mem-iLID, a fast and economic protein purification method.

Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

Functional assessment of the "two-hit" model for neurodevelopmental defects in Drosophila and X. laevis.

We previously identified a deletion on chromosome 16p12.1 that is mostly inherited and associated with multiple neurodevelopmental outcomes, where severely affected probands carried an excess of rare pathogenic variants compared to mildly affected carrier parents. We hypothesized that the 16p12.1 deletion sensitizes the genome for disease, while "second-hits" in the genetic background modulate the phenotypic trajectory. To test this model, we examined how neurodevelopmental defects conferred by knockdown of individual 16p12.1 homologs are modulated by simultaneous knockdown of homologs of "second-hit" genes in Drosophila melanogaster and Xenopus laevis. We observed that knockdown of 16p12.1 homologs affect multiple phenotypic domains, leading to delayed developmental timing, seizure susceptibility, brain alterations, abnormal dendrite and axonal morphology, and cellular proliferation defects. Compared to genes within the 16p11.2 deletion, which has higher de novo occurrence, 16p12.1 homologs were less likely to interact with each other in Drosophila models or a human brain-specific interaction network, suggesting that interactions with "second-hit" genes may confer higher impact towards neurodevelopmental phenotypes. Assessment of 212 pairwise interactions in Drosophila between 16p12.1 homologs and 76 homologs of patient-specific "second-hit" genes (such as ARID1B and CACNA1A), genes within neurodevelopmental pathways (such as PTEN and UBE3A), and transcriptomic targets (such as DSCAM and TRRAP) identified genetic interactions in 63% of the tested pairs. In 11 out of 15 families, patient-specific "second-hits" enhanced or suppressed the phenotypic effects of one or many 16p12.1 homologs in 32/96 pairwise combinations tested. In fact, homologs of SETD5 synergistically interacted with homologs of MOSMO in both Drosophila and X. laevis, leading to modified cellular and brain phenotypes, as well as axon outgrowth defects that were not observed with knockdown of either individual homolog. Our results suggest that several 16p12.1 genes sensitize the genome towards neurodevelopmental defects, and complex interactions with "second-hit" genes determine the ultimate phenotypic manifestation.

Dissociation Process of a MDM2/p53 Complex Investigated by Parallel Cascade Selection Molecular Dynamics and the Markov State Model.

Recently, we efficiently generated dissociation pathways of a protein-ligand complex without applying force bias with parallel cascade selection molecular dynamics (PaCS-MD) and showed that PaCS-MD in combination with the Markov state model (MSM) yielded a binding free energy comparable to experimental values. In this work, we applied the same procedure to a complex of MDM2 protein and the transactivation domain of p53 protein (TAD-p53), the latter of which is known to be very flexible in the unbound state. Using 30 independent MD simulations in PaCS-MD, we successfully generated 25 dissociation pathways of the complex, which showed complete or partial unfolding of the helical region of TAD-p53 during the dissociation process within an average simulation time of 154.8 ± 46.4 ns. The standard binding free energy obtained in combination with one-dimensional-, three-dimensional (3D)- or Cα-MSM was in good agreement with those determined experimentally. Using 3D-MSM based on the center of mass position of TAD-p53 relative to MDM2, the dissociation rate constant was calculated, which was comparable to those measured experimentally. Cα-MSM based on all Cα coordinates of TAD-p53 reproduced the experimentally measured standard binding free energy, and dissociation and association rate constants. We conclude that the combination of PaCS-MD and MSM offers an efficient computational procedure to calculate binding free energies and kinetic rates.

Promotora assisted depression and self-care management among predominantly Latinos with concurrent chronic illness: Safety net care system clinical trial results.

The study evaluated depression and self-care management among patients with diabetes and/or heart disease in a 12-month randomized trial conducted in Los Angeles County Department of Health Services (LAC-DHS) community clinics. We compared LAC-DHS clinic usual care (UC) versus A-Helping-Hand (AHH) intervention in which bilingual promotoras, hired and supervised by the research project, provided 6 weekly psychoeducational sessions followed by boosters. Of 1957 screened, 348 depressed patients (PHQ-9 score≥10) were enrolled, randomized to AHH (n=178) or UC (n=170) after baseline interview assessing mental health, treatment receipt, co-morbid illness, self-care management, and environmental stressors. Comprehensive assessments were repeated at 6 and 12months by an independent interviewer blind to the study group. Patients (85% diabetes, 4% heart disease, 11% both) were predominantly female (85%), Latino (99%), born outside of the US (91%). Study attrition at 12months was 30% (AHH 31%, UC 28%, P=0.51). No baseline characteristics were associated with attrition. Half of AHH patients received 4 or more sessions. Intend-to-treat analysis found study groups did not vary significantly at 6 and 12months. Before-after paired t-tests showed significant improvements in most measures in each group. During the trial, LAC-DHS activated healthcare improvements including depression screening, referral to clinic staff including community health workers (with the same role as the promotoras) to improve patient care management. Both patient groups performed equally well which may be a function of the enhanced healthcare model. Future research should replicate the promotora-integrated care model with other groups and care settings with similar comorbid conditions.

Single-Molecule Investigations on Histone H2A-H2B Dynamics in the Nucleosome.

Nucleosomes impose physical barriers to DNA-templated processes, playing important roles in eukaryotic gene regulation. DNA is packaged into nucleosomes by histone proteins mainly through strong electrostatic interactions that can be modulated by various post-translational histone modifications. Investigating the dynamics of histone dissociation from the nucleosome and how it is altered upon histone modifications is important for understanding eukaryotic gene regulation mechanisms. In particular, histone H2A-H2B dimer displacement in the nucleosome is one of the most important and earliest steps of histone dissociation. Two conflicting hypotheses on the requirement for dimer displacement are that nucleosomal DNA needs to be unwrapped before a dimer can displace and that a dimer can displace without DNA unwrapping. In order to test the hypotheses, we employed three-color single-molecule FRET and monitored in a time-resolved manner the early kinetics of H2A-H2B dimer dissociation triggered by high salt concentration and by histone chaperone Nap1. The results reveal that dimer displacement requires DNA unwrapping in the vast majority of the nucleosomes in the salt-induced case, while dimer displacement precedes DNA unwrapping in >60% of the nucleosomes in the Nap1-mediated case. We also found that acetylation at histone H4K16 or H3K56 affects the kinetics of Nap1-mediated dimer dissociation and facilitates the process both kinetically and thermodynamically. On the basis of these results, we suggest a mechanism by which histone chaperone facilitates H2A-H2B dimer displacement from the histone core without requiring another factor to unwrap the nucleosomal DNA.

Custos controls ß-catenin to regulate head development during vertebrate embryogenesis.

Precise control of the canonical Wnt pathway is crucial in embryogenesis and all stages of life, and dysregulation of this pathway is implicated in many human diseases including cancers and birth defect disorders. A key aspect of canonical Wnt signaling is the cytoplasmic to nuclear translocation of ß-catenin, a process that remains incompletely understood. Here we report the identification of a previously undescribed component of the canonical Wnt signaling pathway termed Custos, originally isolated as a Dishevelled-interacting protein. Custos contains casein kinase phosphorylation sites and nuclear localization sequences. In Xenopus, custos mRNA is expressed maternally and then widely throughout embryogenesis. Depletion or overexpression of Custos produced defective anterior head structures by inhibiting the formation of the Spemann-Mangold organizer. In addition, Custos expression blocked secondary axis induction by positive signaling components of the canonical Wnt pathway and inhibited ß-catenin/TCF-dependent transcription. Custos binds to ß-catenin in a Wnt responsive manner without affecting its stability, but rather modulates the cytoplasmic to nuclear translocation of ß-catenin. This effect on nuclear import appears to be the mechanism by which Custos inhibits canonical Wnt signaling. The function of Custos is conserved as loss-of-function and gain-of-function studies in zebrafish also demonstrate a role for Custos in anterior head development. Our studies suggest a role for Custos in fine-tuning canonical Wnt signal transduction during embryogenesis, adding an additional layer of regulatory control in the Wnt-ß-catenin signal transduction cascade.

Functional clustering of immunoglobulin superfamily proteins with protein-protein interaction information calibrated hidden Markov model sequence profiles.

Secreted and cell-surface-localized members of the immunoglobulin superfamily (IgSF) play central roles in regulating adaptive and innate immune responses and are prime targets for the development of protein-based therapeutics. An essential activity of the ectodomains of these proteins is the specific recognition of cognate ligands, which are often other members of the IgSF. In this work, we provide functional insight for this important class of proteins through the development of a clustering algorithm that groups together extracellular domains of the IgSF with similar binding preferences. Information from hidden Markov model-based sequence profiles and domain architecture is calibrated against manually curated protein interaction data to define functional families of IgSF proteins. The method is able to assign 82% of the 477 extracellular IgSF protein to a functional family, while the rest are either single proteins with unique function or proteins that could not be assigned with the current technology. The functional clustering of IgSF proteins generates hypotheses regarding the identification of new cognate receptor-ligand pairs and reduces the pool of possible interacting partners to a manageable level for experimental validation.

Distribution of BODIPY-labelled phosphatidylethanolamines in lipid bilayers exhibiting different curvatures.

In this paper we have investigated the behaviour of newly synthesised mono-palmitoyl- and dipalmitoyl-phosphatidylethanolamine probes (abbreviated as mPE and dPE, respectively) labelled in the polar headgroup region by either the FL-BODIPY or the 564/570-BODIPY fluorophore and solubilised in lipid systems that exhibit different curvatures. Because of the bulky BODIPY-groups, the monoacyl-form derivatives have a conic-like shape, whereas that for the diacyl derivatives is rather cylindrical. A careful analysis of time-resolved resonance energy transfer experiments by means of analytical models as well as Monte Carlo simulations shows that the mPE derivatives have a comparable affinity to highly curved bilayer regions (torroidal pores formed by magainin-2 in lipid bilayers, or the rims of discoid bicelles) and to planar bilayer regions (i.e. the flat region of lipid bilayers and bicelles). Furthermore, the monoacyl-probes are as compared to the diacyl-probes effectively closer to each other in a lipid bilayer, while none of these probes seems to be randomly distributed. Self-aggregation is most efficiently induced by the larger aromatic 564/570-BODIPY chromophore, but it is suppressed when using the diacyl instead of the monoacyl-form, and/or by attaching BODIPY-groups to the acyl-chain.

In vivo assessment and potential diagnosis of xenobiotics that perturb the thyroid pathway: Proteomic analysis of Xenopus laevis brain tissue following exposure to model T4 inhibitors.

As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression patterns in the brain of the amphibian model (Xenopus laevis) following in vivo exposure to a suite of T4 synthesis inhibitors. We specifically address the application of Two Dimensional Polyacrylamide Gel Electrophoresis (2D PAGE), Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and LC-MS/MS to assess changes in relative protein expression levels. 2D PAGE and iTRAQ proved to be effective complementary techniques for distinguishing protein changes in the developing amphibian brain in response to T4 synthesis inhibition. This information served to evaluate the use of distinctive protein profiles as a potential mechanism to screen chemicals for endocrine activity in anurans. Regulatory pathways associated with proteins expressed as a result of chemical effect are reported. To our knowledge, this is also the first account of the anuran larvae brain proteome characterization using proteomic technologies. Correlation of protein changes to other cellular and organism-level responses will aid in the development of a more rapid and cost-effective, non-mammalian screening assay for thyroid axis-disrupting chemicals.

Xenopus Sox3 activates sox2 and geminin and indirectly represses Xvent2 expression to induce neural progenitor formation at the expense of non-neural ectodermal derivatives.

The SRY-related, HMG box SoxB1 transcription factors are highly homologous, evolutionarily conserved proteins that are expressed in neuroepithelial cells throughout neural development. SoxB1 genes are down-regulated as cells exit the cell-cycle to differentiate and are considered functionally redundant in maintaining neural precursor populations. However, little is known about Sox3 function and its mode of action during primary neurogenesis. Using gain and loss-of-function studies, we analyzed Sox3 function in detail in Xenopus early neural development and compared it to that of Sox2. Through these studies we identified the first targets of a SoxB1 protein during primary neurogenesis. Sox3 functions as an activator to induce expression of the early neural genes, sox2 and geminin in the absence of protein synthesis and to indirectly inhibit the Bmp target Xvent2. As a result, Sox3 increases cell proliferation, delays neurogenesis and inhibits epidermal and neural crest formation to expand the neural plate. Our studies indicate that Sox3 and 2 have many similar functions in this process including the ability to activate expression of geminin in naïve ectodermal explants. However, there are some differences; Sox3 activates the expression of sox2, while Sox2 does not activate expression of sox3 and sox3 is uniquely expressed throughout the ectoderm prior to neural induction suggesting a role in neural competence. With morpholino-mediated knockdown of Sox3, we demonstrate that it is required for induction of neural tissue by BMP inhibition. Together these data indicate that Sox3 has multiple roles in early neural development including as a factor required for nogginmediated neural induction.

Robust, tunable biological oscillations from interlinked positive and negative feedback loops.

A simple negative feedback loop of interacting genes or proteins has the potential to generate sustained oscillations. However, many biological oscillators also have a positive feedback loop, raising the question of what advantages the extra loop imparts. Through computational studies, we show that it is generally difficult to adjust a negative feedback oscillator's frequency without compromising its amplitude, whereas with positive-plus-negative feedback, one can achieve a widely tunable frequency and near-constant amplitude. This tunability makes the latter design suitable for biological rhythms like heartbeats and cell cycles that need to provide a constant output over a range of frequencies. Positive-plus-negative oscillators also appear to be more robust and easier to evolve, rationalizing why they are found in contexts where an adjustable frequency is unimportant.

Interactions of cationic-hydrophobic peptides with lipid bilayers: a Monte Carlo simulation method.

We present a computational model of the interaction between hydrophobic cations, such as the antimicrobial peptide, Magainin2, and membranes that include anionic lipids. The peptide's amino acids were represented as two interaction sites: one corresponds to the backbone alpha-carbon and the other to the side chain. The membrane was represented as a hydrophobic profile, and its anionic nature was represented by a surface of smeared charges. Thus, the Coulombic interactions between the peptide and the membrane were calculated using the Gouy-Chapman theory that describes the electrostatic potential in the aqueous phase near the membrane. Peptide conformations and locations near the membrane, and changes in the membrane width, were sampled at random, using the Metropolis criterion, taking into account the underlying energetics. Simulations of the interactions of heptalysine and the hydrophobic-cationic peptide, Magainin2, with acidic membranes were used to calibrate the model. The calibrated model reproduced structural data and the membrane-association free energies that were measured also for other basic and hydrophobic-cationic peptides. Interestingly, amphipathic peptides, such as Magainin2, were found to adopt two main membrane-associated states. In the first, the peptide resided mostly outside the polar headgroups region. In the second, which was energetically more favorable, the peptide assumed an amphipathic-helix conformation, where its hydrophobic face was immersed in the hydrocarbon region of the membrane and the charged residues were in contact with the surface of smeared charges. This dual behavior provides a molecular interpretation of the available experimental data.

Retinoic acid signaling is essential for pancreas development and promotes endocrine at the expense of exocrine cell differentiation in Xenopus.

How and when the vertebrate endoderm is first subdivided into discrete progenitor cell populations that will give rise to the different major organs, including pancreas and liver, are only poorly understood. We have used Xenopus laevis as a model system to characterize these events, since it is particularly suited to study the early embryonic patterning in vertebrates. Our experimental results support the notion that retinoic acid (RA) functions as an essential endodermal patterning signal in Xenopus and that it acts as early as during gastrulation. As a result of RA treatment, the expression of Sonic Hedgehog (Shh), a known inhibitor of pancreas development in other vertebrate systems, is negatively regulated in the dorsal prepancreatic endoderm. Furthermore, RA is found to promote endocrine at the expense of exocrine differentiation in the dorsal pancreas, correlating with a specific inhibition of Notch signaling activities in this territory. Conversely, RA enhances exocrine marker gene expression in the ventral pancreas.

Sumoylation of Smad4, the common Smad mediator of transforming growth factor-beta family signaling.

Transforming growth factor-beta (TGF-beta) and TGF-beta-related factors regulate cell growth, differentiation, and apoptosis, and play key roles in normal development and tumorigenesis. TGF-beta family-induced changes in gene expression are mediated by serine/threonine kinase receptors at the cell surface and Smads as intracellular effectors. Receptor-activated Smads combine with a common Smad4 to translocate into the nucleus where they cooperate with other transcription factors to activate or repress transcription. The activities of the receptor-activated Smads are controlled by post-translational modifications such as phosphorylation and ubiquitylation. Here we show that Smad4 is modified by sumoylation. Sumoylation of Smad4 was enhanced by the conjugating enzyme Ubc9 and members of the PIAS family of SUMO ligases. A major sumoylation site in Smad4 was localized to Lys-159 in its linker segment with an additional site at Lys-113 in the MH-1 domain. Increased sumoylation in the presence of the PIASy E3 ligase correlated with targeting of Smad4 to subnuclear speckles that contain SUMO-1 and PIASy. Replacement of lysines 159 and 113 by arginines or increased sumoylation enhanced the stability of Smad4, and transcription in mammalian cells and Xenopus embryos. These observations suggest a role for Smad4 sumoylation in the regulation of TGF-beta signaling through Smads.

Expression of three spalt (sal) gene homologues in zebrafish embryos.

Three homologues of the Drosophilaregion-specific homeotic gene spalt (sal) have been isolated in zebrafish, sall1a, sall1b and sall3. Phylogenetic analysis of these genes against known salDNA sequences showed zebrafish sall1aand sall1b to be orthologous to other vertebrate sal-1 genes and zebrafish sall3to be orthologous to other vertebrate sal-3 genes, except Xenopus sall3. Phylogenetic reconstruction suggests that zebrafish sall1a and sall1bresulted from a gene duplication event occurring prior to the divergence of the ray-finned and lobe-finned fish lineages. Analysis of the expression pattern of the zebrafish sal genes shows that sall1a and sall3 share expression domains with both orthologous and non-orthologous vertebrate sal genes. Both are expressed in various regions of the CNS, including in primary motor neurons. Outside of the CNS, sall1a expression is observed in the otic vesicle (ear), heart and in a discrete region of the pronephric ducts. These analyses indicate that orthologies between zebrafish sal genes and other vertebrate sal genes do not imply equivalence of expression pattern and, therefore, that biological functions are not entirely conserved. However we suggest that, like other vertebrate sal genes, zebrafish sal genes have a role in neural development. Also, expression of zebrafish sall1a in the otic vesicle, heart sac and the pronephric ducts of zebrafish embryos is possibly consistent with some of the abnormalities seen in Sall1-deficient mice and in Townes-Brocks Syndrome, a human disorder which is caused by mutations in the human spalt gene SALL1.

Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells.

RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin.

A Monte Carlo study of peptide insertion into lipid bilayers: equilibrium conformations and insertion mechanisms.

The membrane insertion behavior of two peptides, Magainin2 and M2 delta, was investigated by applying the Monte Carlo simulation technique to a theoretical model. The model included many novel aspects, such as a new semi-empirical lipid bilayer model and a new set of semi-empirical transfer energies, which reproduced the experimental insertion behavior of Magainin2 and M2 delta without parameter fitting. Additionally, we have taken into account diminished internal (intramolecular) hydrogen bonding at the N- and C-termini of helical peptides. All simulations were carried out at 305 K, above the membrane thermal phase transition temperature, and at pH 7.0. The peptide equilibrium conformations are discussed for a range of bilayers with tail polarities varying from octanol-like to alkane-like. Probability distributions of the individual amino-acid-residue positions show the dynamic nature of these equilibrium conformations. Two different insertion mechanisms for M2 delta, and a translocation mechanism for Magainin2, are described. A study of the effect of bilayer thickness on M2 delta insertion suggests a critical thickness above which insertion is unfavorable. Additionally, we did not need to use an orientational potential or array of hard cylinders to persuade M2 delta to insert perpendicular to the membrane surface. Instead, we found that diminished internal hydrogen bonding in the helical conformation anchored the termini in the headgroups and resulted in a nearly perpendicular orientation.

A solvent model for simulations of peptides in bilayers. II. Membrane-spanning alpha-helices.

We describe application of the implicit solvation model (see the first paper of this series), to Monte Carlo simulations of several peptides in bilayer- and water-mimetic environments, and in vacuum. The membrane-bound peptides chosen were transmembrane segments A and B of bacteriorhodopsin, the hydrophobic segment of surfactant lipoprotein, and magainin2. Their conformations in membrane-like media are known from the experiments. Also, molecular dynamics study of surfactant lipoprotein with different explicit solvents has been reported (Kovacs, H., A. E. Mark, J. Johansson, and W. F. van Gunsteren. 1995. J. Mol. Biol. 247:808-822). The principal goal of this work is to compare the results obtained in the framework of our solvation model with available experimental and computational data. The findings could be summarized as follows: 1) structural and energetic properties of studied molecules strongly depend on the solvent; membrane-mimetic media significantly promote formation of alpha-helices capable of traversing the bilayer, whereas a polar environment destabilizes alpha-helical conformation via reduction of solvent-exposed surface area and packing; 2) the structures calculated in a membrane-like environment agree with the experimental ones; 3) noticeable differences in conformation of surfactant lipoprotein assessed via Monte Carlo simulation with implicit solvent (this work) and molecular dynamics in explicit solvent were observed; 4) in vacuo simulations do not correctly reproduce protein-membrane interactions, and hence should be avoided in modeling membrane proteins.

Dorsal-ventral patterning during neural induction in Xenopus: assessment of spinal cord regionalization with xHB9, a marker for the motor neuron region.

While the role of the notochord and floor plate in patterning the dorsal-ventral (D/V) axis of the neural tube is clearly established, relatively little is known about the earliest stages of D/V regionalization. In an effort to examine more closely the initial, preneural plate stages of regionalization along the prospective D/V neural axis, we have performed a series of explant experiments employing xHB9, a novel marker of the motor neuron region in Xenopus. Using tissue recombinants and Keller explants we show that direct mesodermal contact is both necessary and sufficient for the initial induction of xHB9 in the motor neuron region. We also show that presumptive neural plate explants removed as early as midgastrulation and cultured in isolation are already specified to express xHB9 but do so in an inappropriate spatial pattern while identical explants are specified to express the floor plate marker vhh-1 with correct spatial patterning. Our data suggest that, in addition to floor plate signaling, continued interactions with the underlying mesoderm through neural tube stages are essential for proper spatial patterning of the motor neuron region.