The early environment impacts many aspects of an individual’s developing phenotype. In particular, early environmental conditions are important for shaping the hypothalamic-pituitary-adrenal (HPA) axis, which coordinates an individual’s stress response. These developmental changes are likely mediated by epigenetic modifications, functional changes to the genome that can alter gene expression in response to environmental variation, resulting in significant phenotypic differences (Kundakovic and Champagne 2015; Richards 2006). Determining how early life variation alters epigenetic modifications (such as DNA methylation) of genes throughout the HPA axis, and how these marks change over time, in wild organisms is important for understanding their potential long-term fitness consequences. Here, I examine DNA methylation modifications in the HPA axis in relation to early environmental variation in free-living house sparrows (Passer domesticus). In Chapter 1, I show a relationship between natural variation in the early environment and DNA methylation marks of numerous genes related to HPA axis function, which in turn predict growth trajectories. In Chapter 2, I show that early life stress in particular impacts DNA methylation in genes critical to HPA axis function, but does so differently depending on the life history stage in which stress is encountered. Finally, in Chapter 3, I find that these early life marks have long-term effects past the developmental period, predicting longevity as well as lifetime reproductive output in a sex-specific manner. Overall, my dissertation adds to a growing understanding of the dynamic role of epigenetic modifications in mediating phenotypic responses to the early life environment in wild birds, and demonstrates the potential long-term fitness outcomes of these changes.

In modern manufacturing industries, many applications require precision motion control of multi-agent systems, like multi-joint robot arms and multi-axis machine tools. Cutter (end effector) should stay as close as possible to the reference trajectory to ensure the quality of the final products. In conventional computer numerical control (CNC), the control unit of each axis is independently designed to achieve the best individual tracking performance. However, this becomes less effective when dealing with multi-axis contour following tasks because of the lack of coordination among axes. This dissertation studies the control of multi-axis machine tools with focus on reducing the contour error. The proposed research explicitly addresses the minimization of contour error and treats the multi-axis machine tool as a multi-input-multi-output (MIMO) system instead of several decoupled single-input-single-output (SISO) systems. New control schemes are developed to achieve superior contour following performance even in the presence of disturbances. This study also extends the applications of the proposed control system from plane contours to regular contours in R3. The effectiveness of the developed control systems is experimentally verified on a micro milling machine.

Liquid crystals have been a major subject of research for the past decades. Aside from the variety of structures they can form, they exhibit a vast range of optical phenomena. Many of these phenomena found applications in technology and became an essential part of it. In this dissertation thesis we continue the line to propose a number of new applications of optical effects in liquid crystals and develop their theoretical framework. One such application is the possibility of beam combining using Orientational Stimulated Scattering in a nematic liquid crystal cell. Our numerical study of the OSS process shows that normally this possibility does not exist. However, we found that if a number of special conditions is satisfied efficient beam combining with OSS can be done. These conditions require a combination of special geometric arrangement of incident beams, their profiles, nematic material, and more. When these conditions are fulfilled, power of the beamlets can be coherently combined into a single beam, with high conversion efficiency while the shape and wave-front of the output beam are still of good quality. We also studied the dynamics of the OSS process itself and observed (in a numerical model) a number of notorious instabilities caused by effects of back-conversion iv process. Additionally, there was found a numerical solitary-wave solution associated with this back-conversion process. As a liquid crystal display application, we consider a nematic liquid crystal layer with the anisotropy axis modulated at a fixed rate in the transverse direction with respect to light propagation direction. If the layer locally constitutes a half-wave plate, then the thinscreen approximation predicts 100% -efficient diffraction of normal incident wave. If this diffracted light is blocked by an aperture only transmitting the zero-th order, the cell is in dark state. If now the periodic structure is washed out by applying voltage across the cell and light passes through the cell undiffracted, the light will pass through the aperture as well and the cell will be in its bright state. Such properties of this periodically aligned nematic layer suggest it as a candidate element in projection display cells. We studied the possibility to implement such layer through anchoring at both surfaces of the cell. It was found that each cell has a thickness threshold for which the periodic structure can exist. The anchored periodic structure cannot exist if thickness of the cell exceeds this threshold. For the case when the periodic structure exists, we found the structure distortion in comparison with the preferable ideal sinusoidal profile. To complete description of the electromechanical properties of the periodic cell, we studied its behavior at Freedericksz transition. Optical performance was successfully described with the coupled-mode theory. While influence of director distortion is shown to be negligibly small, the walk-off effects appear to be larger. In summary, there are good prospects for use of this periodically v aligned cell as a pixel in projection displays but experimental study and optimization need to be performed. In the next part we discuss another modulated liquid crystal structure in which the director periodically swings in the direction of light propagation. The main characteristic of such structure is the presence of bandgap. Cholesteric liquid crystals are known to possess bandgap for one of two circular polarizations of light. However, unlike the cholesterics the bandgap of the proposed structure is independent of polarization of normally incident light. This means that no preparation of light is needed in order for the structure to work in, for example, liquid crystal displays. The polarization universality comes at the cost of bandgap size, whose maximum possible value ∆ωPTN compared to that of cholesterics ∆ωCh is approximately twice smaller: ∆ωPTN ≈ 0.58∆ωCh if modulation profile is sinusoidal, and ∆ωPTN ≈ 0.64∆ωCh if it is rectangular. This structure has not yet been experimentally demonstrated, and we discuss possible ways to make it.

Since the Islamic Revolution in 1979 Iran has employed a deliberate and successful strategy centered around the use and support of various proxies, notably Hezbollah and Hamas who are part of the Iranian led Axis of Resistance. There is a vast literature that has studied different aspects of this Iranian proxy dimension, but relatively little research has been conducted on propaganda as a unifying mechanism. This thesis thus expanded upon the current literature through a content analysis of the propaganda produced by Iran, Hezbollah, and Hamas in order to gain a fuller understanding of how cross-group cohesion has been achieved. The analysis incorporated social identity theory, and identified how the propaganda could be used in accordance with three theoretical dimensions: Identity constructions, Justification for Violence, and Weaponization of the ummah. The findings revealed how cross-group cohesion was achieved through the creation of a dichotomous worldview, consisting of an ingroup that is existentially threatened by an outgroup consisting of the West and Jews. Moreover, they use conspiratorial rhetoric combined with victimhood in order to frame the struggle as an ongoing religious war against Islam. The thesis concludes with a theoretical discussion on the normative assumptions that underpin the propaganda, arguing that the cross-group cohesion is dependent on the creation of shared identities based on victimhood, religious references, and the creation of shared external enemies.

Past work has linked self-injurious behavior (SIB) to a history of traumatic experiences and to problems regulating affect. While this affect dysregulation is conceptualized as occurring at a biological (as well as a behavioral) level, relatively little is known about the biological mechanisms involved. The current study explored whether reactivity of the hypothalamic-pituitary-adrenal (HPA) axis to an interpersonal stressor mediated the relation between trauma and SIB in a sample of 178 18-21 year-old heterosexual dating couples. As predicted, both trauma experience and symptoms positively predicted SIB. While the mediating model was not supported, SIB was associated with an HPA axis response marked by heightened reactivity to interpersonal stress within the context of lower cortisol levels. Trauma symptoms and experience interacted with adult attachment security to predict HPA axis response in different ways for men and women, a compelling set of findings suggesting the importance of contextual factors in the study of trauma and HPA axis function. Future directions for the study of trauma, HPA axis reactivity, and SIB are discussed.

The present work generalizes the necessary condition of univariate symmetry of Patil et al. (2012) to the bivariate setting, develops a test of bivariate symmetry based on it, and generalizes the measure of asymmetry in Patil et al. (2014) to the bivariate setting. In doing so, as a byproduct, it pays attention to the interrelation between central symmetry and symmetry about an axis of a continuous bivariate density function.

Minimally invasive surgery (MIS) has several advantages over traditional methods. Scaling MIS instruments to smaller sizes and increasing their performance will enable surgeons to offer new procedures to a wider range of patients. In this work, two compliant mechanism-based minimally invasive surgical instrument wrist or gripper mechanisms are designed and analyzed.The cylindrical cross-axis flexural pivot (CCAFP) is a single-degree-of-freedom wrist mechanism that could be combined with existing gripper mechanisms to create a multi-degree-of freedom instrument. The simplicity of the CCAFP mechanism facilitates analysis and implementation. The flexures of the CCAFP are integral with the instrument shaft, enabling accessories to be passed through the lumen. The CCAFP is analyzed and determined to be a viable wrist mechanism for MIS instruments based on research results. A finite element (FE) model of the mechanism is created to analyze the force-deflection and strain-deflection relationships. Experimental results are used to verify the FE model. A 3 mm design is created that could undergo an angular deflection of +/- 90 degrees. The addition of cam surfaces to help guide the flexures and limit the maximum stress during deflection is explored. These cam surfaces can be integral to the instrument shaft along with the flexures. A 2 degree-of-freedom (DoF) CCAFP with intersecting axes of rotation is also introduced. The inverted L-Arm gripper compliant mechanism has 2 DoF, one wrist and one gripping. Three challenges associated with using compliant mechanisms in MIS instruments are considered: inadequate performance in compression, large flexure deformations, and a highly variable mechanical advantage. These challenges were resolved in the L-Arm design by inverting the flexures, tailoring flexure geometry and employing nitinol, and integrating pulleys into each jaw of the mechanism. The L-Arm was prototyped at several sizes to demonstrate functionality and scalability. A finite element model of the L-Arm flexure was created to determine the strain-deflection relationship. A fatigue test was completed to characterize nitinol for use in compliant mechanism MIS instruments.These concepts demonstrate the ability of compliant mechanisms to overcome the design and manufacturing challenges associated with MIS instruments at the 3 mm scale. The models and principles included in this work could be used in the application of compliant mechanisms to design new MIS instruments as well as in other areas that employ compliant mechanisms in a cylindrical form factor.

Direct detection of dark matter remains an important outstanding problem since abundant astrophysical evidence points towards its existence, but no experiment has succeeded in detecting it. Axions and axion-like-particles are some of the most compelling candidates for dark matter given their appearance in many theories of physics beyond the Standard Model and their relatively unexplored parameter space compared to other candidates. Recently, the Cosmic Axion Spin Precession Experiment-Electric (CASPEr-e) has used nuclear magnetic resonance (NMR) to search for effective magnetic fields created by axionic dark matter. By decreasing technical noise sources, CASPEr-e is projected to reach the standard quantum limit where spin projection noise is the dominant noise source limiting sensitivity. However, some axion models predict axion couplings to normal matter that would be too small for even a quantum limited CASPEr-e experiment to detect. This creates a need for surpassing the spin projection noise limit in NMR dark matter searches. In this thesis, I explore the prospects for surpassing the quantum limit in NMR by using spin squeezed states, entangled states with variance in one projection reduced below the standard quantum limit. First, I propose an experimental scheme for generating squeezed states by coupling the spins to an off-resonant circuit to create a One-Axis-Twist Hamiltonian. Then, using exact results and numerical simulations, I determine the amount of squeezing that can be achieved given decoherence and noise. Next, I perform modeling to show that squeezing can accelerate dark matter searches despite earlier results that argued squeezing cannot improve experimental sensitivity when subject to decoherence. Finally, I apply these results to the CASPEr-e experiment and show that at axion frequencies near 100MHz, squeezing can speed up the experiment by a factor of up to 30, corresponding to a sensitivity improvement by a factor of over 5.

Sexual reproduction requires meiosis, a specialized cell division program that halves the chromosome number of germ cells in order to generate gametes. Chromosome number reduction is achieved by two successive rounds of cell divisions after a single round of DNA replication. In meiosis I, homologous chromosomes (homologs) recombine to produce at least one reciprocal DNA exchange, called crossover (CO), in each chromosome. COs ensure proper segregation, as the resulting tetrad chromosomes are bisected during the first division to form dyads. In meiosis II, dyads split allowing segregation of single chromatids, resembling mitosis. During prophase I, the earliest stage of meiosis I, each pair of sister chromatids is arranged in series of loops tethered longitudinally by a structure called the chromosome axis, which serves as a scaffold for the machinery that promotes the formation of programmed DNA double-strand breaks (DSBs). Recombination is initiated when the DSBs are resected to produce single-stranded DNA (ssDNA) overhangs that seek out their homologs, promoting pairing. Then the synaptonemal complex (SC) forms, physically linking homolog axes through transverse filaments. In the context of the SC, meiotic recombination repairs the DSBs and turns a small fraction of them into COs. DSB formation will continue on unsynapsed axes and it is only terminated by the complete synapsis of all homolog pairs in pachytene stage. Meiocytes that fail to complete recombination and/or synapsis are eliminated (spermatocytes by mid-pachytene; oocytes from late prophase I, but at or before follicle formation), as these defects can cause chromosomal abnormalities that will be passed down to the offspring, causing severe diseases or death. Our group and others suggest that there are two simultaneous pachytene checkpoints, one that is activated by persistent DSBs and the other by asynapsis. This issue has proven very difficult to approach, since defective DSB formation/repair will inevitably affect synapsis by impairing homology search and strand invasion. The meiosis-specific HORMAD2 protein binds preferentially to unsynapsed axes, where HORMAD2 promotes the recruitment of ATR kinase. The ATR-dependent accumulation of γH2AX (histone H2AX phosphorylated on Ser139) is thought to generate a checkpoint signal from unsynapsed chromosomal regions as part of the synapsis checkpoint. In the case of spermatocytes ATR activity is concentrated to the non-homologous regions of sex chromosomes, which remain unsynapsed. This leads to γH2AX accumulation and subsequent transcriptional silencing of the sex chromosomes. This mechanism is called meiotic sex chromosome inactivation (MSCI) and is essential for the survival of spermatocytes, since the expression of sex chromosome-linked genes is toxic to spermatocytes beyond mid pachytene. Despite the importance of HORMAD2, the mechanism for its interaction with the unsynapsed chromosomes was unknown. Furthermore, it remained untested if HORMAD2 binding to the axis is actually required for the synapsis checkpoint. To address whether HORMAD2 binding to the axis is required for its function, I generated a mouse strain that expresses an altered version of the constitutive chromosome axis component SYCP2. The mutant SYCP2Δex16 lacks a short peptide sequence, called closure motif, which is predicted to bind HORMAD2. Immunofluorescence (IF) in spermatocytes showed that the localization of HORMAD2 to the chromosome axis is lost in Sycp2Δex16/Δex16, while axis formation is not impaired. Consistently, immunoblotting showed that HORMAD2 was depleted from insoluble fractions (chromatin-rich) of Sycp2Δex16/Δex16 testis extracts, while present in the soluble and total fractions. These results confirmed that the closure motif of SYCP2 serves as anchor for HORMAD2 on meiotic chromosome axis. Sycp2Δex16/Δex16 males are infertile. IF staining of cryosections of testes showed a complete loss of spermatocytes beyond pachytene. The number of cells undergoing apoptosis increased relative to the wild type, most of them in stage IV of the epithelium cycle, which corresponds to mid-pachytene. Thus, the onset of arrest coincides with the activation of the checkpoint that prevents asynaptic/DSB repair defective meiocytes from progressing beyond mid-pachytene. Nonetheless, global SC formation, and patterns of DSB formation and recombination foci in Sycp2Δex16/Δex16 are similar to the wild type. By early pachytene, the last stage reached by Sycp2Δex16/Δex16 spermatocytes, most cells have completed synapsis and DSBs are repaired. Only a small percentage of pachytene cells show a degree of incomplete synapsis. Although this suggests a synapsis-enhancing role of HORMAD2, it is thought to be concomitant to the MSCI failure observed in this mutant. In Sycp2Δex16/Δex16, 83.7% of pachytene spermatocytes have an abnormal distribution of γH2AX on sex chromosomes, accompanied by impaired accumulation of ATR and BRCA1 (another MSCI-promoting protein). The phenotype of Sycp2Δex16/Δex16 suggests a misregulation of pachytene checkpoint functions due to loss of HORMAD2-dependent ATR signaling from unsynapsed axes, and I propose that this misregulation accounts for the meiotic arrest phenotype. Females do not require MSCI, as their XX chromosomes can synapse. Accordingly, Sycp2Δex16/Δex16 females are fertile. Nonetheless, previous reports indicate that HORMAD2 has a role in sensing asynapsis, but not persistent DSB, as part of a checkpoint for quality control in oocytes. As expected, Sycp2Δex16/Δex16 cannot rescue oocytes in a DSB repair and synapsis defective background. Sycp2Δex16/Δex16 females have higher oocyte numbers than wild type. This could suggest that oocytes with defective synapsis that are usually culled in wild type ovaries, are not eliminated in Sycp2Δex16/Δex16, supporting HORMAD2 role in signaling asynapsis. Further testing in a synapsis defective background is underway. Sycp2Δex16/Δex16 seems to phenocopy Hormad2-/-, supporting a model where HORMAD2 binding to the unsynapsed axis is required for enabling its checkpoint-activating functions.:List of figures I List of tables II List of abbreviations III Acknowledgments V 1. Introduction 1 1.1. Meiosis and gametogenesis in mouse 1 1.2. Prophase of the first meiotic division 3 1.2.1. Meiotic recombination 4 1.2.2. The chromosome axis and the synaptonemal complex 5 1.2.3. The interplay between DSBs and synaptonemal complex formation 8 1.3. Surveillance mechanisms in prophase I 9 1.3.1. HORMAD1 and HORMAD2 in the male pachytene checkpoint 11 1.3.2. HORMAD1 and HORMAD2 in the female prophase checkpoint 12 1.4. HORMAD1/2 interaction with the chromosome axis 14 1.5. Aim of the project 15 2. Materials and methods 16 2.1. Generation of a mouse model lacking the closure motif 16 2.1.1. Generation of a mutant using CRISPR-Cas9 system 16 2.1.2. Identification of mutant candidates by PCR and HMA 17 2.1.3. Genomic DNA sequencing for the selection of founders 19 2.1.4. cDNA sequencing 20 2.2. Mice 21 2.2.1 Mice from a homogenized genetic background 21 2.3. Methods for the fixation of tissues 22 2.3.1. Nuclear surface spread spermatocytes 22 2.3.2. No-spin sucrose spreads 23 2.3.3. Testis fixation and cryosectioning 23 2.3.4. Ovary fixation and cryosectioning 23 2.3.4. Fixed Wild Type Cells (FWTC) 24 2.4 Staging 24 2.4.1. Staging of spermatocytes from nuclear surface spreads 24 2.4.2. Staging of epithelial cycles in cross-sections of seminiferous tubules 25 2.4.3. Staging of oocytes 26 2.5. Protein extraction 27 2.5.1. Total protein extraction and western blotting 27 2.5.2. Fractionation assay and western blotting 27 2.6. Immunofluorescence microscopy 29 2.6.1. Staining conditions 29 2.6.2. Imaging 30 3. Results 31 3.1. HORMAD2 recruitment to the axis requires the closure motif of SYCP2 31 3.1.1. Generation of a mouse mutant lacking the closure motif 31 3.1.2. Axial HORMAD2 localization is lost in Sycp2Δex16/Δex16 spermatocytes 34 3.1.3. HORMAD2 is present in Sycp2Δex16/Δex16 spermatocytes but greatly decreased from the chromatin-rich fraction 36 3.2. HORMAD1 localization to the axis seems to be independent of the closure motif of SYCP2 37 3.3. Loss of HORMAD2 localization to the axis causes infertility in males 38 3.3.1. Low levels of H1t signal indicate that Sycp2Δex16/Δex16 spermatocytes do not progress beyond mid-pachytene 39 3.3.2. Defect in spermatogenesis of Sycp2Δex16/Δex16 is caused by mid-pachytene arrest 41 3.4. Axis formation is not impaired in Sycp2Δex16/Δex16 spermatocytes 44 3.5. HORMAD2 localization to the axis plays a minor role in SC formation 45 3.5.1. In Sycp2Δex16/Δex16 spermatocytes, autosomal SC formation does not seem impaired, but abnormal XY synapsis is observed 45 3.5.2. IHO1 is properly removed from axes in Sycp2Δex16/Δex16 and Hormad2-/- spermatocytes 48 3.5.3. Synapsis at early pachytene is mildly defective in Sycp2Δex16/Δex16 and Hormad2-/- spermatocytes 50 3.6. HORMAD2 localization to the axis is not essential for DSB repair 52 3.7. Loss of HORMAD2-dependent ATR signaling from unsynapsed axes causes a misregulation of pachytene checkpoint 55 3.7.1. Abnormal sex body formation in Sycp2Δex16/Δex16 is an indicator of defective ATR signaling on unsynapsed sex chromosomes 55 3.7.2. ATR localization is affected by the loss of HORMAD2 localization to the axes 58 3.8. HORMAD2 seems to be required for a checkpoint mechanism in females that is activated by persistent asynapsis 60 3.8.1. HORMAD2 is not essential for fertility in females 60 3.8.3. HORMAD2 localization to the axis is not required for the elimination of oocytes with persistent DSB 60 4. Discussion 63 4.1. The closure motif in SYCP2 is the binding site of HORMAD2 to the unsynapsed axes 63 4.1.1 The role of HORMAD1 in the recruitment of HORMAD2 to the chromosome axis 64 4.2. Loss of HORMAD2 localization to the axes causes mid-pachytene arrest 65 4.2.1 Persistent DSBs and/or asynapsis are unlikely to trigger the mid-pachytene arrest in Sycp2Δex16/Δex16 spermatocytes 66 4.3. Loss of HORMAD2 from the unsynapsed axes impairs the recruitment of ATR activity on sex chromosomes, triggering the elimination by the mid-pachytene checkpoint 67 4.3.1 Increased asynapsis of sex chromosomes is most likely an effect of defective ATR signaling 68 4.3.2 ATR-dependent silencing of sex chromosomes is required for survival of spermatocytes beyond pachytene 69 4.4. HORMAD2 localization to the axis has a limited role in the prophase checkpoint in females 69 4.4.1 HORMAD2 seems to be required for a checkpoint mechanism in females that is activated by persistent asynapsis 70 4.4.2 HORMAD2 localization to the axis is not required for the elimination of oocytes with persistent DSB 70 4.4.3 HORMAD2 might play a role in the checkpoint that monitors unrepaired DSBs during female prophase 71 4.5. Final remarks 72 5. Summary 73 5. Zusammenfassung 75 References 78

Ion channels and pumps play critical roles during sea urchin development including mediating the blocks to polyspermy, regulating left-right and dorsal-ventral axis specification, directing ventral PMC migration, and controlling biomineralization of the larval skeleton. We performed a screen of pharmacological ion channel inhibitors, and we chose two inhibitors to investigate further. First, we found that tricaine, a potent inhibitor of voltage-gated sodium channels (VGSCs), induces aberrant skeletal patterning in Lytechinus variegatus larvae. The larval skeleton is secreted by the primary mesenchyme cells (PMCs), which migrate within the blastocoel into a stereotypical pattern. We show that VGSC activity is required for normal PMC migration and skeletal patterning. Timed inhibitor studies identified VGSC activity as specifically required from early gastrula to the onset of late gastrula for normal skeletal patterning. Tricaine inhibits the voltage-gated sodium channel LvScn5a which is strongly expressed in the developing nervous system in pluteus larvae. We found that exogenous expression of an anesthetic-insensitive version of LvScn5a is sufficient to rescue hallmark tricaine-mediated skeletal patterning defects, demonstrating the specificity of the inhibitor. LvScn5a exhibits a ventrolateral ectodermal expression domain in gastrulating embryos that is spatiotemporally congruent with triradiate formation in the ventrolateral PMC clusters at the onset of skeletogenesis. This ectodermal territory normally expresses the patterning cue Wnt5, and we find that the expression of Wnt5 is dramatically spatially expanded by tricaine treatment. We also observe ectopic PMC clusters in tricaine-treated embryos. We found that knockdown of Wnt5 expression is sufficient to rescue tricaine-mediated skeletal patterning defects. These results are consistent with a model in which LvScn5a activity in the ventrolateral ectoderm functions to spatially restrict the expression of the ectodermal patterning cue Wnt5 that in turn induces PMC cluster formation. Together, these findings show that spatially restricted sodium channel activity regulates ectodermal cue expression that, in turn, regulates PMC differentiation and skeletal morphogenesis. Second, we show that V-type H⁺ ATPase (VHA) activity is required for specification of the dorsal-ventral (DV) axis. DV specification is controlled by the TGF-β signal Nodal that specifies the ventral territory and indirectly activates dorsal specification via induction of BMP 2/4 expression. Nodal expression occurs downstream of p38 MAPK, which is transiently, asymmetrically inactive on the presumptive dorsal side of the blastula embryo. VHA activity is required for that transient inactivation of p38 MAPK, and it is required for the subsequent spatial restriction of Nodal expression. We show that VHA inhibition is sufficient to induce global Nodal expression during the blastula stage, resulting in ventralization of the embryo. We show that this phenotype can be rescued by experimentally imposing asymmetric Nodal expression at the 4-cell stage. We discover a VHA-dependent voltage gradient across the DV axis and find that VHA activity is required for hypoxia inducible factor (HIF) activation. We show that neither hyperpolarization nor HIF activation is sufficient to perturb DV specification, which implicates a third unknown pathway connecting VHA activity and p38 MAPK symmetry breaking. These results are consistent with a model in which dorsal VHA activity is required to inhibit Nodal expression and signaling, potentially via dorsal p38 MAPK inhibition. Together, these studies demonstrate that ion channels are required for both DV specification and for normal skeletal patterning.