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

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 properties1,2. 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 materials3-8, 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.

The role of Niemann-Pick type C2 in zebrafish embryonic development.

Niemann-Pick disease type C (NPC) is a rare, fatal, neurodegenerative lysosomal disease caused by mutations of either NPC1 or NPC2. NPC2 is a soluble lysosomal protein that functions in coordination with NPC1 to efflux cholesterol from the lysosomal compartment. Mutations of either gene result in the accumulation of unesterified cholesterol and other lipids in the late endosome/lysosome, and reduction of cellular cholesterol bioavailability. Zygotic null npc2m/m zebrafish showed significant unesterified cholesterol accumulation at larval stages, a reduction in body size, and motor and balance defects in adulthood. However, the phenotype at embryonic stages was milder than expected, suggesting a possible role of maternal Npc2 in embryonic development. Maternal-zygotic npc2m/m zebrafish exhibited significant developmental defects, including defective otic vesicle development/absent otoliths, abnormal head/brain development, curved/twisted body axes and no circulating blood cells, and died by 72 hpf. RNA-seq analysis conducted on 30 hpf npc2+/m and MZnpc2m/m embryos revealed a significant reduction in the expression of notch3 and other downstream genes in the Notch signaling pathway, suggesting that impaired Notch3 signaling underlies aspects of the developmental defects observed in MZnpc2m/m zebrafish.

Tunable spin and valley excitations of correlated insulators in Γ-valley moiré bands.

Moiré superlattices formed from transition metal dichalcogenides support a variety of quantum electronic phases that are highly tunable using applied electromagnetic fields. While the valley degree of freedom affects optoelectronic properties in the constituent transition metal dichalcogenides, it has yet to be fully explored in moiré systems. Here we establish twisted double-bilayer WSe2 as an experimental platform to study electronic correlations within Γ-valley moiré bands. Through local and global electronic compressibility measurements, we identify charge-ordered phases at multiple integer and fractional moiré fillings. By measuring the magnetic field dependence of their energy gaps and the chemical potential upon doping, we reveal spin-polarized ground states with spin-polaron quasiparticle excitations. In addition, an applied displacement field induces a metal-insulator transition driven by tuning between Γ- and K-valley moiré bands. Our results demonstrate control over the spin and valley character of the correlated ground and excited states in this system.

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

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.

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

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.

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

Vertebrate mesoderm and endoderm formation requires signaling by Nodal-related ligands from the TGFß 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.

Mapping twist-tuned multiband topology in bilayer WSe2.

Semiconductor moiré superlattices have been shown to host a wide array of interaction-driven ground states. However, twisted homobilayers have been difficult to study in the limit of large moiré wavelengths, where interactions are most dominant. In this study, we conducted local electronic compressibility measurements of twisted bilayer WSe2 (tWSe2) at small twist angles. We demonstrated multiple topological bands that host a series of Chern insulators at zero magnetic field near a "magic angle" around 1.23°. Using a locally applied electric field, we induced a topological quantum-phase transition at one hole per moiré unit cell. Our work establishes the topological phase diagram of a generalized Kane-Mele-Hubbard model in tWSe2, demonstrating a tunable platform for strongly correlated topological phases.

Uncovering the spin ordering in magic-angle graphene via edge state equilibration.

The flat bands in magic-angle twisted bilayer graphene (MATBG) provide an especially rich arena to investigate interaction-driven ground states. While progress has been made in identifying the correlated insulators and their excitations at commensurate moiré filling factors, the spin-valley polarizations of the topological states that emerge at high magnetic field remain unknown. Here we introduce a technique based on twist-decoupled van der Waals layers that enables measurement of their electronic band structure and-by studying the backscattering between counter-propagating edge states-the determination of the relative spin polarization of their edge modes. We find that the symmetry-broken quantum Hall states that extend from the charge neutrality point in MATBG are spin unpolarized at even integer filling factors. The measurements also indicate that the correlated Chern insulator emerging from half filling of the flat valence band is spin unpolarized and suggest that its conduction band counterpart may be spin polarized.

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

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.

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

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.

1/4 is the new 1/2 when topology is intertwined with Mottness.

In non-interacting systems, bands from non-trivial topology emerge strictly at half-filling and exhibit either the quantum anomalous Hall or spin Hall effects. Here we show using determinantal quantum Monte Carlo and an exactly solvable strongly interacting model that these topological states now shift to quarter filling. A topological Mott insulator is the underlying cause. The peak in the spin susceptibility is consistent with a possible ferromagnetic state at T = 0. The onset of such magnetism would convert the quantum spin Hall to a quantum anomalous Hall effect. While such a symmetry-broken phase typically is accompanied by a gap, we find that the interaction strength must exceed a critical value for this to occur. Hence, we predict that topology can obtain in a gapless phase but only in the presence of interactions in dispersive bands. These results explain the recent quarter-filled quantum anomalous Hall effects seen in moiré systems.

Comparing Kundalini Yoga, cognitive behavioral therapy, and stress education for generalized anxiety disorder: Anxiety and depression symptom outcomes.

Increasingly, individuals with anxiety disorders are seeking mind-body interventions (e.g., yoga), but their effectiveness is unclear. This report summarizes seven additional, secondary outcomes measuring anxiety and depression symptoms from a study of 226 adults with generalized anxiety disorder who were randomized to 12-week Kundalini Yoga, Cognitive-Behavior Therapy (CBT) or stress education (control). At post-treatment, participants receiving CBT displayed significantly lower symptom severity, compared to those in the control group, on 6 of the 7 measures. Participants who received Yoga (vs. those in the control group) displayed lower symptom severity on 3 of the 7 measures. No significant differences were detected between participants receiving CBT vs those receiving Yoga. At the 6-month follow-up, participants from the CBT continued to display lower symptoms than the control group.

Spin skyrmion gaps as signatures of strong-coupling insulators in magic-angle twisted bilayer graphene.

The flat electronic bands in magic-angle twisted bilayer graphene (MATBG) host a variety of correlated insulating ground states, many of which are predicted to support charged excitations with topologically non-trivial spin and/or valley skyrmion textures. However, it has remained challenging to experimentally address their ground state order and excitations, both because some of the proposed states do not couple directly to experimental probes, and because they are highly sensitive to spatial inhomogeneities in real samples. Here, using a scanning single-electron transistor, we observe thermodynamic gaps at even integer moiré filling factors at low magnetic fields. We find evidence of a field-tuned crossover from charged spin skyrmions to bare particle-like excitations, suggesting that the underlying ground state belongs to the manifold of strong-coupling insulators. From the spatial dependence of these states and the chemical potential variation within the flat bands, we infer a link between the stability of the correlated ground states and local twist angle and strain. Our work advances the microscopic understanding of the correlated insulators in MATBG and their unconventional excitations.

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

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.

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

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.

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

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.

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

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.

Pre-gastrula expression of zebrafish extraembryonic genes.

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.

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

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.

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

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.