SummaryINNER NO OUTER (INO) expression is limited to the abaxial cell layer of the incipient and developing outer integument in Arabidopsis ovules. Using deletion analysis of the previously de®ned INO promoter (P-INO), at least three distinct regions that contribute to the endogenous INO expression pattern were identi®ed. One such positive element, designated POS9, which comprises at least three distinct subelements, was found to include suf®cient information to duplicate the INO expression pattern when four or more copies were used in conjunction with a heterologous minimal promoter. While known regulators of INO, including INO, SUPERMAN, BELL1, and AINTEGUMENTA, did not detectably interact with POS9 in yeast one-hybrid assays, two groups of proteins that interact speci®cally with POS9 were identi®ed in one-hybrid library screens. Members of one group include C2H2 zinc ®nger motifs. Members of the second group contain a novel, conserved DNA-binding region and were designated the BASIC PENTACYSTEINE (BPC) proteins on the basis of conserved features of this region. The BPC proteins are nuclear localized and speci®cally bind in vitro to GA dinucleotide repeats located within POS9. The widespread expression patterns of the BPCs and the large number of GA repeat potential target sequences in the Arabidopsis genome indicate that BPC proteins may affect expression of genes involved in a variety of plant processes.
SUMMARYKANADI (KAN) transcription factors promote abaxial cell fate throughout plant development and are required for organ formation during embryo, leaf, carpel and ovule development. ABERRANT TESTA SHAPE (ATS, or KAN4) is necessary during ovule development to maintain the boundary between the two ovule integuments and to promote inner integument growth. Yeast two-hybrid assays identified ETTIN (ETT, or AUXIN RESPONSE FACTOR 3) as a transcription factor that could physically interact with ATS. ATS and ETT were shown to physically interact in vivo in transiently transformed tobacco epidermal cells using bimolecular fluorescence complementation. ATS and ETT were found to share an overlapping expression pattern during Arabidopsis ovule development and loss of either gene resulted in congenital fusion of the integuments and altered seed morphology. We hypothesize that in wild-type ovules a physical interaction between ATS and ETT allows these proteins to act in concert to define the boundary between integument primordia. We further show protein-protein interaction in yeast between ETT and KAN1, a paralog of ATS. Thus, a direct physical association between ETT and KAN proteins underpins their previously described common role in polarity establishment and organogenesis. We propose that ETT-KAN protein complex(es) constitute part of an auxin-dependent regulatory module that plays a conserved role in a variety of developmental contexts. ETT cDNA without the stop codon was amplified from leaf cDNA and cloned into pENTR/D-Topo to create pDK74. pAA29 was Gateway cloned into pDEST-GBKT7 (Rossignol et al., 2007) creating pAA36 (BD-KAN2). pDK132 was Gateway cloned into pDEST-GADT7 creating plasmid pDK136 (AD-ETT). ETT cDNA was cloned as an NdeI/XhoI fragment from pDK136 into pGBKT7 using NdeI/SalI to create pDK137 (BD-ETT). ARF4 cDNA was amplified from Col leaf cDNA and cloned into pENTR/D-Topo, creating pAA30. pAA30 was Gateway cloned into pDEST-GADT7 to create pAA42 (AD-ARF4). KAN1 cDNA was amplified and inserted as a BamHI/PstI fragment into pLITMUS28 to create pLMK37 and transferred as a BamHI/PstI fragment into: (1) pGAD424, creating pLMK44 (AD-KAN1); and (2) pAS2, creating pLMK46 (BD-KAN1).A 700 bp subclone of ETT (pDK73) cDNA was generated from pAS13 as described (Sessions et al., 1997). ATS-eGFP (pDK77) was created by Gateway cloning pAA34 into pDH51-GW-eGFP (Zhong et al., 2008). ETT-eGFP (pDK80) was created by Gateway cloning pDK74 into pDH51-GW-eGFP. Subclones for ATS-YFPc (pCG51) and ETT-YFPn (pCG54) were created by amplifying ATS or ETT cDNA without stop codons and inserting the resulting fragments into pJET1.2 (Fermentas) to form pCG45 and pCG46, respectively. The cDNAs were inserted into 2X35S-SPYCE or 2X35S-SPYNE vectors (Walter et al., 2004), respectively, as XhoI/XmaI fragments into these same sites forming pCG47 and pCG50. The resulting expression cassettes were transferred into pMLBART (Gleave, 1992) as NotI fragments, producing pCG51and pCG54. Gateway reactions were performed using LR Clonase II (Invitro...
Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos was strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle function.
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 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.
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