Biomedical subjects

Yau-Hung Chen

Publications and source records attributed to Yau-Hung Chen.

11 recordsLinked to original sources

PubMed2007

Multiple upstream modules regulate zebrafish myf5 expression.

BACKGROUND: Myf5 is one member of the basic helix-loop-helix family of transcription factors, and it functions as a myogenic factor that is important for the specification and differentiation of muscle cells. The expression of myf5 is somite- and stage-dependent during embryogenesis through a delicate regulation. However, this complex regulatory mechanism of myf5 is not clearly understood. RESULTS: We isolated a 156-kb bacterial artificial chromosome clone that includes an upstream 80-kb region and a downstream 70-kb region of zebrafish myf5 and generated a transgenic line carrying this 156-kb segment fused to a green fluorescent protein (GFP) reporter gene. We find strong GFP expression in the most rostral somite and in the presomitic mesoderm during segmentation stages, similar to endogenous myf5 expression. Later, the GFP signals persist in caudal somites near the tail bud but are down-regulated in the older, rostral somites. During the pharyngula period, we detect GFP signals in pectoral fin buds, dorsal rostral myotomes, hypaxial myotomes, and inferior oblique and superior oblique muscles, a pattern that also corresponds well with endogenous myf5 transcripts. To characterize the specific upstream cis-elements that regulate this complex and dynamic expression pattern, we also generated several transgenic lines that harbor various lengths within the upstream 80-kb segment. We find that (1) the -80 kb/-9977 segment contains a fin and cranial muscle element and a notochord repressor; (2) the -9977/-6213 segment contains a strong repressive element that does not include the notochord-specific repressor; (3) the -6212/-2938 segment contains tissue-specific elements for bone and spinal cord; (4) the -2937/-291 segment contains an eye enhancer, and the -2937/-2457 segment is required for notochord and myocyte expression; and (5) the -290/-1 segment is responsible for basal transcription in somites and the presomitic mesoderm. CONCLUSION: We suggest that the cell lineage-specific expression of myf5 is delicately orchestrated by multiple modules within the distal upstream region. This study provides an insight to understand the molecular control of myf5 and myogenesis in the zebrafish.

Animals↗

PubMed2006

Myogenic regulatory factors Myf5 and Myod function distinctly during craniofacial myogenesis of zebrafish.

The functions of Myf5 and Myod are well known in trunk myogenesis. However, the roles that Myf5 and Myod play during craniofacial myogenesis are far from well known. We observed that zebrafish myf5 was detected in the primordia of the obliques, lateral rectus, sternohyoideus, and pharyngeal mesoderm cores. In contrast, myod transcripts were expressed in all head muscle precursors at later stages. Knockdown of myf5 revealed that Myf5 was required for the development of the obliques, lateral rectus, sternohyoideus, and all pharyngeal muscles, whereas knockdown of myod proved that Myod was required for the development of superior rectus, medial rectus, inferior rectus, lateral rectus, and the ventral pharyngeal muscles. myod mRNA did not rescue the loss of the cranial muscle caused by injecting myf5-morpholino, or vice versa, suggesting that the functions of Myf5 and Myod were not redundant in head paraxial mesoderm, a finding different from their functions in trunk myogenesis. Myf5, but not Myod, was required for the forward migration of myf5-positive oblique precursors. All evidences reveal that Myf5 and Myod function independently during cranial myogenesis. On the basis of the expression patterns of myf5 and myod, we propose a model to present how Myf5 and Myod are involved in head myogenesis of zebrafish.

Animals↗

PubMed2006

Spatiotemporal expression of zebrafish keratin 18 during early embryogenesis and the establishment of a keratin 18:RFP transgenic line.

Zebrafish cytokeratin 18 (K18) is one of the type I keratin genes expressed the earliest after amputation of the zebrafish fin, but its spatiotemporal expression during early development is unclear. Whole-mount in situ hybridization revealed that k18 was a maternally inherited gene and that its expression is restricted to the single layer of enveloping cells on the surface of embryos during the gastrula stage. At later stages, K18 expression was detected in the epithelial cells, pronephric duct, digestive tract, dorsal aorta, and fins. By using microinjection, we generated the transgenic line Tg(k18(2.9):RFP), which carries an upstream 2.9-kb segment of k18 gene fused with a red fluorescent protein (RFP) reporter. The spatiotemporal distributions of red fluorescent signal of Tg(k18(2.9):RFP) line correlated well with endogenous k18 transcripts detected by whole-mount in situ hybridization, indicating that this line is capable of recapitulating endogenous k18 expression patterns. We noticed that the red fluorescence appeared strongly in the dorsal, pectoral, pelvic, anal, and caudal fins when transgenic fish became adults. Interestingly, we also found that when F1 female from the Tg(k18(2.9):RFP) line were mated with wild-type males, 100% (326/326) of F2 offspring expressed red fluorescence at the one-cell stage. In contrast, when F1 male from the Tg(k18(2.9):RFP) line were mated with wild-type females, only 49.8% (138/277) of F2 embryos exhibited red fluorescence. On the basis of these findings, we suggest that the transcript of zebrafish K18 is inherited as a maternal effect. We believe that Tg(k18(2.9):RFP) fish should be an excellent experimental animal for studying the zygotic regulatory mechanism of k18.

Animals↗

PubMed2006

A keratin 18 transgenic zebrafish Tg(k18(2.9):RFP) treated with inorganic arsenite reveals visible overproliferation of epithelial cells.

Inorganic arsenic has strong human carcinogenic potential, but the availability of an animal model to study toxicity is extremely limited. Here, we used the transgenic zebrafish line Tg(k18(2.9):RFP) as an animal model to study arsenite toxicity. This line was chosen because the red fluorescent protein (RFP) is expressed in stratified epithelia (including skin), due to the RFP reporter driven by the promoter of the zebrafish keratin 18 gene. We titrated doses of inorganic arsenite for zebrafish embryos and found that arsenite exposure at 50 microM for 120 h was suitable for mimicking a long-term, chronic effect. When embryos derived from Tg(k18(2.9):RFP) adults were treated with this arsenite dose and time of exposure, abnormal phenotypes were not noticeable under the light microscope. However, arsenic keratosis was visible in the epithelial cells under the fluorescent microscope. Morphological defects became more severe with increased dose and exposure duration, suggesting that the severity of skin lesions was dose- and time-dependent. Histochemical examination of keratosis after 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) staining showed that the epithelial cells overproliferated after treatment with arsenite. Therefore, this Tg(k18(2.9):RFP) zebrafish line is an excellent model for studying toxicity induced by inorganic arsenite and may have potential for studying other environmental pollutants.

Animals↗

PubMed2006

Foxd3 mediates zebrafish myf5 expression during early somitogenesis.

Myf5, one of the basic helix-loop-helix transcription factors, controls muscle differentiation and is expressed in somites during early embryogenesis. However, the transcription factors bound to the cis-elements of myf5 are poorly understood. In this study, we used the yeast one-hybrid assay and found that Forkhead box d3 (Foxd3) interacted specifically with the -82/-62 cassette, a key element directing somite-specific expression of myf5. The dual-luciferase assay revealed that the expression of Foxd3 potently transactivated the myf5 promoter. Knocking down foxd3 with morpholino oligonucleotide (MO) resulted in a dramatic down-regulation of myf5 in somites and adaxial cells but not in the presomitic mesoderm. On the other hand, myod expression remained unchanged in foxd3 morphants. Foxd3 mediation of myf5 expression is stage-dependent, maintaining myf5 expression in the somites and adaxial cells during the 7- to 18-somite stage. Furthermore, in the pax3 morphant, the expression of foxd3 was down-regulated greatly and the expression of myf5 was similar to that of the foxd3 morphant. Co-injection of foxd3 mRNA and pax3-MO1 greatly restored the expression of myf5 in the somites and adaxial cells, suggesting that pax3 induces foxd3 expression, which then induces the expression of myf5. This report is the first study to show that Foxd3, a well-known regulator in neural crest development, is also involved in myf5 regulation.

Animals↗

PubMed2006

Epidermis-restricted expression of zebrafish cytokeratin II is controlled by a -141/+85 minimal promoter, and cassette -141/-111 is essential for driving the tissue specificity.

We isolated a 2.3 kb DNA segment from the upstream region of the zebrafish cytokeratin II (zfCKII) gene. Transgenic embryos, produced by using a series of 5' deletions linked to the red fluorescent protein (RFP) reporter, showed that the -141/+85 segment of zfCKII directed RFP expression in epidermal cells, whereas the -111/+85 segment did not. When -141/-111 was deleted from -355/+85 and microinjected into one-celled embryos, no fluorescence was observed at later stages, indicating that the -141/-111 segment is required for green fluorescent protein expression in epidermal cells. Furthermore, when a putative KLF-binding site at -119/-117 was mutated, RFP expression rates and intensities were reduced dramatically, although still observed, suggesting that -119/-117 within -141/-111 is a key cis-element for controlling epidermis-specific expression of the zfCKII gene. Finally, we generated a zebrafish transgenic line, Tg(zfCKII(2.3):RFP), which carries an upstream 2.3 kb regulatory region of the zfCKII gene fused with RFP. The expression pattern in the epidermal cells of Tg(zfCKII(2.3):RFP) fish recapitulated that of the endogenous gene. F2 embryos derived from Tg(zfCKII(2.3):RFP) males crossed with wild-type females revealed that the earliest onset of RFP expression was at the sphere stage, indicating that this transgenic approach can be used for studying zygotic expression of maternally inherited genes.

Animals↗

PubMed2005

A 23-amino acid motif spanning the basic domain targets zebrafish myogenic regulatory factor myf5 into nucleolus.

Myf5 is a nuclear protein and one of the basic helix-loop-helix (bHLH) myogenic factors that play an important role in muscle specification and differentiation. The motif responsible for the nuclear translocation of Myf5 was unknown. Using on-line monitoring of EGFP (enhanced green fluorescent protein)-tagged zebrafish Myf5 translocation, we demonstrated that Myf5-EGFP protein resided in the nucleoplasm and nucleolus of zebrafish fibroblast cell lines (ZEM2S and ZF4), mammalian nonmuscle cell line (COS1), and muscle cell lines (RD and C2C12). In contrast, zebrafish MyoD-EGFP was localized in the nucleus but did not condense in the nucleolus. Using indirect immunofluorescent staining, we determined that zebrafish Myf5 was colocalized with nucleophosmin/B23, a nucleolus protein. Deletion analysis revealed that amino acid residues 60 to 82 (60KRKASTVDRRRAATMRERRRLKK82) of Myf5 were sufficient and necessary for nucleolus targeting. A GST pulldown assay followed by Western analysis showed that nucleolin/C23 could be pulled down specifically by GST-Myf5, but not by GST-MyoD. Based on these findings, we propose that the distinct functions of Myf5 and MyoD may result from their differential binding affinity to nucleolin/C23.

Animals↗

PubMed2004

Novel cis-element in intron 1 represses somite expression of zebrafish myf-5.

Myf-5 is a basic helix-loop-helix (bHLH) transcription factor that controls muscle differentiation. During early embryogenesis, myf-5 expression is transient, somite- and stage-specific. However, the negative regulation of myf-5 is poorly understood. We constructed a plasmid [(-9977/-1)/E1/I1/E2/GFP] that contains the sequence -9977 to -1, exon 1 (E1), intron 1 (I1), and exon 2 (E2) of zebrafish (Danio rerio) myf-5 and a reporter GFP gene. This plasmid was microinjected into zebrafish zygotes. Surprisingly, the somite-specific expression rate of reporter GFP in the transgenic embryos was extremely low (2%, n=392), compared to that of (-9977/-1)/GFP (92%, n=210). Dramatic repression of myf-5 expression was also observed in embryos microinjected with plasmids in which the sequence -8600/-1, -2937/-1 or -290/-1 was linked to E1/I1/E2/GFP. Thus, intron 1 contains a silencer that specifically represses the activity of myf-5. Functional analysis of intron 1 showed a strong, negative, cis-regulatory element was located at +502/+835. Its function was orientation- and position-dependent. The repressive capability of this silencer was completely dependent on two core motifs, IE1 (+502/+527) and IE2 (+816/+835), and a 156-bp spanning sequence that lies between them. This is the first study to identify a novel, cis-acting silencer in intron 1 that is crucial to negatively regulating zebrafish myf-5 expression.

Animals↗

PubMed2003

Novel regulatory sequence -82/-62 functions as a key element to drive the somite-specificity of zebrafish myf-5.

Myf-5, a transcription factor that controls muscle differentiation, is expressed in somites during early embryogenesis. However, gene regulation of myf-5 is poorly understood and detailed functional analysis of the regulatory cis-elements is needed. In zebrafish, the myf-5 upstream sequence from -82 to -62 (-82/-62) was fused with a basal promoter and transferred to fertilized zebrafish eggs. The -82/-62 cassette drove green fluorescent protein (GFP) reporter gene expression specifically in the somites. Moreover, GFP signals were detected exclusively in the somites of 28-hpf embryos derived from eggs injected with pCMV-5x(-82/-62), which contained five copies of the -82/-62 cassette inserted within cytomegalovirus promoter/enhancer. Thus, the -82/-62 cassette, conserved in mouse myf-5, functions to drive somite-specific expression and to repress nonspecific expression during the early development of zebrafish embryos. Mutated sequence analysis of -82/-62 cassette showed that the -70/-62 sequence was the key element for controlling myf-5 specificity. The putative CCAAT-like box, located at -66/-62, could not direct somite-specific expression. A DNA-protein complex was specifically formed between the -70/-62 probe and embryonic nuclear extracts. We conclude that the -70/-62 motif is essential for controlling somite-specific expression and the CCAAT-like box is essential for activating gene transcription.

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PubMed2002

Functional analysis of the proximal promoter regions of fish rhodopsin and myf-5 genes using transgenesis.

Little is known about the cis-acting elements controlling transcriptional activities of fish rhodopsin and myf-5 genes. Transgenic medaka and zebrafish were used to characterize promoters of carp (Cyprinus carpio) rhodopsin gene (cRh) and zebrafish (Danio rerio) myf-5 gene (myf-5), respectively. Transgenic medaka carrying different lengths of cRh upstream fragments fused with enhanced green fluorescence protein (EGFP) gene revealed several functional regions. Both upstream regions from -1261 to -163 bp (-1261/-163) and -163/-138 contributed to enhance the retina-specific activities of cRh. The cNRE (-75/-63) and CSE (-52/-46) motifs located at the cRh proximal promoter were sufficient to drive the transgene expressed in retinae. The -73/-68 within cNRE was also proved to be essential for the integrity of the minimal cis-regulatory elements. Regarding the myf-5, transgene expression in zebrafish embryos injected with different deletion fragments of zebrafish myf-5 upstream sequences showed that the -2776/-2456 and -1046/-844 regions contributed to the enhancer function. The -290/-154 region might be involved in controlling the translocation of progenitor muscle cells. Moreover, the upstream -82/-1 region was identified as a minimal promoter required for somite-specific expression. The -82/-62 region was the most critical sequence for myf-5 specificity.

Journal Article↗

PubMed2002

Treatment with Myf5-morpholino results in somite patterning and brain formation defects in zebrafish.

Myf-5 is a stage-dependent transcription factor associated with somitogenesis. To study its biological functions in zebrafish, we injected the Myf5-morpholinos ZMF-MO (antisense nucleotides 28 to 52) and ZMF-OTHER (antisense nucleotides 3 to 27) into zebrafish embryos to establish a myf-5 gene knockdown. No phenotypic abnormalities were observed following injection with 0.2 ng of ZMF-MO, but defects were displayed in 2 of 118 (1.7%) surviving embryos injected with 1 ng ZMF-MO. Morphological defects became more severe with increased dosages: 105 of 270 (38.9%) surviving embryos injected with 4.5 ng of ZMF-MO displayed such abnormalities as the absence of eyes or brains in addition to the following low-dosage defects in 24 hpf embryos: longitudinal yolk sacs, incomplete epiboly coverage, abnormal and suspended tail buds, diffused somite boundaries, and head shrinkage. Similar results were observed in the 4.5 ng ZMF-OTHER injection group. However, when fish were co-injected with 4.5 ng ZMF-MO and 4.5 ng myf-5 mRNA, abnormality rates decreased from 49.6% to 5.5%. Our results show that the brain krox20 gene was down-regulated at rhombomere 3; the pax2.1 gene was completely down-regulated; myoD was expressed normally; myogenin was substantially down-regulated in whole somites; and desmin was partly inhibited in newly forming somites. Our conclusion is that zebrafish Myf-5 may play important roles in brain formation and in the convergence and extension of shield epiblasts and tail buds during early embryogenesis, in addition to its well-understood role as a muscle regulatory factor in somites.

Animals↗