Biomedical subjects

Cheng-Yung Lin

Publications and source records attributed to Cheng-Yung Lin.

7 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.

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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.

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PubMed2006

Biomolecular imaging based on far-red fluorescent protein with a high two-photon excitation action cross section.

Received October 14, 2005; revised January 7, 2006; accepted January 9, 2006; posted January 12, 2006 (Doc. ID 65391) The two-photon excitation action cross section of Hc-Red fluorescent proteins (Hc-RFPs) is measured and found to be of the same order as that of enhanced green fluorescent proteins. With a 618 nm emission wavelength in the far-red region and with an excitation wavelength around 1200 nm, Hc-RPF-based two-photon fluorescence microscopy (2PFM) can offer deep penetration capability inside live samples and is ideal for in vivo gene expression study and biomolecular imaging in live objects. In vivo 2PFM of the developing heart deep inside a transgenic zebrafish embryo tagged by Hc-RFP is also successfully demonstrated.

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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.

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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.

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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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PubMedDate not supplied

Noninvasive harmonics optical microscopy for long-term observation of embryonic nervous system development in vivo.

Nervous system development is a complicated dynamic process, and many mechanisms remain unknown. By utilizing endogenous second-harmonic-generation as the contrast of polarized nerve fibers and third-harmonic-generation (THG) to reveal morphological changes, we have successfully observed the vertebrate embryonic nervous development from the very beginning based on a 1230-nm light source. The dynamic development of the nerve system within a live zebrafish embryo can be recorded continuously more than 20 hr without fluorescence markers. Since the THG process is not limited by the time of gene expression and differentiation as fluorescence-based techniques are, the observable stages can be advanced to the very beginning of the development process. The complete three-dimensional brain development from a neural plate to a neural tube can be uncovered with a submicron lateral resolution. We have, for the first time, also reported the generation of SHG from myelinated nerve fibers and the outer segment of the photoreceptors with a stacked membrane structure. Our study clearly indicates the fact that higher-harmonics-based optical microscopy has the strong potential to long-term in vivo study of the nervous system, including genetic disorders of the nervous system, axon pathfinding, neural regeneration, neural repair, and neural stem cell development.

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