The Ancient Origins of Neural Substrates for Land Walking.

Walking is the predominant locomotor behavior expressed by land-dwelling vertebrates, but it is unknown when the neural circuits that are essential for limb control first appeared. Certain fish species display walking-like behaviors, raising the possibility that the underlying circuitry originated in primitive marine vertebrates. We show that the neural substrates of bipedalism are present in the little skate Leucoraja erinacea, whose common ancestor with tetrapods existed ∼420 million years ago. Leucoraja exhibits core features of tetrapod locomotor gaits, including left-right alternation and reciprocal extension-flexion of the pelvic fins. Leucoraja also deploys a remarkably conserved Hox transcription factor-dependent program that is essential for selective innervation of fin/limb muscle. This network encodes peripheral connectivity modules that are distinct from those used in axial muscle-based swimming and has apparently been diminished in most modern fish. These findings indicate that the circuits that are essential for walking evolved through adaptation of a genetic regulatory network shared by all vertebrates with paired appendages. VIDEO ABSTRACT.

Structural Titration of Slo2.2, a Na+-Dependent K+ Channel.

The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of structures of Slo2.2, a neuronal Na+-activated K+ channel, as a function of the Na+ concentration. We find that Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Na+ concentration. An open conformation emerges from an ensemble of closed conformations in a highly Na+-dependent manner, without evidence of Na+-dependent intermediates. In other words, channel opening is a highly concerted, switch-like process. The midpoint of the structural titration matches that of the functional titration. A maximum open conformation probability approaching 1.0 and maximum functional open probability approaching 0.7 imply that, within the class of open channels, there is a subclass that is not permeable to ions.

Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars.

Parrot feathers contain red, orange, and yellow polyene pigments called psittacofulvins. Budgerigars are parrots that have been extensively bred for plumage traits during the last century, but the underlying genes are unknown. Here we use genome-wide association mapping and gene-expression analysis to map the Mendelian blue locus, which abolishes yellow pigmentation in the budgerigar. We find that the blue trait maps to a single amino acid substitution (R644W) in an uncharacterized polyketide synthase (MuPKS). When we expressed MuPKS heterologously in yeast, yellow pigments accumulated. Mass spectrometry confirmed that these yellow pigments match those found in feathers. The R644W substitution abolished MuPKS activity. Furthermore, gene-expression data from feathers of different bird species suggest that parrots acquired their colors through regulatory changes that drive high expression of MuPKS in feather epithelia. Our data also help formulate biochemical models that may explain natural color variation in parrots. VIDEO ABSTRACT.

Bending gradients: how the intestinal stem cell gets its home.

We address the mechanism by which adult intestinal stem cells (ISCs) become localized to the base of each villus during embryonic development. We find that, early in gut development, proliferating progenitors expressing ISC markers are evenly distributed throughout the epithelium, in both the chick and mouse. However, as the villi form, the putative stem cells become restricted to the base of the villi. This shift in the localization is driven by mechanically influenced reciprocal signaling between the epithelium and underlying mesenchyme. Buckling forces physically distort the shape of the morphogenic field, causing local maxima of epithelial signals, in particular Shh, at the tip of each villus. This induces a suite of high-threshold response genes in the underlying mesenchyme to form a signaling center called the "villus cluster." Villus cluster signals, notably Bmp4, feed back on the overlying epithelium to ultimately restrict the stem cells to the base of each villus.

X-ray structure of acid-sensing ion channel 1-snake toxin complex reveals open state of a Na(+)-selective channel.

Acid-sensing ion channels (ASICs) detect extracellular protons produced during inflammation or ischemic injury and belong to the superfamily of degenerin/epithelial sodium channels. Here, we determine the cocrystal structure of chicken ASIC1a with MitTx, a pain-inducing toxin from the Texas coral snake, to define the structure of the open state of ASIC1a. In the MitTx-bound open state and in the previously determined low-pH desensitized state, TM2 is a discontinuous α helix in which the Gly-Ala-Ser selectivity filter adopts an extended, belt-like conformation, swapping the cytoplasmic one-third of TM2 with an adjacent subunit. Gly 443 residues of the selectivity filter provide a ring of three carbonyl oxygen atoms with a radius of ∼3.6 Å, presenting an energetic barrier for hydrated ions. The ASIC1a-MitTx complex illuminates the mechanism of MitTx action, defines the structure of the selectivity filter of voltage-independent, sodium-selective ion channels, and captures the open state of an ASIC.

RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks.

The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-ß expression during virus infection, the expression level of IFN-ß in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.

SMPD3 expression is spatially regulated in the developing embryo by SOXE factors.

During epithelial-to-mesenchymal transition (EMT), significant rearrangements occur in plasma membrane protein and lipid content that are important for membrane function and acquisition of cell motility. To gain insight into how neural crest cells regulate their lipid content at the transcriptional level during EMT, here we identify critical enhancer sequences that regulate the expression of SMPD3, a gene responsible for sphingomyelin hydrolysis to produce ceramide and necessary for neural crest EMT. We uncovered three enhancer regions within the first intron of the SMPD3 locus that drive reporter expression in distinct spatial and temporal domains, together collectively recapitulating the expression domains of endogenous SMPD3 within the ectodermal lineages. We further dissected one enhancer that is specifically active in the migrating neural crest. By mutating putative transcriptional input sites or knocking down upstream regulators, we find that the SOXE-family transcription factors SOX9 and SOX10 regulate the expression of SMPD3 in migrating neural crest cells. Further, ChIP-seq and nascent transcription analysis reveal that SOX10 directly regulates expression of an SMPD3 enhancer specific to migratory neural crest cells. Together these results shed light on how core components of developmental gene regulatory networks interact with metabolic effector genes to control changes in membrane lipid content.

TMEM8C-mediated fusion is regionalized and regulated by NOTCH signalling during foetal myogenesis.

The location and regulation of fusion events within skeletal muscles during development remain unknown. Using the fusion marker myomaker (Mymk), named TMEM8C in chicken, as a readout of fusion, we identified a co-segregation of TMEM8C-positive cells and MYOG-positive cells in single-cell RNA-sequencing datasets of limbs from chicken embryos. We found that TMEM8C transcripts, MYOG transcripts and the fusion-competent MYOG-positive cells were preferentially regionalized in central regions of foetal muscles. We also identified a similar regionalization for the gene encoding the NOTCH ligand JAG2 along with an absence of NOTCH activity in TMEM8C+ fusion-competent myocytes. NOTCH function in myoblast fusion had not been addressed so far. We analysed the consequences of NOTCH inhibition for TMEM8C expression and myoblast fusion during foetal myogenesis in chicken embryos. NOTCH inhibition increased myoblast fusion and TMEM8C expression and released the transcriptional repressor HEYL from the TMEM8C regulatory regions. These results identify a regionalization of TMEM8C-dependent fusion and a molecular mechanism underlying the fusion-inhibiting effect of NOTCH in foetal myogenesis. The modulation of NOTCH activity in the fusion zone could regulate the flux of fusion events.

The proofreading exonuclease of leading-strand DNA polymerase epsilon prevents replication fork collapse at broken template strands.

Leading-strand DNA replication by polymerase epsilon (Polϵ) across single-strand breaks (SSBs) causes single-ended double-strand breaks (seDSBs), which are repaired via homology-directed repair (HDR) and suppressed by fork reversal (FR). Although previous studies identified many molecules required for hydroxyurea-induced FR, FR at seDSBs is poorly understood. Here, we identified molecules that specifically mediate FR at seDSBs. Because FR at seDSBs requires poly(ADP ribose)polymerase 1 (PARP1), we hypothesized that seDSB/FR-associated molecules would increase tolerance to camptothecin (CPT) but not the PARP inhibitor olaparib, even though both anti-cancer agents generate seDSBs. Indeed, we uncovered that Polϵ exonuclease and CTF18, a Polϵ cofactor, increased tolerance to CPT but not olaparib. To explore potential functional interactions between Polϵ exonuclease, CTF18, and PARP1, we created exonuclease-deficient POLE1exo-/-, CTF18-/-, PARP1-/-, CTF18-/-/POLE1exo-/-, PARP1-/-/POLE1exo-/-, and CTF18-/-/PARP1-/- cells. Epistasis analysis indicated that Polϵ exonuclease and CTF18 were interdependent and required PARP1 for CPT tolerance. Remarkably, POLE1exo-/- and HDR-deficient BRCA1-/- cells exhibited similar CPT sensitivity. Moreover, combining POLE1exo-/- with BRCA1-/- mutations synergistically increased CPT sensitivity. In conclusion, the newly identified PARP1-CTF18-Polϵ exonuclease axis and HDR act independently to prevent fork collapse at seDSBs. Olaparib inhibits this axis, explaining the pronounced cytotoxic effects of olaparib on HDR-deficient cells.

Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers.

m6A methylation provides an essential layer of regulation in organismal development, and is aberrant in a range of cancers and neuro-pathologies. The information encoded by m6A methylation is integrated into existing RNA regulatory networks by RNA binding proteins that recognise methylated sites, the m6A readers. m6A readers include a well-characterised class of dedicated proteins, the YTH proteins, as well as a broader group of multi-functional regulators where recognition of m6A is only partially understood. Molecular insight in this recognition is essential to build a mechanistic understanding of global m6A regulation. In this study, we show that the reader IMP1 recognises the m6A using a dedicated hydrophobic platform that assembles on the methyl moiety, creating a stable high-affinity interaction. This recognition is conserved across evolution and independent from the underlying sequence context but is layered upon the strong sequence specificity of IMP1 for GGAC RNA. This leads us to propose a concept for m6A regulation where methylation plays a context-dependent role in the recognition of selected IMP1 targets that is dependent on the cellular concentration of available IMP1, differing from that observed for the YTH proteins.

The ciliary protein C2cd3 is required for mandibular musculoskeletal tissue patterning.

The mandible is composed of several musculoskeletal tissues including bone, cartilage, and tendon that require precise patterning to ensure structural and functional integrity. Interestingly, most of these tissues are derived from one multipotent cell population called cranial neural crest cells (CNCCs). How CNCCs are properly instructed to differentiate into various tissue types remains nebulous. To better understand the mechanisms necessary for the patterning of mandibular musculoskeletal tissues we utilized the avian mutant talpid2 (ta2) which presents with several malformations of the facial skeleton including dysplastic tendons, mispatterned musculature, and bilateral ectopic cartilaginous processes extending off Meckel's cartilage. We found an ectopic epithelial BMP signaling domain in the ta2 mandibular prominence (MNP) that correlated with the subsequent expansion of SOX9+ cartilage precursors. These findings were validated with conditional murine models suggesting an evolutionarily conserved mechanism for CNCC-derived musculoskeletal patterning. Collectively, these data support a model in which cilia are required to define epithelial signal centers essential for proper musculoskeletal patterning of CNCC-derived mesenchyme.

Structural basis of Focal Adhesion Kinase activation on lipid membranes.

Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions.

Independent loss events of a functional tetherin gene in galliform birds.

IMPORTANCE: Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.

A flowchart for adequate controls in virus-based monosynaptic tracing experiments identified Cre-independent leakage of the TVA receptor in RΦGT mice.

BACKGROUND: A pseudotyped modified rabies virus lacking the rabies glycoprotein (G-protein), which is crucial for transsynaptic spread, can be used for monosynaptic retrograde tracing. By coupling the pseudotyped virus with transgene expression of the G-protein and the avian leukosis and sarcoma virus subgroup A receptor (TVA), which is necessary for cell entry of the virus, researchers can investigate specific neuronal populations. Responder mouse lines, like the RΦGT mouse line, carry the genes encoding the G-protein and TVA under Cre-dependent expression. These mouse lines are valuable tools because they reduce the number of viral injections needed compared to when using helper viruses. Since RΦGT mice do not express Cre themselves, introducing the pseudotyped rabies virus into their brain should not result in viral cell entry or spread. RESULTS: We present a straightforward flowchart for adequate controls in tracing experiments, which we employed to demonstrate Cre-independent expression of TVA in RΦGT mice. CONCLUSIONS: Our observations revealed TVA leakage, indicating that RΦGT mice should be used with caution for transgene expression of TVA. Inaccurate tracing outcomes may occur if TVA is expressed in the absence of Cre since background leakage leads to nonspecific cell entry. Moreover, conducting appropriate control experiments can identify the source of potential caveats in virus-based neuronal tracing experiments.

Manipulation of a social signal affects DNA methylation of a stress-related gene in a free-living bird.

Social status directly affects the health of humans and other animals. Low status individuals receive more antagonistic encounters, have fewer supportive relationships and have worse health outcomes. However, the physiological and cellular processes that mediate the relationship between the social environment and health are incompletely known. Epigenetic regulation of the hypothalamic-pituitary-adrenal (HPA) axis, the neuroendocrine pathway that activates in response to stressors, may be one process that is sensitive to the social environment. Here, we experimentally manipulated plumage, a key social signal in female tree swallows (Tachycineta bicolor) and quantified methylation of four genes in the HPA axis before and after treatment. We found that dulling the white breast plumage affected methylation in one gene, CRHR1; however, the effect depended on the original brightness of the bird. Methylation in this gene was correlated with baseline corticosterone levels, suggesting that DNA methylation of CRHR1 helps regulate glucocorticoid production in this species. Methylation in two other genes, FKBP5 and GR, changed over the course of the experiment, independent of treatment. These results show that methylation of these genes is labile into adulthood and suggest that epigenetic regulation of the HPA axis could help birds respond to current environmental conditions.

Ig Enhancers Increase RNA Polymerase II Stalling at Somatic Hypermutation Target Sequences.

Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

The antioxidant enzyme Prdx1 controls neuronal differentiation by thiol-redox-dependent activation of GDE2.

The six-transmembrane protein GDE2 controls the onset and progression of spinal motor neuron differentiation through extracellular glycerophosphodiester phosphodiesterase metabolism. Although this process is likely to be tightly regulated, the relevant mechanisms that modulate its activity are unknown. Here we show that the antioxidant scavenger peroxiredoxin1 (Prdx1) interacts with GDE2, and that loss of Prdx1 causes motor neuron deficits analogous to GDE2 ablation. Prdx1 cooperates with GDE2 to drive motor neuron differentiation, and this synergy requires Prdx1 thiol-dependent catalysis. Prdx1 activates GDE2 through reduction of an intramolecular disulfide bond that bridges its intracellular N- and C-terminal domains. GDE2 variants incapable of disulfide bond formation acquire independence from Prdx1 and are potent inducers of motor neuron differentiation. These findings define Prdx1 as a pivotal regulator of GDE2 activity and suggest roles for coupled thiol-redox-dependent cascades in controlling neuronal differentiation in the spinal cord.

PrimPol Is Required for Replicative Tolerance of G Quadruplexes in Vertebrate Cells.

G quadruplexes (G4s) can present potent blocks to DNA replication. Accurate and timely replication of G4s in vertebrates requires multiple specialized DNA helicases and polymerases to prevent genetic and epigenetic instability. Here we report that PrimPol, a recently described primase-polymerase (PrimPol), plays a crucial role in the bypass of leading strand G4 structures. While PrimPol is unable to directly replicate G4s, it can bind and reprime downstream of these structures. Disruption of either the catalytic activity or zinc-finger of PrimPol results in extreme G4-dependent epigenetic instability at the BU-1 locus in avian DT40 cells, indicative of extensive uncoupling of the replicative helicase and polymerase. Together, these observations implicate PrimPol in promoting restart of DNA synthesis downstream of, but closely coupled to, G4 replication impediments.

Structural Analysis of dsRNA Binding to Anti-viral Pattern Recognition Receptors LGP2 and MDA5.

RIG-I and MDA5 sense virus-derived short 5'ppp blunt-ended or long dsRNA, respectively, causing interferon production. Non-signaling LGP2 appears to positively and negatively regulate MDA5 and RIG-I signaling, respectively. Co-crystal structures of chicken (ch) LGP2 with dsRNA display a fully or semi-closed conformation depending on the presence or absence of nucleotide. LGP2 caps blunt, 3' or 5' overhang dsRNA ends with 1 bp longer overall footprint than RIG-I. Structures of 1:1 and 2:1 complexes of chMDA5 with short dsRNA reveal head-to-head packing rather than the polar head-to-tail orientation described for long filaments. chLGP2 and chMDA5 make filaments with a similar axial repeat, although less co-operatively for chLGP2. Overall, LGP2 resembles a chimera combining a MDA5-like helicase domain and RIG-I like CTD supporting both stem and end binding. Functionally, RNA binding is required for LGP2-mediated enhancement of MDA5 activation. We propose that LGP2 end-binding may promote nucleation of MDA5 oligomerization on dsRNA.

Primary sex determination in birds depends on DMRT1 dosage, but gonadal sex does not determine adult secondary sex characteristics.

In birds, males are the homogametic sex (ZZ) and females the heterogametic sex (ZW). Primary sex determination is thought to depend on a sex chromosome gene dosage mechanism, and the most likely sex determinant is the Z chromosome gene Doublesex and Mab-3-Related Transcription factor 1 (DMRT1). To clarify this issue, we used a CRISPR-Cas9-based monoallelic targeting approach and sterile surrogate hosts to generate birds with targeted mutations in the DMRT1 gene. The resulting chromosomally male (ZZ) chicken with a single functional copy of DMRT1 developed ovaries in place of testes, demonstrating the avian sex-determining mechanism is based on DMRT1 dosage. These ZZ ovaries expressed typical female markers and showed clear evidence of follicular development. However, these ZZ adult birds with an ovary in place of testes were indistinguishable in appearance to wild-type adult males, supporting the concept of cell-autonomous sex identity (CASI) in birds. In experiments where estrogen synthesis was blocked in control ZW embryos, the resulting gonads developed as testes. In contrast, if estrogen synthesis was blocked in ZW embryos that lacked DMRT1, the gonads invariably adopted an ovarian fate. Our analysis shows that DMRT1 is the key sex determination switch in birds and that it is essential for testis development, but that production of estrogen is also a key factor in primary sex determination in chickens, and that this production is linked to DMRT1 expression.