Production and characterization of monoclonal antibodies to Xenopus proteins.

Monoclonal antibodies are powerful and versatile tools that enable the study of proteins in diverse contexts. They are often utilized to assist with identification of subcellular localization and characterization of the function of target proteins of interest. However, because there can be considerable sequence diversity between orthologous proteins in Xenopus and mammals, antibodies produced against mouse or human proteins often do not recognize Xenopus counterparts. To address this issue, we refined existing mouse monoclonal antibody production protocols to generate antibodies against Xenopus proteins of interest. Here, we describe several approaches for the generation of useful mouse anti-Xenopus antibodies to multiple Xenopus proteins and their validation in various experimental approaches. These novel antibodies are now available to the research community through the Developmental Study Hybridoma Bank (DSHB).

Bop1 is required to establish precursor domains of craniofacial tissues.

Bop1 can promote cell proliferation and is a component of the Pes1-Bop1-WDR12 (PeBoW) complex that regulates ribosomal RNA processing and biogenesis. In embryos, however, bop1 mRNA is highly enriched in the neural plate, cranial neural crest and placodes, and potentially may interact with Six1, which also is expressed in these tissues. Recent work demonstrated that during development, Bop1 is required for establishing the size of the tadpole brain, retina and cranial cartilages, as well as controlling neural tissue gene expression levels. Herein, we extend this work by assessing the effects of Bop1 knockdown at neural plate and larval stages. Loss of Bop1 expanded neural plate gene expression domains (sox2, sox11, irx1) and reduced neural crest (foxd3, sox9), placode (six1, sox11, irx1, sox9) and epidermal (dlx5) expression domains. At larval stages, Bop1 knockdown reduced the expression of several otic vesicle genes (six1, pax2, irx1, sox9, dlx5, otx2, tbx1) and branchial arch genes that are required for chondrogenesis (sox9, tbx1, dlx5). The latter was not the result of impaired neural crest migration. Together these observations indicate that Bop1 is a multifunctional protein that in addition to its well-known role in ribosomal biogenesis functions during early development to establish the craniofacial precursor domains.

Mcrs1 is required for branchial arch and cranial cartilage development.

Mcrs1 is a multifunctional protein that is critical for many cellular processes in a wide range of cell types. Previously, we showed that Mcrs1 binds to the Six1 transcription factor and reduces the ability of the Six1-Eya1 complex to upregulate transcription, and that Mcrs1 loss-of-function leads to the expansion of several neural plate genes, reduction of neural border and pre-placodal ectoderm (PPR) genes, and pleiotropic effects on various neural crest (NC) genes. Because the affected embryonic structures give rise to several of the cranial tissues affected in Branchio-otic/Branchio-oto-renal (BOR) syndrome, herein we tested whether these gene expression changes subsequently alter the development of the proximate precursors of BOR affected structures - the otic vesicles (OV) and branchial arches (BA). We found that Mcrs1 is required for the expression of several OV genes involved in inner ear formation, patterning and otic capsule cartilage formation. Mcrs1 knockdown also reduced the expression domains of many genes expressed in the larval BA, derived from either NC or PPR, except for emx2, which was expanded. Reduced Mcrs1 also diminished the length of the expression domain of tbx1 in BA1 and BA2 and interfered with cranial NC migration from the dorsal neural tube; this subsequently resulted in defects in the morphology of lower jaw cartilages derived from BA1 and BA2, including the infrarostral, Meckel's, and ceratohyal as well as the otic capsule. These results demonstrate that Mcrs1 plays an important role in processes that lead to the formation of craniofacial cartilages and its loss results in phenotypes consistent with reduced Six1 activity associated with BOR.

MMP14 is required for delamination of chick neural crest cells independently of its catalytic activity.

Matrix metalloproteinases have a broad spectrum of substrates ranging from extracellular matrix components and adhesion molecules to chemokines and growth factors. Despite being mostly secreted, MMPs have been detected in the cytosol, the mitochondria or the nucleus. Although most of the attention is focused on their role in matrix remodeling, the diversity of their substrates and their complex trafficking open the possibility for non-canonical functions. Yet in vivo examples and experimental demonstration of the physiological relevance of such activities are rare. Here, we have used chick neural crest (NC) cells, a highly migratory stem cell population likened to invasive cancer cells, as a model for physiological epithelial-mesenchymal transition (EMT). We demonstrate that MMP14 is required for NC delamination. Interestingly, this role is independent of its cytoplasmic tail and of its catalytic activity. Our in vivo data indicate that, in addition to being a late pro-invasive factor, MMP14 is also likely to be an early player, owing to its role in EMT.

Using Xenopus to discover new candidate genes involved in BOR and other congenital hearing loss syndromes.

Hearing in infants is essential for brain development, acquisition of verbal language skills, and development of social interactions. Therefore, it is important to diagnose hearing loss soon after birth so that interventions can be provided as early as possible. Most newborns in the United States are screened for hearing deficits and commercially available next-generation sequencing hearing loss panels often can identify the causative gene, which may also identify congenital defects in other organs. One of the most prevalent autosomal dominant congenital hearing loss syndromes is branchio-oto-renal syndrome (BOR), which also presents with defects in craniofacial structures and the kidney. Currently, mutations in three genes, SIX1, SIX5, and EYA1, are known to be causative in about half of the BOR patients that have been tested. To uncover new candidate genes that could be added to congenital hearing loss genetic screens, we have combined the power of Drosophila mutants and protein biochemical assays with the embryological advantages of Xenopus, a key aquatic animal model with a high level of genomic similarity to human, to identify potential Six1 transcriptional targets and interacting proteins that play a role during otic development. We review our transcriptomic, yeast 2-hybrid, and proteomic approaches that have revealed a large number of new candidates. We also discuss how we have begun to identify how Six1 and co-factors interact to direct developmental events necessary for normal otic development.

Trim-Away mediated knock down uncovers a new function for Lbh during gastrulation of Xenopus laevis.

We previously identified the protein Lbh as necessary for cranial neural crest (CNC) cell migration in Xenopus through the use of morpholinos. However, Lbh is a maternally deposited protein and morpholinos achieve knockdowns through prevention of translation. In order to investigate the role of Lbh in earlier embryonic events, we employed the new technique "Trim-Away" to degrade this maternally deposited protein. Trim-Away utilizes the E3 ubiquitin ligase trim21 to degrade proteins targeted with an antibody and was developed in mammalian systems. Our results show that Xenopus is amenable to the Trim-Away technique. We also show that early knockdown of Lbh in Xenopus results in defects in gastrulation that present with a decrease in fibronectin matrix assembly, an increased in mesodermal cell migration and decrease in endodermal cell cohesion. We further show that the technique is also effective on a second abundant maternal protein PACSIN2. We discuss potential advantages and limit of the technique in Xenopus embryos as well as the mechanism of gastrulation inhibition.

Mcrs1 interacts with Six1 to influence early craniofacial and otic development.

The Six1 transcription factor plays a major role in craniofacial development. Mutations in SIX1 and its co-factor, EYA1, are causative for about 50% of Branchio-otic/Branchio-oto-renal syndrome (BOR) patients, who are characterized by variable craniofacial, otic and renal malformations. We previously screened for other proteins that might interact with Six1 to identify additional genes that may play a role in BOR, and herein characterize the developmental role of one of them, Microspherule protein 1 (Mcrs1). We found that in cultured cells, Mcrs1 bound to Six1 and in both cultured cells and embryonic ectoderm reduced Six1-Eya1 transcriptional activation. Knock-down of Mcrs1 in embryos caused an expansion of the domains of neural plate genes and two genes expressed in both the neural plate and neural crest (zic1, zic2). In contrast, two other genes expressed in pre-migratory neural crest (foxd3, sox9) were primarily reduced. Cranial placode genes showed a mixture of expanded and diminished expression domains. At larval stages, loss of Mcrs1 resulted in a significant reduction of otic vesicle gene expression concomitant with a smaller otic vesicle volume. Experimentally increasing Mcrs1 above endogenous levels favored the expansion of neural border and neural crest gene domains over cranial placode genes; it also reduced otic vesicle gene expression but not otic vesicle volume. Co-expression of Mcrs1 and Six1 as well as double knock-down and rescue experiments establish a functional interaction between Mcrs1 and Six1 in the embryo, and demonstrate that this interaction has an important role in the development of craniofacial tissues including the otic vesicle.

Xenopus ADAM19 regulates Wnt signaling and neural crest specification by stabilizing ADAM13.

During vertebrate gastrulation, canonical Wnt signaling induces the formation of neural plate border (NPB). Wnt is also thought to be required for the subsequent specification of neural crest (NC) lineage at the NPB, but the direct evidence is lacking. We found previously that the disintegrin metalloproteinase ADAM13 is required for Wnt activation and NC induction in Xenopus Here, we report that knockdown of ADAM13 or its close paralog ADAM19 severely downregulates Wnt activity at the NPB, inhibiting NC specification without affecting earlier NPB formation. Surprisingly, ADAM19 functions nonproteolytically in NC specification by interacting with ADAM13 and inhibiting its proteasomal degradation. Ectopic expression of stabilized ADAM13 mutants that function independently of ADAM19 can induce the NC marker/specifier snail2 in the future epidermis via Wnt signaling. These results unveil the essential roles of a novel protease-protease interaction in regulating a distinct wave of Wnt signaling, which directly specifies the NC lineage.

Cut loose and run: The complex role of ADAM proteases during neural crest cell development.

ADAM metalloproteases have been shown to play critical roles during development. In this review, we will describe functional evidence that implicates ADAM proteins during the genesis, migration and differentiation of neural crest cells. We will restrict our analysis to the transmembrane ADAMs as other reviews have addressed the role of extracellular metalloproteases (Christian et al. [2013] Critical Reviews in Biochemistry and Molecular Biology 48:544-560). This review will describe advances that have been obtained mainly through the use of two vertebrate model systems, the frog, and avian embryos. The role of the principal substrates of ADAMs, the cadherins, has been extensively described in other reviews, most recently in (Cousin [1997] Mechanisms of Development 148:79-88; Taneyhill and Schiffmacher [2017] Genesis, 55). The function of ADAMs in the migration of other cell types, including the immune system, wound healing and cancer has been described previously in (Dreymueller et al. [2017] Mediators of Inflammation 2017: 9621724). Our goal is to illustrate both the importance of ADAMs in controlling neural crest behavior and how neural crest cells have helped us understand the molecular interactions, substrates, and functions of ADAM proteins in vivo.

Pa2G4 is a novel Six1 co-factor that is required for neural crest and otic development.

Mutations in SIX1 and in its co-factor, EYA1, underlie Branchiootorenal Spectrum disorder (BOS), which is characterized by variable branchial arch, otic and kidney malformations. However, mutations in these two genes are identified in only half of patients. We screened for other potential co-factors, and herein characterize one of them, Pa2G4 (aka Ebp1/Plfap). In human embryonic kidney cells, Pa2G4 binds to Six1 and interferes with the Six1-Eya1 complex. In Xenopus embryos, knock-down of Pa2G4 leads to down-regulation of neural border zone, neural crest and cranial placode genes, and concomitant expansion of neural plate genes. Gain-of-function leads to a broader neural border zone, expanded neural crest and altered cranial placode domains. In loss-of-function assays, the later developing otocyst is reduced in size, which impacts gene expression. In contrast, the size of the otocyst in gain-of-function assays is not changed but the expression domains of several otocyst genes are reduced. Together these findings establish an interaction between Pa2G4 and Six1, and demonstrate that it has an important role in the development of tissues affected in BOS. Thereby, we suggest that pa2g4 is a potential candidate gene for BOS.

Active site-adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9.

Caspase-9 (casp-9) is an initiator caspase and plays a central role in activating apoptotic cell death. Control of all caspases is tightly regulated by a series of phosphorylation events enacted by several different kinases. Caspase-9 is the most heavily phosphorylated of all caspases, with phosphorylation of at least 11 distinct residues in all three caspase-9 domains by nine kinases. Caspase-9 phosphorylation by the non-receptor tyrosine kinase c-Abl at Tyr-153 reportedly leads to caspase-9 activation. All other phosphorylation events on caspases have been shown to block proteolytic function by a number of mechanisms, so we sought to unravel the molecular mechanism of the putative caspase-9 activation by phosphorylation. Surprisingly, we observed no evidence for Tyr-153 phosphorylation of caspase-9 in vitro or in cells, suggesting that Tyr-153 is not phosphorylated by c-Abl. Instead, we identified a new site for c-Abl-mediated phosphorylation, Tyr-397. This residue is adjacent to the caspase-9 active site but, as a member of the second shell, not a residue that directly contacts substrate. Our results further indicate that Tyr-397 is the dominant site of c-Abl phosphorylation both in vitro and upon c-Abl activation in cells. Of note, phosphorylation at this site inhibits caspase-9 activity, and the bulk of the added phosphate moiety appeared to directly block substrate binding. c-Abl plays both proapoptotic and prosurvival roles, and our findings suggest that c-Abl's effects on caspase-9 activity promote the prosurvival mode.

ADAM13 cleavage of cadherin-11 promotes CNC migration independently of the homophilic binding site.

The cranial neural crest (CNC) is a highly motile population of cells that is responsible for forming the face and jaw in all vertebrates and perturbing their migration can lead to craniofacial birth defects. Cell motility requires a dynamic modification of cell-cell and cell-matrix adhesion. In the CNC, cleavage of the cell adhesion molecule cadherin-11 by ADAM13 is essential for cell migration. This cleavage generates a shed extracellular fragment of cadherin-11 (EC1-3) that possesses pro-migratory activity via an unknown mechanism. Cadherin-11 plays an important role in modulating contact inhibition of locomotion (CIL) in the CNC to regulate directional cell migration. Here, we show that while the integral cadherin-11 requires the homophilic binding site to promote CNC migration in vivo, the EC1-3 fragment does not. In addition, we show that increased ADAM13 activity or expression of the EC1-3 fragment increases CNC invasiveness in vitro and blocks the repulsive CIL response in colliding cells. This activity requires the presence of an intact homophilic binding site on the EC1-3 suggesting that the cleavage fragment may function as a competitive inhibitor of cadherin-11 adhesion in CIL but not to promote cell migration in vivo.

The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity.

Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity.

Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases.

Xenopus Cadherin-11 (Xcad-11) is expressed when cranial neural crest cells (CNC) acquire motility. However, its function in stimulating cell migration is poorly understood. Here, we demonstrate that Xcad-11 initiates filopodia and lamellipodia formation, which is essential for CNC to populate pharyngeal pouches. We identified the cytoplasmic tail of Xcad-11 as both necessary and sufficient for proper CNC migration as long as it was linked to the plasma membrane. Our results showing that guanine nucleotide exchange factor (GEF)-Trio binds to Xcad-11 and can functionally substitute for it like constitutively active forms of RhoA, Rac, and cdc42 unravel a novel cadherin function.

Antibody responses in blood and saliva post COVID-19 bivalent booster do not reveal an Omicron BA.4/BA.5- specific response.

Introduction: Current SARS-CoV-2 strains continue to mutate and attempt to evade the antibody response elicited by previous exposures and vaccinations. In September of 2022, the first updated SARS-CoV-2 vaccines, designed to create immune responses specific for the variants circulating in 2022, were approved. These new vaccines, known commonly as the bivalent boost(er), include mRNA that encodes both the original Wuhan-Hu-1 spike protein as well as the spike protein specific to the Omicron BA.4 and BA.5 variants. Methods: We recruited volunteers from University of Massachusetts student, faculty and staff members to provide samples of blood and saliva at four different time points, including pre-boost and three times post boost and analyzed samples for antibody production as well as neutralization of virus. Results: Our data provide a comprehensive analysis of the antibody response following a single dose of the bivalent boost over a 6-month period and support previous findings that the response induced after the bivalent boost does not create a strong BA.4/BA.5-specific antibody response. Conclusion: We found no evidence of a specific anti-BA.4/BA.5 response developing over time, including in a sub-population of individuals who become infected after a single dose of the bivalent booster. Additionally, we present data that support the use of saliva samples as a reliable alternative to blood for antibody detection against specific SARS-CoV-2 antigens.

ADAM13 function is required in the 3 dimensional context of the embryo during cranial neural crest cell migration in Xenopus laevis.

The cranial neural crest (CNC) is a population of cells that arises from the lateral part of the developing brain, migrates ventrally and coordinates the entire craniofacial development of vertebrates. Many molecules are involved in CNC migration including the transmembrane metalloproteases ADAM13 and 19. We have previously shown that these ADAMs cleave a number of extracellular proteins and modify the transcription of a number of genes, and that both of these activities are important for cell migration. Here we show that the knock down of ADAM13 inhibits CNC migration in vivo but not in vitro, indicating that ADAM13 function is required in the 3-dimentional context of the embryo. We further show that the migration of CNC that do not express ADAM13 and ADAM19 can be rescued in vivo by co-grafting wild type CNC. Furthermore, the migration of CNC lacking ADAM13 can be rescued by mechanically separating the CNC from the surrounding ectoderm and mesoderm. Finally, we show that ADAM13 function is autonomous to CNC tissue, as the migration of morphant CNC can only be rescued by ADAM13 expression in the CNC and not the surrounding tissues. Together our results suggest that ADAM13 changes CNC interaction with the extracellular environment and that this change is necessary for their migration in vivo.

ADAM11 a novel regulator of Wnt and BMP4 signaling in neural crest and cancer.

Cranial neural crest (CNC) cells are induced at the border of the neural plate by a combination of FGF, Wnt, and BMP4 signaling. CNC then migrate ventrally and invade ventral structures where they contribute to craniofacial development. Here we show that a non-proteolytic ADAM, Adam11, originally identified as a putative tumor suppressor binds to proteins of the Wnt and BMP4 signaling pathway. Mechanistic studies concerning these non-proteolytic ADAM lack almost entirely. We show that Adam11 positively regulates BMP4 signaling while negatively regulating ß-catenin activity. By modulating these pathways, Adam11 controls the timing of neural tube closure and the proliferation and migration of CNC. Using both human tumor data and mouse B16 melanoma cells, we further show that ADAM11 levels similarly correlate with Wnt or BMP4 activation levels. We propose that ADAM11 preserve naïve cells by maintaining low Sox3 and Snail/Slug levels through stimulation of BMP4 and repression of Wnt signaling, while loss of ADAM11 results in increased Wnt signaling, increased proliferation and early epithelium to mesenchyme transition.

ADAM11 a novel regulator of Wnt and BMP4 signaling in neural crest and cancer.

Introduction: Cranial neural crest (CNC) cells are induced at the border of the neural plate by a combination of FGF, Wnt, and BMP4 signaling. CNC then migrate ventrally and invade ventral structures where they contribute to craniofacial development. Methods: We used loss and gain of function experiments to determine phenotypes associated with the perturbation of Adam11 expression in Xenopus Laevis. Mass spectrometry to identify partners of Adam11 and changes in protein expression in CNC lacking Adam11. We used mouse B16 melanoma to test the function of Adam11 in cancer cells, and published database analysis to study the expression of ADAM11 in human tumors. Results: Here we show that a non-proteolytic ADAM, Adam11, originally identified as a putative tumor suppressor binds to proteins of the Wnt and BMP4 signaling pathway. Mechanistic studies concerning these non-proteolytic ADAM lack almost entirely. We show that Adam11 positively regulates BMP4 signaling while negatively regulating ß-catenin activity. In vivo, we show that Adam11 influences the timing of neural tube closure and the proliferation and migration of CNC. Using both human tumor data and mouse B16 melanoma cells, we further show that ADAM11 levels similarly correlate with Wnt or BMP4 activation levels. Discussion: We propose that ADAM11 preserves naïve cells by maintaining low Sox3 and Snail/Slug levels through stimulation of BMP4 and repression of Wnt signaling, while loss of ADAM11 results in increased Wnt signaling, increased proliferation and early epithelium to mesenchyme transition.

Essential role of Mg2+ in mouse preimplantation embryo development revealed by TRPM7 chanzyme-deficient gametes.

TRPM7 (transient receptor potential cation channel subfamily M member 7) is a chanzyme with channel and kinase domains essential for embryo development. Using gamete-specific Trpm7-null lines, we report that TRPM7-mediated Mg2+ influx is indispensable for reaching the blastocyst stage. TRPM7 is expressed dynamically from gametes to blastocysts; displays stage-specific localization on the plasma membrane, cytoplasm, and nucleus; and undergoes cleavage that produces C-terminal kinase fragments. TRPM7 underpins Mg2+ homeostasis, and excess Mg2+ but not Zn2+ or Ca2+ overcomes the arrest of Trpm7-null embryos; expressing Trpm7 mRNA restores development, but mutant versions fail or are partially rescued. Transcriptomic analyses of Trpm7-null embryos reveal an abundance of oxidative stress-pathway genes, confirmed by mitochondrial dysfunction, and a reduction in transcription factor networks essential for proliferation; Mg2+ supplementation corrects these defects. Hence, TRPM7 underpins Mg2+ homeostasis in preimplantation embryos, prevents oxidative stress, and promotes gene expression patterns necessary for developmental progression and cell-lineage specification.

Zmym4 is required for early cranial gene expression and craniofacial cartilage formation.

Introduction: The Six1 transcription factor plays important roles in the development of cranial sensory organs, and point mutations underlie craniofacial birth defects. Because Six1's transcriptional activity can be modulated by interacting proteins, we previously screened for candidate interactors and identified zinc-finger MYM-containing protein 4 (Zmym4) by its inclusion of a few domains with a bona fide cofactor, Sine oculis binding protein (Sobp). Although Zmym4 has been implicated in regulating early brain development and certain cancers, its role in craniofacial development has not previously been described. Methods: We used co-immunoprecipitation and luciferase-reporter assays in cultured cells to test interactions between Zmym4 and Six1. We used knock-down and overexpression of Zmym4 in embryos to test for its effects on early ectodermal gene expression, neural crest migration and craniofacial cartilage formation. Results: We found no evidence that Zmym4 physically or transcriptionally interacts with Six1 in cultured cells. Nonetheless, knockdown of endogenous Zmym4 in embryos resulted in altered early cranial gene expression, including those expressed in the neural border, neural plate, neural crest and preplacodal ectoderm. Experimentally increasing Zmym4 levels had minor effects on neural border or neural plate genes, but altered the expression of neural crest and preplacodal genes. At larval stages, genes expressed in the otic vesicle and branchial arches showed reduced expression in Zmym4 morphants. Although we did not detect defects in neural crest migration into the branchial arches, loss of Zmym4 resulted in aberrant morphology of several craniofacial cartilages. Discussion: Although Zmym4 does not appear to function as a Six1 transcriptional cofactor, it plays an important role in regulating the expression of embryonic cranial genes in tissues critical for normal craniofacial development.