Cell lineage-guided mass spectrometry reveals increased energy metabolism and reactive oxygen species in the vertebrate organizer.

Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been mostly limited to transcripts and a few proteins, the latter due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog (X. laevis), a popular model of development, has long been known to be the origin of signals that pattern the mesoderm and central nervous system. Molecular screens of the SMO have identified several genes responsible for the ability of the SMO to establish the body axis. Nonetheless, a comprehensive study of proteins and metabolites produced specifically in the SMO and their functional roles has been lacking. Here, we pioneer a deep discovery proteomic and targeted metabolomic screen of the SMO in comparison to the remainder of the embryo using high-resolution mass spectrometry (HRMS). Quantification of ~4,600 proteins and a panel of targeted metabolites documented differential expression for 460 proteins and multiple intermediates of energy metabolism in the SMO. Upregulation of oxidative phosphorylation and redox regulatory proteins gave rise to elevated oxidative stress and an accumulation of reactive oxygen species in the SMO. Imaging experiments corroborated these findings, discovering enrichment of hydrogen peroxide in the SMO. Chemical perturbation of the redox gradient perturbed mesoderm involution during early gastrulation. HRMS expands the bioanalytical toolbox of cell and developmental biology, providing previously unavailable information on molecular classes to challenge and refine our classical understanding of the Organizer and its function during early patterning of the embryo.

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.

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.

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.

Time-resolved quantitative proteomic analysis of the developing Xenopus otic vesicle reveals putative congenital hearing loss candidates.

Over 200 genes are known to underlie human congenital hearing loss (CHL). Although transcriptomic approaches have identified candidate regulators of otic development, little is known about the abundance of their protein products. We used a multiplexed quantitative mass spectrometry-based proteomic approach to determine protein abundances over key stages of Xenopus otic morphogenesis to reveal a dynamic expression of cytoskeletal, integrin signaling, and extracellular matrix proteins. We correlated these dynamically expressed proteins to previously published lists of putative downstream targets of human syndromic hearing loss genes: SIX1 (BOR syndrome), CHD7 (CHARGE syndrome), and SOX10 (Waardenburg syndrome). We identified transforming growth factor beta-induced (Tgfbi), an extracellular integrin-interacting protein, as a putative target of Six1 that is required for normal otic vesicle formation. Our findings demonstrate the application of this Xenopus dataset to understanding the dynamic regulation of proteins during otic development and to discovery of additional candidates for human CHL.

The sulfotransferase XB5850668.L is required to apportion embryonic ectodermal domains.

BACKGROUND: Members of the sulfotransferase superfamily (SULT) influence the activity of a wide range of hormones, neurotransmitters, metabolites and xenobiotics. However, their roles in developmental processes are not well characterized even though they are expressed during embryogenesis. We previously found in a microarray screen that Six1 up-regulates LOC100037047, which encodes XB5850668.L, an uncharacterized sulfotransferase. RESULTS: Since Six1 is required for patterning the embryonic ectoderm into its neural plate, neural crest, preplacodal and epidermal domains, we used loss- and gain-of function assays to characterize the role of XB5850668.L during this process. Knockdown of endogenous XB5850668.L resulted in the reduction of epidermal, neural crest, cranial placode and otic vesicle gene expression domains, concomitant with neural plate expansion. Increased levels had minimal effects, but infrequently expanded neural plate and neural crest gene domains, and infrequently reduced cranial placode and otic vesicle gene domains. Mutation of two key amino acids in the sulfotransferase catalytic domain required for PAPS binding and enzymatic activity tended to reduce the effects of overexpressing the wild-type protein. CONCLUSIONS: Our analyses indicates that XB5850668.L is a member of the SULT2 family that plays important roles in patterning the embryonic ectoderm. Some aspects of its influence likely depend on sulfotransferase activity.

Duplicated zebrafish (Danio rerio) inositol phosphatases inpp5ka and inpp5kb diverged in expression pattern and function.

One hurdle in the development of zebrafish models of human disease is the presence of multiple zebrafish orthologs resulting from whole genome duplication in teleosts. Mutations in inositol polyphosphate 5-phosphatase K (INPP5K) lead to a syndrome characterized by variable presentation of intellectual disability, brain abnormalities, cataracts, muscle disease, and short stature. INPP5K is a phosphatase acting at position 5 of phosphoinositides to control their homeostasis and is involved in insulin signaling, cytoskeletal regulation, and protein trafficking. Previously, our group and others have replicated the human phenotypes in zebrafish knockdown models by targeting both INPP5K orthologs inpp5ka and inpp5kb. Here, we show that inpp5ka is the more closely related orthologue to human INPP5K. While both inpp5ka and inpp5kb mRNA expression levels follow a similar trend in the developing head, eyes, and tail, inpp5ka is much more abundantly expressed in these tissues than inpp5kb. In situ hybridization revealed a similar trend, also showing unique localization of inpp5kb in the pineal gland and retina indicating different transcriptional regulation. We also found that inpp5kb has lost its catalytic activity against its preferred substrate, PtdIns(4,5)P2. Since most human mutations are missense changes disrupting phosphatase activity, we propose that loss of inpp5ka alone can be targeted to recapitulate the human presentation. In addition, we show that the function of inpp5kb has diverged from inpp5ka and may play a novel role in the zebrafish.

Advances in Understanding the Pathogenesis of Craniofacial Birth Defects.

Each year approximately 35% of babies are born with craniofacial abnormalities of the skull, jaws, ears, and/or teeth, which in turn can lead to problems in feeding, hearing, and sight [...].

Normal Table of Xenopus development: a new graphical resource.

Normal tables of development are essential for studies of embryogenesis, serving as an important resource for model organisms, including the frog Xenopus laevis. Xenopus has long been used to study developmental and cell biology, and is an increasingly important model for human birth defects and disease, genomics, proteomics and toxicology. Scientists utilize Nieuwkoop and Faber's classic 'Normal Table of Xenopus laevis (Daudin)' and accompanying illustrations to enable experimental reproducibility and reuse the illustrations in new publications and teaching. However, it is no longer possible to obtain permission for these copyrighted illustrations. We present 133 new, high-quality illustrations of X. laevis development from fertilization to metamorphosis, with additional views that were not available in the original collection. All the images are available on Xenbase, the Xenopus knowledgebase (http://www.xenbase.org/entry/zahn.do), for download and reuse under an attributable, non-commercial creative commons license. Additionally, we have compiled a 'Landmarks Table' of key morphological features and marker gene expression that can be used to distinguish stages quickly and reliably (https://www.xenbase.org/entry/landmarks-table.do). This new open-access resource will facilitate Xenopus research and teaching in the decades to come.

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.

Retinoic Acid is Required for Normal Morphogenetic Movements During Gastrulation.

Retinoic acid (RA) is a central regulatory signal that controls numerous developmental processes in vertebrate embryos. Although activation of Hox expression is considered one of the earliest functions of RA signaling in the embryo, there is evidence that embryos are poised to initiate RA signaling just before gastrulation begins, and manipulations of the RA pathway have been reported to show gastrulation defects. However, which aspects of gastrulation are affected have not been explored in detail. We previously showed that partial inhibition of RA biosynthesis causes a delay in the rostral migration of some of the earliest involuting cells, the leading edge mesendoderm (LEM) and the prechordal mesoderm (PCM). Here we identify several detrimental gastrulation defects resulting from inhibiting RA biosynthesis by three different treatments. RA reduction causes a delay in the progression through gastrulation as well as the rostral migration of the goosecoid-positive PCM cells. RA inhibition also hampered the elongation of explanted dorsal marginal zones, the compaction of the blastocoel, and the length of Brachet's cleft, all of which indicate an effect on LEM/PCM migration. The cellular mechanisms underlying this deficit were shown to include a reduced deposition of fibronectin along Brachet's cleft, the substrate for their migration, as well as impaired separation of the blastocoel roof and involuting mesoderm, which is important for the formation of Brachet's cleft and successful LEM/PCM migration. We further show reduced non-canonical Wnt signaling activity and altered expression of genes in the Ephrin and PDGF signaling pathways, both of which are required for the rostral migration of the LEM/PCM, following RA reduction. Together, these experiments demonstrate that RA signaling performs a very early function critical for the progression of gastrulation morphogenetic movements.

Repressive Interactions Between Transcription Factors Separate Different Embryonic Ectodermal Domains.

The embryonic ectoderm is composed of four domains: neural plate, neural crest, pre-placodal region (PPR) and epidermis. Their formation is initiated during early gastrulation by dorsal-ventral and anterior-posterior gradients of signaling factors that first divide the embryonic ectoderm into neural and non-neural domains. Next, the neural crest and PPR domains arise, either via differential competence of the neural and non-neural ectoderm (binary competence model) or via interactions between the neural and non-neural ectoderm tissues to produce an intermediate neural border zone (NB) (border state model) that subsequently separates into neural crest and PPR. Many previous gain- and loss-of-function experiments demonstrate that numerous TFs are expressed in initially overlapping zones that gradually resolve into patterns that by late neurula stages are characteristic of each of the four domains. Several of these studies suggested that this is accomplished by a combination of repressive TF interactions and competence to respond to local signals. In this study, we ectopically expressed TFs that at neural plate stages are characteristic of one domain in a different domain to test whether they act cell autonomously as repressors. We found that almost all tested TFs caused reduced expression of the other TFs. At gastrulation these effects were strictly within the lineage-labeled cells, indicating that the effects were cell autonomous, i.e., due to TF interactions within individual cells. Analysis of previously published single cell RNAseq datasets showed that at the end of gastrulation, and continuing to neural tube closure stages, many ectodermal cells express TFs characteristic of more than one neural plate stage domain, indicating that different TFs have the opportunity to interact within the same cell. At neurula stages repression was observed both in the lineage-labeled cells and in adjacent cells not bearing detectable lineage label, suggesting that cell-to-cell signaling has begun to contribute to the separation of the domains. Together, these observations directly demonstrate previous suggestions in the literature that the segregation of embryonic ectodermal domains initially involves cell autonomous, repressive TF interactions within an individual cell followed by the subsequent advent of non-cell autonomous signaling to neighbors.

Selective disruption of trigeminal sensory neurogenesis and differentiation in a mouse model of 22q11.2 deletion syndrome.

22q11.2 Deletion Syndrome (22q11DS) is a neurodevelopmental disorder associated with cranial nerve anomalies and disordered oropharyngeal function, including pediatric dysphagia. Using the LgDel 22q11DS mouse model, we investigated whether sensory neuron differentiation in the trigeminal ganglion (CNgV), which is essential for normal orofacial function, is disrupted. We did not detect changes in cranial placode cell translocation or neural crest migration at early stages of LgDel CNgV development. However, as the ganglion coalesces, proportions of placode-derived LgDel CNgV cells increase relative to neural crest cells. In addition, local aggregation of placode-derived cells increases and aggregation of neural crest-derived cells decreases in LgDel CNgV. This change in cell-cell relationships was accompanied by altered proliferation of placode-derived cells at embryonic day (E)9.5, and premature neurogenesis from neural crest-derived precursors, reflected by an increased frequency of asymmetric neurogenic divisions for neural crest-derived precursors by E10.5. These early differences in LgDel CNgV genesis prefigure changes in sensory neuron differentiation and gene expression by postnatal day 8, when early signs of cranial nerve dysfunction associated with pediatric dysphagia are observed in LgDel mice. Apparently, 22q11 deletion destabilizes CNgV sensory neuron genesis and differentiation by increasing variability in cell-cell interaction, proliferation and sensory neuron differentiation. This early developmental divergence and its consequences may contribute to oropharyngeal dysfunction, including suckling, feeding and swallowing disruptions at birth, and additional orofacial sensory/motor deficits throughout life.

Xenopus Explants and Transplants.

There is a long tradition of testing the developmental potential and competency of different regions of the embryo by transplanting tissue to novel locations or by growing isolated tissue in explant culture. The protocols introduced here describe several types of embryological manipulations in Xenopus that are particularly useful for analyzing tissue inductive signals, cell migration, and organogenesis. These techniques draw upon classical approaches that are quite easy to perform in both Xenopus laevis and Xenopus tropicalis embryos.

Generation of a new six1-null line in Xenopus tropicalis for study of development and congenital disease.

The vertebrate Six (Sine oculis homeobox) family of homeodomain transcription factors plays critical roles in the development of several organs. Six1 plays a central role in cranial placode development, including the precursor tissues of the inner ear, as well as other cranial sensory organs and the kidney. In humans, mutations in SIX1 underlie some cases of Branchio-oto-renal (BOR) syndrome, which is characterized by moderate-to-severe hearing loss. We utilized CRISPR/Cas9 technology to establish a six1 mutant line in Xenopus tropicalis that is available to the research community. We demonstrate that at larval stages, the six1-null animals show severe disruptions in gene expression of putative Six1 target genes in the otic vesicle, cranial ganglia, branchial arch, and neural tube. At tadpole stages, six1-null animals display dysmorphic Meckel's, ceratohyal, and otic capsule cartilage morphology. This mutant line will be of value for the study of the development of several organs as well as congenital syndromes that involve these tissues.

Sobp modulates the transcriptional activation of Six1 target genes and is required during craniofacial development.

Branchio-oto-renal syndrome (BOR) is a disorder characterized by hearing loss, and craniofacial and/or renal defects. Variants in the transcription factor Six1 and its co-factor Eya1, both of which are required for otic development, are linked to BOR. We previously identified Sobp as a potential Six1 co-factor, and SOBP variants in mouse and humans cause otic phenotypes; therefore, we asked whether Sobp interacts with Six1 and thereby may contribute to BOR. Co-immunoprecipitation and immunofluorescence experiments demonstrate that Sobp binds to and colocalizes with Six1 in the cell nucleus. Luciferase assays show that Sobp interferes with the transcriptional activation of Six1+Eya1 target genes. Experiments in Xenopus embryos that either knock down or increase expression of Sobp show that it is required for formation of ectodermal domains at neural plate stages. In addition, altering Sobp levels disrupts otic vesicle development and causes craniofacial cartilage defects. Expression of Xenopus Sobp containing the human variant disrupts the pre-placodal ectoderm similar to full-length Sobp, but other changes are distinct. These results indicate that Sobp modifies Six1 function and is required for vertebrate craniofacial development, and identify Sobp as a potential candidate gene for BOR.

Mutations in SIX1 Associated with Branchio-oto-Renal Syndrome (BOR) Differentially Affect Otic Expression of Putative Target Genes.

Several single-nucleotide mutations in SIX1 underlie branchio-otic/branchio-oto-renal (BOR) syndrome, but the clinical literature has not been able to correlate different variants with specific phenotypes. We previously assessed whether variants in either the cofactor binding domain (V17E, R110W) or the DNA binding domain (W122R, Y129C) might differentially affect early embryonic gene expression, and found that each variant had a different combination of effects on neural crest and placode gene expression. Since the otic vesicle gives rise to the inner ear, which is consistently affected in BOR, herein we focused on whether the variants differentially affected the otic expression of genes previously found to be likely Six1 targets. We found that V17E, which does not bind Eya cofactors, was as effective as wild-type Six1 in reducing most otic target genes, whereas R110W, W122R and Y129C, which bind Eya, were significantly less effective. Notably, V17E reduced the otic expression of prdm1, whereas R110W, W122R and Y129C expanded it. Since each mutant has defective transcriptional activity but differs in their ability to interact with Eya cofactors, we propose that altered cofactor interactions at the mutated sites differentially interfere with their ability to drive otic gene expression, and these differences may contribute to patient phenotype variability.

Altering metabolite distribution at Xenopus cleavage stages affects left-right gene expression asymmetries.

The left-right (L-R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in the lateral plate mesoderm. However, in some animals, an earlier differential distribution of molecules and cell division patterns initiate or at least influence L-R patterning. Using single-cell high-resolution mass spectrometry, we previously reported a limited number of small molecule (metabolite) concentration differences between left and right dorsal-animal blastomeres of the eight-cell Xenopus embryo. Herein, we examined whether altering the distribution of some of these molecules influenced early events in L-R patterning. Using lineage tracing, we found that injecting right-enriched metabolites into the left cell caused its descendant cells to disperse in patterns that varied from those in control gastrulae; this did not occur when left-enriched metabolites were injected into the right cell. At later stages, injecting left-enriched metabolites into the right cell perturbed the expression of genes known to: (a) be required for the formation of the gastrocoel roof plate (foxj1); (b) lead to the asymmetric expression of Nodal (dand5/coco); or (c) result from asymmetrical nodal expression (pitx2). Despite these perturbations in gene expression, we did not observe heterotaxy in heart or gut looping at tadpole stages. These studies indicate that altering metabolite distribution at cleavage stages at the concentrations tested in this study impacts the earliest steps of L-R gene expression that then can be compensated for during organogenesis.