Frem1 activity is regulated by Sonic hedgehog signaling in the cranial neural crest mesenchyme during midfacial morphogenesis.

BACKGROUND: Frem1 has been linked to human face shape variation, dysmorphology, and malformation, but little is known about its regulation and biological role in facial development. RESULTS: During midfacial morphogenesis in mice, we observed Frem1 expression in the embryonic growth centers that form the median upper lip, nose, and palate. Expansive spatial gradients of Frem1 expression in the cranial neural crest cell (cNCC) mesenchyme of these tissues suggested transcriptional regulation by a secreted morphogen. Accordingly, Frem1 expression paralleled that of the conserved Sonic Hedgehog (Shh) target gene Gli1 in the cNCC mesenchyme. Suggesting direct transcriptional regulation by Shh signaling, we found that Frem1 expression is induced by SHH ligand stimulation or downstream pathway activation in cNCCs and observed GLI transcription factor binding at the Frem1 transcriptional start site during midfacial morphogenesis. Finally, we found that FREM1 is sufficient to induce cNCC proliferation in a concentration-dependent manner and that Shh pathway antagonism reduces Frem1 expression during pathogenesis of midfacial hypoplasia. CONCLUSIONS: By demonstrating that the Shh signaling pathway regulates Frem1 expression in cNCCs, these findings provide novel insight into the mechanisms underlying variation in midfacial morphogenesis.

TurboID functions as an efficient biotin ligase for BioID applications in Xenopus embryos.

BioID is a proximity labeling strategy whose goal is to identify in vivo protein-protein interactions. The central components of this strategy are modified biotin ligase enzymes that promiscuously add biotin groups to proteins in close proximity. The transferred biotin group provides a powerful tag for purification and thus identification of interacting proteins. While a variety of modified biotin ligases were created for BioID, the original enzymes were inefficient, required long incubation times, and high intracellular biotin concentrations for protein labeling. These limitations hinder the application of BioID in contexts such as developing embryos where processes such as cell division and cell fate decisions occur rapidly. Recently, a new biotin ligase called TurboID was developed that addressed many of the deficiencies of previous enzymes. In this paper we compare TurboID to the BioID2 biotin ligase in developing Xenopus embryos. We find that the TurboID enzyme has several advantages over the BioID2 enzyme. TurboID labels proteins efficiently without the addition of additional biotin and occurs at a range of temperatures compatible with the culturing of Xenopus embryos. Biotinylation events occurred rapidly and were limited by TurboID expression and not its activity. Thus, TurboID is an efficient tool for BioID applications in Xenopus embryos and its use should facilitate the identification of interacting proteins in specific networks and complexes during Xenopus development.

A single KH domain in Bicaudal-C links mRNA binding and translational repression functions to maternal development.

Bicaudal-C (Bicc1) is a conserved RNA-binding protein that represses the translation of selected mRNAs to control development. In Xenopus embryos, Bicc1 binds and represses specific maternal mRNAs to control anterior-posterior cell fates. However, it is not known how Bicc1 binds its RNA targets or how binding affects Bicc1-dependent embryogenesis. Focusing on the KH domains, we analyzed Bicc1 mutants for their ability to bind RNA substrates in vivo and in vitro Analyses of these Bicc1 mutants demonstrated that a single KH domain, KH2, was crucial for RNA binding in vivo and in vitro, while the KH1 and KH3 domains contributed minimally. The Bicc1 mutants were also assayed for their ability to repress translation, and results mirrored the RNA-binding data, with KH2 being the only domain essential for repression. Finally, maternal knockdown and rescue experiments indicated that the KH domains were essential for the regulation of embryogenesis by Bicc1. These data advance our understanding of how Bicc1 selects target mRNAs and provide the first direct evidence that the RNA binding functions of Bicc1 are essential for both Bicc1-dependent translational repression and maternal vertebrate development.

A gradient of maternal Bicaudal-C controls vertebrate embryogenesis via translational repression of mRNAs encoding cell fate regulators.

Vertebrate Bicaudal-C (Bicc1) has important biological roles in the formation and homeostasis of multiple organs, but direct experiments to address the role of maternal Bicc1 in early vertebrate embryogenesis have not been reported. Here, we use antisense phosphorothioate-modified oligonucleotides and the host-transfer technique to eliminate specifically maternal stores of both bicc1 mRNA and Bicc1 protein from Xenopus laevis eggs. Fertilization of these Bicc1-depleted eggs produced embryos with an excess of dorsal-anterior structures and overexpressed organizer-specific genes, indicating that maternal Bicc1 is crucial for normal embryonic patterning of the vertebrate embryo. Bicc1 is an RNA-binding protein with robust translational repression function. Here, we show that the maternal mRNA encoding the cell-fate regulatory protein Wnt11b is a direct target of Bicc1-mediated repression. It is well established that the Wnt signaling pathway is crucial to vertebrate embryogenesis. Thus, the work presented here links the molecular function of Bicc1 in mRNA target-specific translation repression to its biological role in the maternally controlled stages of vertebrate embryogenesis.

Limiting Ago protein restricts RNAi and microRNA biogenesis during early development in Xenopus laevis.

We show that, in Xenopus laevis oocytes and early embryos, double-stranded exogenous siRNAs cannot function as microRNA (miRNA) mimics in either deadenylation or guided mRNA cleavage (RNAi). Instead, siRNAs saturate and inactivate maternal Argonaute (Ago) proteins, which are present in low amounts but are needed for Dicer processing of pre-miRNAs at the midblastula transition (MBT). Consequently, siRNAs impair accumulation of newly made miRNAs, such as the abundant embryonic pre-miR-427, but inhibition dissipates upon synthesis of zygotic Ago proteins after MBT. These effects of siRNAs, which are independent of sequence, result in morphological defects at later stages of development. The expression of any of several exogenous human Ago proteins, including catalytically inactive Ago2 (Ago2mut), can overcome the siRNA-mediated inhibition of miR-427 biogenesis and function. However, expression of wild-type, catalytically active hAgo2 is required to elicit RNAi in both early embryos and oocytes using either siRNA or endogenous miRNAs as guides. The lack of endogenous Ago2 endonuclease activity explains why these cells normally are unable to support RNAi. Expression of catalytically active exogenous Ago2, which appears not to perturb normal Xenopus embryonic development, can now be exploited for RNAi in this vertebrate model organism.

Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development.

The selective translation of maternal mRNAs encoding cell-fate determinants drives the earliest decisions of embryogenesis that establish the vertebrate body plan. This chapter will discuss studies in Xenopus laevis that provide insights into mechanisms underlying this translational control. Xenopus has been a powerful model organism for many discoveries relevant to the translational control of maternal mRNAs because of the large size of its oocytes and eggs that allow for microinjection of molecules and the relative ease of manipulating the oocyte to egg transition (maturation) and fertilization in culture. Consequently, many key studies have focused on the expression of maternal mRNAs during the oocyte to egg transition (the meiotic cell cycle) and the rapid cell divisions immediately following fertilization. This research has made seminal contributions to our understanding of translational regulatory mechanisms, but while some of the mRNAs under consideration at these stages encode cell-fate determinants, many encode cell cycle regulatory proteins that drive these early cell cycles. In contrast, while maternal mRNAs encoding key developmental (i.e., cell-fate) regulators that function after the first cleavage stages may exploit aspects of these foundational mechanisms, studies reveal that these mRNAs must also rely on distinct and, as of yet, incompletely understood mechanisms. These findings are logical because the functions of such developmental regulatory proteins have requirements distinct from cell cycle regulators, including becoming relevant only after fertilization and then only in specific cells of the embryo. Indeed, key maternal cell-fate determinants must be made available in exquisitely precise amounts (usually low), only at specific times and in specific cells during embryogenesis. To provide an appreciation for the regulation of maternal cell-fate determinant expression, an overview of the maternal phase of Xenopus embryogenesis will be presented. This section will be followed by a review of translational mechanisms operating in oocytes, eggs, and early cleavage-stage embryos and conclude with a discussion of how the regulation of key maternal cell-fate determinants at the level of translation functions in Xenopus embryogenesis. A key theme is that the molecular asymmetries critical for forming the body axes are established and further elaborated upon by the selective temporal and spatial regulation of maternal mRNA translation.

Important Role of Asparagines in Coupling the Pore and Votage-Sensor Domain in Voltage-Gated Sodium Channels.

Voltage-gated sodium (NaV) channels contain an α-subunit incorporating the channel's pore and gating machinery composed of four homologous domains (DI-DIV), with a pore domain formed by the S5 and S6 segments and a voltage-sensor domain formed by the S1-S4 segments. During a membrane depolarization movement, the S4s in the voltage-sensor domains exert downstream effects on the S6 segments to control ionic conductance through the pore domain. We used lidocaine, a local anesthetic and antiarrhythmic drug, to probe the role of conserved Asn residues in the S6s of DIII and DIV in NaV1.5 and NaV1.4. Previous studies have shown that lidocaine binding to the pore domain causes a decrease in the maximum gating (Qmax) charge of ∼38%, and three-fourths of this decrease results from the complete stabilization of DIII-S4 (contributing a 30% reduction in Qmax) and one-fourth is due to partial stabilization of DIV-S4 (a reduction of 8-10%). Even though substitutions for the Asn in DIV-S6 in NaV1.5, N1764A and N1764C, produce little ionic current in transfected mammalian cells, they both express robust gating currents. Anthopleurin-A toxin, which inhibits movement of DIV-S4, still reduced Qmax by nearly 30%, a value similar to that observed in wild-type channels, in both N1764A and N1764C. By applying lidocaine and measuring the gating currents, we demonstrated that Asn residues in the S6s of DIII and DIV are important for coupling their pore domains to their voltage-sensor domains, and that Ala and Cys substitutions for Asn in both S6s result in uncoupling of the pore domains from their voltage-sensor domains. Similar observations were made for NaV1.4, although substitutions for Asn in DIII-S6 showed somewhat less uncoupling.

Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

Bicaudal-C spatially controls translation of vertebrate maternal mRNAs.

The Xenopus Cripto-1 protein is confined to the cells of the animal hemisphere during early embryogenesis where it regulates the formation of anterior structures. Cripto-1 protein accumulates only in animal cells because cripto-1 mRNA in cells of the vegetal hemisphere is translationally repressed. Here, we show that the RNA binding protein, Bicaudal-C (Bic-C), functioned directly in this vegetal cell-specific repression. While Bic-C protein is normally confined to vegetal cells, ectopic expression of Bic-C in animal cells repressed a cripto-1 mRNA reporter and associated with endogenous cripto-1 mRNA. Repression by Bic-C required its N-terminal domain, comprised of multiple KH motifs, for specific binding to relevant control elements within the cripto-1 mRNA and a functionally separable C-terminal translation repression domain. Bic-C-mediated repression required the 5' CAP and translation initiation factors, but not a poly(A) tail or the conserved SAM domain within Bic-C. Bic-C-directed immunoprecipitation followed by deep sequencing of associated mRNAs identified multiple Bic-C-regulated mRNA targets, including cripto-1 mRNA, providing new insights and tools for understanding the role of Bic-C in vertebrate development.

Poly(A)-binding proteins are functionally distinct and have essential roles during vertebrate development.

Translational control of many mRNAs in developing metazoan embryos is achieved by alterations in their poly(A) tail length. A family of cytoplasmic poly(A)-binding proteins (PABPs) bind the poly(A) tail and can regulate mRNA translation and stability. However, despite the extensive biochemical characterization of one family member (PABP1), surprisingly little is known about their in vivo roles or functional relatedness. Because no information is available in vertebrates, we address their biological roles, establishing that each of the cytoplasmic PABPs conserved in Xenopus laevis [PABP1, embryonic PABP (ePABP), and PABP4] is essential for normal development. Morpholino-mediated knockdown of PABP1 or ePABP causes both anterior and posterior phenotypes and embryonic lethality. In contrast, depletion of PABP4 results mainly in anterior defects and lethality at later stages. Unexpectedly, cross-rescue experiments reveal that neither ePABP nor PABP4 can fully rescue PABP1 depletion, establishing that PABPs have distinct functions. Comparative analysis of the uncharacterized PABP4 with PABP1 and ePABP shows that it shares a mechanistically conserved core role in promoting global translation. Consistent with this analysis, each morphant displays protein synthesis defects, suggesting that their roles in mRNA-specific translational regulation and/or mRNA decay, rather than global translation, underlie the functional differences between PABPs. Domain-swap experiments reveal that the basis of the functional specificity is complex, involving multiple domains of PABPs, and is conferred, at least in part, by protein-protein interactions.

Light-inducible protein degradation in E. coli with the LOVdeg tag.

Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light-controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVdeg, a tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVdeg by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVdeg tag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVdeg system. Finally, we use the LOVdeg tag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVdeg tag system and introduce a powerful new tool for bacterial optogenetics.

An ALK2 inhibitor, BLU-782, prevents heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.

Transcriptional integration of Wnt and Nodal pathways in establishment of the Spemann organizer.

Signaling inputs from multiple pathways are essential for the establishment of distinct cell and tissue types in the embryo. Therefore, multiple signals must be integrated to activate gene expression and confer cell fate, but little is known about how this occurs at the level of target gene promoters. During early embryogenesis, Wnt and Nodal signals are required for formation of the Spemann organizer, which is essential for germ layer patterning and axis formation. Signaling by both Wnt and Nodal pathways is required for the expression of multiple organizer genes, suggesting that integration of these signals is required for organizer formation. Here, we demonstrate transcriptional cooperation between the Wnt and Nodal pathways in the activation of the organizer genes Goosecoid (Gsc), Cerberus (Cer), and Chordin (Chd). Combined Wnt and Nodal signaling synergistically activates transcription of these organizer genes. Effectors of both pathways occupy the Gsc, Cer and Chd promoters and effector occupancy is enhanced with active Wnt and Nodal signaling. This suggests that, at organizer gene promoters, a stable transcriptional complex containing effectors of both pathways forms in response to combined Wnt and Nodal signaling. Consistent with this idea, the histone acetyltransferase p300 is recruited to organizer promoters in a Wnt and Nodal effector-dependent manner. Taken together, these results offer a mechanism for spatial and temporal restriction of organizer gene transcription by the integration of two major signaling pathways, thus establishing the Spemann organizer domain.

Outward stabilization of the voltage sensor in domain II but not domain I speeds inactivation of voltage-gated sodium channels.

To determine the roles of the individual S4 segments in domains I and II to activation and inactivation kinetics of sodium current (INa) in NaV1.5, we used a tethered biotin and avidin approach after a site-directed cysteine substitution was made in the second outermost Arg in each S4 (DI-R2C and DII-R2C). We first determined the fraction of gating charge contributed by the individual S4's to maximal gating current (Qmax), and found that the outermost Arg residue in each S4 contributed ∼19% to Qmax with minimal contributions by other arginines. Stabilization of the S4's in DI-R2C and DII-R2C was confirmed by measuring the expected reduction in Qmax. In DI-R2C, stabilization resulted in a decrease in peak INa of ∼45%, while its peak current-voltage (I-V) and voltage-dependent Na channel availability (SSI) curves were nearly unchanged from wild type (WT). In contrast, stabilization of the DII-R2C enhanced activation with a negative shift in the peak I-V relationship by -7 mV and a larger -17 mV shift in the voltage-dependent SSI curve. Furthermore, its INa decay time constants and time-to-peak INa became more rapid than WT. An explanation for these results is that the depolarized conformation of DII-S4, but not DI-S4, affects the receptor for the inactivation particle formed by the interdomain linker between DIII and IV. In addition, the leftward shifts of both activation and inactivation and the decrease in Gmax after stabilization of the DII-S4 support previous studies that showed ß-scorpion toxins trap the voltage sensor of DII in an activated conformation.

Inhibition of Btk with CC-292 provides early pharmacodynamic assessment of activity in mice and humans.

Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in autoimmune disease and B cell malignancies. Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development and activation through the BCR signaling pathway and represents a new target for diseases characterized by inappropriate B cell activity. N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292) is a highly selective, covalent Btk inhibitor and a sensitive and quantitative assay that measures CC-292-Btk engagement has been developed. This translational pharmacodynamic assay has accompanied CC-292 through each step of drug discovery and development. These studies demonstrate the quantity of Btk bound by CC-292 correlates with the efficacy of CC-292 in vitro and in the collagen-induced arthritis model of autoimmune disease. Recently, CC-292 has entered human clinical trials with a trial design that has provided rapid insight into safety, pharmacokinetics, and pharmacodynamics. This first-in-human healthy volunteer trial has demonstrated that a single oral dose of 2 mg/kg CC-292 consistently engaged all circulating Btk protein and provides the basis for rational dose selection in future clinical trials. This targeted covalent drug design approach has enabled the discovery and early clinical development of CC-292 and has provided support for Btk as a valuable drug target for B-cell mediated disorders.

Selective irreversible inhibition of a protease by targeting a noncatalytic cysteine.

Designing selective inhibitors of proteases has proven problematic, in part because pharmacophores that confer potency exploit the conserved catalytic apparatus. We developed a fundamentally different approach by designing irreversible inhibitors that target noncatalytic cysteines that are structurally unique to a target in a protein family. We have successfully applied this approach to the important therapeutic target HCV protease, which has broad implications for the design of other selective protease inhibitors.

Embryonic poly(A)-binding protein (ePAB) phosphorylation is required for Xenopus oocyte maturation.

Oocyte maturation and early embryonic development require the cytoplasmic polyadenylation and concomitant translational activation of stored maternal mRNAs. ePAB [embryonic poly(A)-binding protein, also known as ePABP and PABPc1-like] is a multifunctional post-transcriptional regulator that binds to poly(A) tails. In the present study we find that ePAB is a dynamically modified phosphoprotein in Xenopus laevis oocytes and show by mutation that phosphorylation at a four residue cluster is required for oocyte maturation. We further demonstrate that these phosphorylations are critical for cytoplasmic polyadenylation, but not for ePAB's inherent ability to promote translation. Our results provide the first insight into the role of post-translational modifications in regulating PABP protein activity in vivo.

An optogenetic toolkit for light-inducible antibiotic resistance.

Antibiotics are a key control mechanism for synthetic biology and microbiology. Resistance genes are used to select desired cells and regulate bacterial populations, however their use to-date has been largely static. Precise spatiotemporal control of antibiotic resistance could enable a wide variety of applications that require dynamic control of susceptibility and survival. Here, we use light-inducible Cre recombinase to activate expression of drug resistance genes in Escherichia coli. We demonstrate light-activated resistance to four antibiotics: carbenicillin, kanamycin, chloramphenicol, and tetracycline. Cells exposed to blue light survive in the presence of lethal antibiotic concentrations, while those kept in the dark do not. To optimize resistance induction, we vary promoter, ribosome binding site, and enzyme variant strength using chromosome and plasmid-based constructs. We then link inducible resistance to expression of a heterologous fatty acid enzyme to increase production of octanoic acid. These optogenetic resistance tools pave the way for spatiotemporal control of cell survival.

Light inducible protein degradation in E. coli with the LOVdeg tag.

Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVdeg, a tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVdeg by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVdeg tag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVdeg system. Finally, we use the LOVdeg tag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVdeg tag system, and introduce a powerful new tool for bacterial optogenetics.

Introduction to Engineering Biology: A Conceptual Framework for Teaching Synthetic Biology.

As the impacts of engineering biology grow, it is important to introduce the field early and in an accessible way. However, teaching engineering biology poses challenges, such as limited representation of the field in widely used scientific textbooks or curricula, and the interdisciplinary nature of the subject. We have created an adaptable curriculum module that can be used by anyone to teach the basic principles and applications of engineering biology. The module consists of a versatile, concept-based slide deck designed by experts across engineering biology to cover key topic areas. Starting with the design, build, test, and learn cycle, the slide deck covers the framework, core tools, and applications of the field at an undergraduate level. The module is available for free on a public website and can be used in a stand-alone fashion or incorporated into existing curricular materials. Our aim is that this modular, accessible slide deck will improve the ease of teaching current engineering biology topics and increase public engagement with the field.