and by Heat-Shock Protein 90 (HSP90) activity. Therefore, Sqt may confer an evolutionary advantage by protecting early-stage embryos against detrimental interacting alleles and environmental challenges.
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http://dx.doi.org/10.1016/j.ydbio.2007.05.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1994576PMC
August 2007

Lefty antagonism of Squint is essential for normal gastrulation.

Curr Biol 2002 Dec;12(24):2129-35

Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.

Activities of a variety of signaling proteins that regulate embryogenesis are limited by endogenous antagonists. The zebrafish Nodal-related ligands, Squint and Cyclops, and their antagonists, Lefty1 and Lefty2, belong to the TGFbeta-related protein superfamily, whose members have widespread biological activities. Among other activities, Nodals direct the formation of most mesendoderm. By inducing their own transcription and that of the Lefties, Nodal signals establish positive and negative autoregulatory loops. To investigate how these autoregulatory pathways regulate development, we depleted zebrafish embryos of Lefty1 and/or Lefty2 by using antisense morpholino oligonucleotides. Loss of Lefty1 causes aberrations during somitogenesis stages, including left-right patterning defects, whereas Lefty2 depletion has no obvious consequences. Depletion of both Lefty1 and Lefty2, by contrast, causes unchecked Nodal signaling, expansion of mesendoderm, and loss of ectoderm. The expansion of mesendoderm correlates with an extended period of rapid cellular internalization and a failure of deep-cell epiboly. The gastrulation defects of embryos depleted of Lefty1 and Lefty2 result from the deregulation of Squint signaling. In contrast, deregulation of Cyclops does not affect morphology or the transcription of Nodal target genes during gastrulation. Furthermore, we find that Cyclops is specifically required for the maintenance of lefty1 and lefty2 transcription.
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http://dx.doi.org/10.1016/s0960-9822(02)01361-1DOI Listing
December 2002

Zebrafish mutants identify an essential role for laminins in notochord formation.

Development 2002 Jul;129(13):3137-46

Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

Basement membranes are thought to be essential for organ formation, providing the scaffold on which individual cells organize to form complex tissues. Laminins are integral components of basement membranes. To understand the development of a simple vertebrate organ, we have used positional cloning to characterize grumpy and sleepy, two zebrafish loci known to control notochord formation, and find that they encode laminin beta1 and laminin gamma1, respectively. Removal of either chain results in the dramatic loss of laminin 1 staining throughout the embryo and prevents formation of the basement membrane surrounding the notochord. Notochord cells fail to differentiate and many die by apoptosis. By transplantation, we demonstrate that, for both grumpy and sleepy, notochord differentiation can be rescued by exogenous sources of the missing laminin chain, although notochordal sources are also sufficient for rescue. These results demonstrate a clear in vivo requirement for laminin beta1 and laminin gamma1 in the formation of a specific vertebrate organ and show that laminin or the laminin-dependent basement membrane is essential for the differentiation of chordamesoderm to notochord.
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July 2002