Keeping development on time: Insights into post-transcriptional mechanisms driving oscillatory gene expression during vertebrate segmentation.

Biological time keeping, or the duration and tempo at which biological processes occur, is a phenomenon that drives dynamic molecular and morphological changes that manifest throughout many facets of life. In some cases, the molecular mechanisms regulating the timing of biological transitions are driven by genetic oscillations, or periodic increases and decreases in expression of genes described collectively as a "molecular clock." In vertebrate animals, molecular clocks play a crucial role in fundamental patterning and cell differentiation processes throughout development. For example, during early vertebrate embryogenesis, the segmentation clock regulates the patterning of the embryonic mesoderm into segmented blocks of tissue called somites, which later give rise to axial skeletal muscle and vertebrae. Segmentation clock oscillations are characterized by rapid cycles of mRNA and protein expression. For segmentation clock oscillations to persist, the transcript and protein molecules of clock genes must be short-lived. Faithful, rhythmic, genetic oscillations are sustained by precise regulation at many levels, including post-transcriptional regulation, and such mechanisms are essential for proper vertebrate development. This article is categorized under: RNA Export and Localization > RNA Localization RNA Turnover and Surveillance > Regulation of RNA Stability Translation > Regulation.

SIX1 reprograms myogenic transcription factors to maintain the rhabdomyosarcoma undifferentiated state.

Rhabdomyosarcoma (RMS) is a pediatric muscle sarcoma characterized by expression of the myogenic lineage transcription factors (TFs) MYOD1 and MYOG. Despite high expression of these TFs, RMS cells fail to terminally differentiate, suggesting the presence of factors that alter their functions. Here, we demonstrate that the developmental TF SIX1 is highly expressed in RMS and critical for maintaining a muscle progenitor-like state. SIX1 loss induces differentiation of RMS cells into myotube-like cells and impedes tumor growth in vivo. We show that SIX1 maintains the RMS undifferentiated state by controlling enhancer activity and MYOD1 occupancy at loci more permissive to tumor growth over muscle differentiation. Finally, we demonstrate that a gene signature derived from SIX1 loss correlates with differentiation status and predicts RMS progression in human disease. Our findings demonstrate a master regulatory role of SIX1 in repression of RMS differentiation via genome-wide alterations in MYOD1 and MYOG-mediated transcription.

Mutations in MYLPF Cause a Novel Segmental Amyoplasia that Manifests as Distal Arthrogryposis.

We identified ten persons in six consanguineous families with distal arthrogryposis (DA) who had congenital contractures, scoliosis, and short stature. Exome sequencing revealed that each affected person was homozygous for one of two different rare variants (c.470G>T [p.Cys157Phe] or c.469T>C [p.Cys157Arg]) affecting the same residue of myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF). In a seventh family, a c.487G>A (p.Gly163Ser) variant in MYLPF arose de novo in a father, who transmitted it to his son. In an eighth family comprised of seven individuals with dominantly inherited DA, a c.98C>T (p.Ala33Val) variant segregated in all four persons tested. Variants in MYLPF underlie both dominant and recessively inherited DA. Mylpf protein models suggest that the residues associated with dominant DA interact with myosin whereas the residues altered in families with recessive DA only indirectly impair this interaction. Pathological and histological exam of a foot amputated from an affected child revealed complete absence of skeletal muscle (i.e., segmental amyoplasia). To investigate the mechanism for this finding, we generated an animal model for partial MYLPF impairment by knocking out zebrafish mylpfa. The mylpfa mutant had reduced trunk contractile force and complete pectoral fin paralysis, demonstrating that mylpf impairment most severely affects limb movement. mylpfa mutant muscle weakness was most pronounced in an appendicular muscle and was explained by reduced myosin activity and fiber degeneration. Collectively, our findings demonstrate that partial loss of MYLPF function can lead to congenital contractures, likely as a result of degeneration of skeletal muscle in the distal limb.

Zebrafish rbm8a and magoh mutants reveal EJC developmental functions and new 3'UTR intron-containing NMD targets.

Many post-transcriptional mechanisms operate via mRNA 3'UTRs to regulate protein expression, and such controls are crucial for development. We show that homozygous mutations in two zebrafish exon junction complex (EJC) core genes rbm8a and magoh leads to muscle disorganization, neural cell death, and motor neuron outgrowth defects, as well as dysregulation of mRNAs subjected to nonsense-mediated mRNA decay (NMD) due to translation termination ≥ 50 nts upstream of the last exon-exon junction. Intriguingly, we find that EJC-dependent NMD also regulates a subset of transcripts that contain 3'UTR introns (3'UI) < 50 nts downstream of a stop codon. Some transcripts containing such stop codon-proximal 3'UI are also NMD-sensitive in cultured human cells and mouse embryonic stem cells. We identify 167 genes that contain a conserved proximal 3'UI in zebrafish, mouse and humans. foxo3b is one such proximal 3'UI-containing gene that is upregulated in zebrafish EJC mutant embryos, at both mRNA and protein levels, and loss of foxo3b function in EJC mutant embryos significantly rescues motor axon growth defects. These data are consistent with EJC-dependent NMD regulating foxo3b mRNA to control protein expression during zebrafish development. Our work shows that the EJC is critical for normal zebrafish development and suggests that proximal 3'UIs may serve gene regulatory function in vertebrates.

Pumilio response and AU-rich elements drive rapid decay of Pnrc2-regulated cyclic gene transcripts.

Vertebrate segmentation is regulated by the segmentation clock, a biological oscillator that controls periodic formation of somites, or embryonic segments, which give rise to many mesodermal tissue types. This molecular oscillator generates cyclic gene expression with the same periodicity as somite formation in the presomitic mesoderm (PSM), an area of mesenchymal cells that give rise to mature somites. Molecular components of the clock include the Hes/her family of genes that encode transcriptional repressors, but additional genes cycle. Cyclic gene transcripts are cleared rapidly, and clearance depends upon the pnrc2 (proline-rich nuclear receptor co-activator 2) gene that encodes an mRNA decay adaptor. Previously, we showed that the her1 3'UTR confers instability to otherwise stable transcripts in a Pnrc2-dependent manner, however, the molecular mechanism(s) by which cyclic gene transcripts are cleared remained largely unknown. To identify features of the her1 3'UTR that are critical for Pnrc2-mediated decay, we developed an array of transgenic inducible reporter lines carrying different regions of the 3'UTR. We find that the terminal 179 nucleotides (nts) of the her1 3'UTR are necessary and sufficient to confer rapid instability. Additionally, we show that the 3'UTR of another cyclic gene, deltaC (dlc), also confers Pnrc2-dependent instability. Motif analysis reveals that both her1 and dlc 3'UTRs contain terminally-located Pumilio response elements (PREs) and AU-rich elements (AREs), and we show that the PRE and ARE in the last 179 â€‹nts of the her1 3'UTR drive rapid turnover of reporter mRNA. Finally, we show that mutation of Pnrc2 residues and domains that are known to facilitate interaction of human PNRC2 with decay factors DCP1A and UPF1 reduce the ability of Pnrc2 to restore normal cyclic gene expression in pnrc2 mutant embryos. Our findings suggest that Pnrc2 interacts with decay machinery components and cooperates with Pumilio (Pum) proteins and ARE-binding proteins to promote rapid turnover of cyclic gene transcripts during somitogenesis.

Cell fusion is differentially regulated in zebrafish post-embryonic slow and fast muscle.

Skeletal muscle fusion occurs during development, growth, and regeneration. To investigate how muscle fusion compares among different muscle cell types and developmental stages, we studied muscle cell fusion over time in wild-type, myomaker (mymk), and jam2a mutant zebrafish. Using live imaging, we show that embryonic myoblast elongation and fusion correlate tightly with slow muscle cell migration. In wild-type embryos, only fast muscle fibers are multinucleate, consistent with previous work showing that the cell fusion regulator gene mymk is specifically expressed throughout the embryonic fast muscle domain. However, by 3 weeks post-fertilization, slow muscle fibers also become multinucleate. At this late-larval stage, mymk is not expressed in muscle fibers, but is expressed in small cells near muscle fibers. Although previous work showed that both mymk and jam2a are required for embryonic fast muscle cell fusion, we observe that muscle force and function is almost normal in mymk and jam2a mutant embryos, despite the lack of fast muscle multinucleation. We show that genetic requirements change post-embryonically, with jam2a becoming much less important by late-larval stages and mymk now required for muscle fusion and growth in both fast and slow muscle cell types. Correspondingly, adult mymk mutants perform poorly in sprint and endurance tests compared to wild-type and jam2a mutants. We show that adult mymk mutant muscle contains small mononucleate myofibers with average myonuclear domain size equivalent to that in wild type adults. The mymk mutant fibers have decreased Laminin expression and increased numbers of Pax7-positive cells, suggesting that impaired fiber growth and active regeneration contribute to the muscle phenotype. Our findings identify several aspects of muscle fusion that change with time in slow and fast fibers as zebrafish develop beyond embryonic stages.

Muscle precursor cell movements in zebrafish are dynamic and require Six family genes.

Muscle precursors need to be correctly positioned during embryonic development for proper body movement. In zebrafish, a subset of hypaxial muscle precursors from the anterior somites undergo long-range migration, moving away from the trunk in three streams to form muscles in distal locations such as the fin. We mapped long-distance muscle precursor migrations with unprecedented resolution using live imaging. We identified conserved genes necessary for normal precursor motility (six1a, six1b, six4a, six4b and met). These genes are required for movement away from somites and later to partition two muscles within the fin bud. During normal development, the middle muscle precursor stream initially populates the fin bud, then the remainder of this stream contributes to the posterior hypaxial muscle. When we block fin bud development by impairing retinoic acid synthesis or Fgfr function, the entire stream contributes to the posterior hypaxial muscle indicating that muscle precursors are not committed to the fin during migration. Our findings demonstrate a conserved muscle precursor motility pathway, identify dynamic cell movements that generate posterior hypaxial and fin muscles, and demonstrate flexibility in muscle precursor fates.

tbx6l and tbx16 are redundantly required for posterior paraxial mesoderm formation during zebrafish embryogenesis.

BACKGROUND: T-box genes encode a large transcription factor family implicated in many aspects of development. We are focusing on two related zebrafish T-box genes, tbx6l and tbx16, that are expressed in highly overlapping patterns in embryonic paraxial mesoderm. tbx16 mutants are deficient in trunk, but not tail, somites; we explored whether presence of tail somites in tbx16 mutants was due to compensatory function provided by the tbx6l gene. RESULTS: We generated two zebrafish tbx6l mutant alleles. Loss of tbx6l has no apparent effect on embryonic development, nor does tbx6l loss enhance the phenotype of two other T-box gene mutants, ta and tbx6, or of the mesp family gene mutant msgn1. In contrast, loss of tbx6l function dramatically enhances the paraxial mesoderm deficiency of tbx16 mutants. CONCLUSIONS: These data demonstrate that tbx6l and tbx16 genes function redundantly to direct tail somite development. tbx6l single mutants develop normally because tbx16 fully compensates for loss of tbx6l function. However, tbx6l only partially compensates for loss of tbx16 function. These results resolve the question of why loss of function of tbx16 gene, which is expressed throughout the ventral and paraxial mesoderm, profoundly affects somite development in the trunk but not the tail. Developmental Dynamics 246:759-769, 2017. © 2017 Wiley Periodicals, Inc.

Pnrc2 regulates 3'UTR-mediated decay of segmentation clock-associated transcripts during zebrafish segmentation.

Vertebrate segmentation is controlled by the segmentation clock, a molecular oscillator that regulates gene expression and cycles rapidly. The expression of many genes oscillates during segmentation, including hairy/Enhancer of split-related (her or Hes) genes, which encode transcriptional repressors that auto-inhibit their own expression, and deltaC (dlc), which encodes a Notch ligand. We previously identified the tortuga (tor) locus in a zebrafish forward genetic screen for genes involved in cyclic transcript regulation and showed that cyclic transcripts accumulate post-splicing in tor mutants. Here we show that cyclic mRNA accumulation in tor mutants is due to loss of pnrc2, which encodes a proline-rich nuclear receptor co-activator implicated in mRNA decay. Using an inducible in vivo reporter system to analyze transcript stability, we find that the her1 3'UTR confers Pnrc2-dependent instability to a heterologous transcript. her1 mRNA decay is Dicer-independent and likely employs a Pnrc2-Upf1-containing mRNA decay complex. Surprisingly, despite accumulation of cyclic transcripts in pnrc2-deficient embryos, we find that cyclic protein is expressed normally. Overall, we show that Pnrc2 promotes 3'UTR-mediated decay of developmentally-regulated segmentation clock transcripts and we uncover an additional post-transcriptional regulatory layer that ensures oscillatory protein expression in the absence of cyclic mRNA decay.

Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish.

Satellite cells, also known as muscle stem cells, are responsible for skeletal muscle growth and repair in mammals. Pax7 and Pax3 transcription factors are established satellite cell markers required for muscle development and regeneration, and there is great interest in identifying additional factors that regulate satellite cell proliferation, differentiation, and/or skeletal muscle regeneration. Due to the powerful regenerative capacity of many zebrafish tissues, even in adults, we are exploring the regenerative potential of adult zebrafish skeletal muscle. Here, we show that adult zebrafish skeletal muscle contains cells similar to mammalian satellite cells. Adult zebrafish satellite-like cells have dense heterochromatin, express Pax7 and Pax3, proliferate in response to injury, and show peak myogenic responses 4-5 days post-injury (dpi). Furthermore, using a pax7a-driven GFP reporter, we present evidence implicating satellite-like cells as a possible source of new muscle. In lieu of central nucleation, which distinguishes regenerating myofibers in mammals, we describe several characteristics that robustly identify newly-forming myofibers from surrounding fibers in injured adult zebrafish muscle. These characteristics include partially overlapping expression in satellite-like cells and regenerating myofibers of two RNA-binding proteins Rbfox2 and Rbfoxl1, known to regulate embryonic muscle development and function. Finally, by analyzing pax7a; pax7b double mutant zebrafish, we show that Pax7 is required for adult skeletal muscle repair, as it is in the mouse.

Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development.

The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver and pancreas. Here, we interrogate the role of Nr5a2 at multiple developmental stages using genetic and chemical approaches and uncover novel pleiotropic requirements during zebrafish liver and pancreas development. Zygotic loss of nr5a2 in a targeted genetic null mutant disrupted the development of the exocrine pancreas and liver, while leaving the endocrine pancreas intact. Loss of nr5a2 abrogated exocrine pancreas markers such as trypsin, while pancreas progenitors marked by ptf1a or pdx1 remained unaffected, suggesting a role for Nr5a2 in regulating pancreatic acinar cell differentiation. In the developing liver, Nr5a2 regulates hepatic progenitor outgrowth and differentiation, as nr5a2 mutants exhibited reduced hepatoblast markers hnf4α and prox1 as well as differentiated hepatocyte marker fabp10a. Through the first in vivo use of Nr5a2 chemical antagonist Cpd3, the iterative requirement for Nr5a2 for exocrine pancreas and liver differentiation was temporally elucidated: chemical inhibition of Nr5a2 function during hepatopancreas progenitor specification was sufficient to disrupt exocrine pancreas formation and enhance the size of the embryonic liver, suggesting that Nr5a2 regulates hepatic vs. pancreatic progenitor fate choice. Chemical inhibition of Nr5a2 at a later time during pancreas and liver differentiation was sufficient to block the formation of mature acinar cells and hepatocytes. These findings define critical iterative and pleiotropic roles for Nr5a2 at distinct stages of pancreas and liver organogenesis, and provide novel perspectives for interpreting the role of Nr5a2 in disease.

Pharyngeal morphogenesis requires fras1-itga8-dependent epithelial-mesenchymal interaction.

Both Fras1 and Itga8 connect mesenchymal cells to epithelia by way of an extracellular 'Fraser protein complex' that functions in signaling and adhesion; these proteins are vital to the development of several vertebrate organs. We previously found that zebrafish fras1 mutants have craniofacial defects, specifically, shortened symplectic cartilages and cartilage fusions that spare joint elements. During a forward mutagenesis screen, we identified a new zebrafish mutation, b1161, that we show here disrupts itga8, as confirmed using CRISPR-generated itga8 alleles. fras1 and itga8 single mutants and double mutants have similar craniofacial phenotypes, a result expected if loss of either gene disrupts function of the Fraser protein complex. Unlike fras1 mutants or other Fraser-related mutants, itga8 mutants do not show blistered tail fins. Thus, the function of the Fraser complex differs in the craniofacial skeleton and the tail fin. Focusing on the face, we find that itga8 mutants consistently show defective outpocketing of a late-forming portion of the first pharyngeal pouch, and variably express skeletal defects, matching previously characterized fras1 mutant phenotypes. In itga8 and fras1 mutants, skeletal severity varies markedly between sides, indicating that both mutants have increased developmental instability. Whereas fras1 is expressed in epithelia, we show that itga8 is expressed complementarily in facial mesenchyme. Paired with the observed phenotypic similarity, this expression indicates that the genes function in epithelial-mesenchymal interactions. Similar interactions between Fras1 and Itga8 have previously been found in mouse kidney, where these genes both regulate Nephronectin (Npnt) protein abundance. We find that zebrafish facial tissues express both npnt and the Fraser gene fibrillin2b (fbn2b), but their transcript levels do not depend on fras1 or itga8 function. Using a revertible fras1 allele, we find that the critical window for fras1 function in the craniofacial skeleton is between 1.5 and 3 days post fertilization, which coincides with the onset of fras1-dependent and itga8-dependent morphogenesis. We propose a model wherein Fras1 and Itga8 interact during late pharyngeal pouch morphogenesis to sculpt pharyngeal arches through epithelial-mesenchymal interactions, thereby stabilizing the developing craniofacial skeleton.

In vivo assessment of contractile strength distinguishes differential gene function in skeletal muscle of zebrafish larvae.

The accessible genetics and extensive skeletal musculature of the zebrafish make it a versatile and increasingly used model for studying muscle contraction. We here describe the development of an in vivo assay for measuring the contractile force of intact zebrafish at the larval stage. In addition, as proof of applicability, we have used this assay to quantify contractile strength of zebrafish larvae in a morphant model of deranged rbfox function. Average maximum tetanic (180 Hz) whole body forces produced by wild-type larvae at 2, 3, 4, and 5 days postfertilization amounted to 3.0, 7.2, 9.1, and 10.8 mN, respectively. To compare at potentially different stages of muscle development, we developed an immunohistological assay for empirically determining the cross-sectional area of larval trunk skeletal muscle to quantify muscle-specific force per cross-sectional area. At 4-5 days postfertilization, specific force amounts to ∼ 300 mN/mm(2), which is similar to fully developed adult mammalian skeletal muscle. We used these assays to measure contractile strength in zebrafish singly or doubly deficient for two rbfox paralogs, rbfox1l and rbfox2, which encode RNA-binding factors shown previously to modulate muscle function and muscle-specific splicing. We found rbfox2 morphants produce maximal tetanic forces similar to wild-type larvae, whereas rbfox1l morphants demonstrate significantly impaired function. rbfox1l/rbfox2 morphants are paralyzed, and their lack of contractile force production in our assay suggests that paralysis is a muscle-autonomous defect. These quantitative functional results allow measurement of muscle-specific phenotypes independent of neural input.

Dynamics of the slowing segmentation clock reveal alternating two-segment periodicity.

The formation of reiterated somites along the vertebrate body axis is controlled by the segmentation clock, a molecular oscillator expressed within presomitic mesoderm (PSM) cells. Although PSM cells oscillate autonomously, they coordinate with neighboring cells to generate a sweeping wave of cyclic gene expression through the PSM that has a periodicity equal to that of somite formation. The velocity of each wave slows as it moves anteriorly through the PSM, although the dynamics of clock slowing have not been well characterized. Here, we investigate segmentation clock dynamics in the anterior PSM in developing zebrafish embryos using an in vivo clock reporter, her1:her1-venus. The her1:her1-venus reporter has single-cell resolution, allowing us to follow segmentation clock oscillations in individual cells in real-time. By retrospectively tracking oscillations of future somite boundary cells, we find that clock reporter signal increases in anterior PSM cells and that the periodicity of reporter oscillations slows to about ∼1.5 times the periodicity in posterior PSM cells. This gradual slowing of the clock in the anterior PSM creates peaks of clock expression that are separated at a two-segment periodicity both spatially and temporally, a phenomenon we observe in single cells and in tissue-wide analyses. These results differ from previous predictions that clock oscillations stop or are stabilized in the anterior PSM. Instead, PSM cells oscillate until they incorporate into somites. Our findings suggest that the segmentation clock may signal somite formation using a phase gradient with a two-somite periodicity.

A visualizable chain-terminating inhibitor of glycosaminoglycan biosynthesis in developing zebrafish.

Heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAG) are proteoglycan-associated polysaccharides with essential functions in animals. They have been studied extensively by genetic manipulation of biosynthetic enzymes, but chemical tools for probing GAG function are limited. HS and CS possess a conserved xylose residue that links the polysaccharide chain to a protein backbone. Here we report that, in zebrafish embryos, the peptide-proximal xylose residue can be metabolically replaced with a chain-terminating 4-azido-4-deoxyxylose (4-XylAz) residue by administration of UDP-4-azido-4-deoxyxylose (UDP-4-XylAz). UDP-4-XylAz disrupted both HS and CS biosynthesis and caused developmental abnormalities reminiscent of GAG biosynthesis and laminin mutants. The azide substituent of protein-bound 4-XylAz allowed for rapid visualization of the organismal sites of chain termination in vivo through bioorthogonal reaction with fluorescent cyclooctyne probes. UDP-4-XylAz therefore complements genetic tools for studies of GAG function in zebrafish embryogenesis.

Single-cell-resolution imaging of the impact of Notch signaling and mitosis on segmentation clock dynamics.

Vertebrate body segmentation is controlled by the segmentation clock, a molecular oscillator involving transcriptional oscillations of cyclic genes in presomitic mesoderm cells. The rapid and highly dynamic nature of this oscillating system has proved challenging for study at the single-cell level. We achieved visualization of clock activity with a cellular level of resolution in living embryos, allowing direct comparison of oscillations in neighbor cells. We provide direct evidence that presomitic mesoderm cells oscillate asynchronously in zebrafish Notch pathway mutants. By tracking oscillations in mitotic cells, we reveal that a robust cell-autonomous, Notch-independent mechanism resumes oscillations after mitosis. Finally, we find that cells preferentially divide at a certain oscillation phase, likely reducing the noise generated by cell division in cell synchrony and suggesting an intriguing relationship between the mitotic cycle and clock oscillation.