Cheng-Yung Lin

In vivo developmental biology study using noninvasive multi-harmonic generation microscopy

Morphological changes and complex developmental processes inside vertebrate embryos are difficult to observe noninvasively with millimeter-penetration and sub-micrometer-resolution at the same time. By using higher harmonic generation, including second and third harmonics, as the microscopic contrast mechanism, optical noninvasiveness can be achieved due to the virtual-level-transition characteristic. The intrinsic nonlinearity of harmonic generations provides optical sectioning capability while the selected 1230-nm near-infrared light source provides the deeppenetration ability. The complicated development within a ~1.5-mm thick zebrafish (Danio rerio) embryo from initial cell proliferation, gastrulation, to tissue formation can all be observed clearly in vivo without any treatment on the live specimen.

Multiple upstream modules regulate zebrafish myf5 expression

BackgroundMyf5 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.ResultsWe 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.ConclusionWe 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.

Real-time second-harmonic-generation microscopy based on a 2-GHz repetition rate Ti:sapphire laser.

The problem of weak harmonic generation signal intensity limited by photodamage probability in optical microscopy and spectroscopy could be resolved by increasing the repetition rate of the excitation light source. Here we demonstrate the first photomultiplier-based real-time second-harmonic-generation microscopy taking advantage of the strongly enhanced nonlinear signal from a high-repetition-rate Ti:sapphire laser. We also demonstrate that the photodamage possibility in common biological tissues can be efficiently reduced with this high repetition rate laser at a much higher average power level compared to the commonly used ~80- MHz repetition rate lasers.

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.

The transcription factor Six1a plays an essential role in the craniofacial myogenesis of zebrafish

Transcription factor Six1a plays important roles in morphogenesis, organogenesis, and cell differentiation. However, the role of Six1a during zebrafish cranial muscle development is still unclear. Here, we demonstrated that Six1a was required for sternohyoideus, medial rectus, inferior rectus, and all pharyngeal arch muscle development. Although Six1a was also necessary for myod and myogenin expression in head muscles, it did not affect myf5 expression in cranial muscles that originate from head mesoderm. Overexpression of myod enabled embryos to rescue all the defects in cranial muscles induced by injection of six1a-morpholino (MO), suggesting that myod is directly downstream of six1a in controlling craniofacial myogenesis. However, overexpression of six1a was unable to rescue arch muscle defects in the tbx1- and myf5-morphants, suggesting that six1a is only involved in myogenic maintenance, not its initiation, during arch muscle myogenesis. Although the craniofacial muscle defects caused by pax3-MO phenocopied those induced by six1a-MO, injection of six1a, myod or myf5 mRNA did not rescue the cranial muscle defects in pax3 morphants, suggesting that six1a and pax3 do not function in the same regulatory network. Therefore, we proposed four putative regulatory pathways to understand how six1a distinctly interacts with either myf5 or myod during zebrafish craniofacial muscle development.

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.

Novel intronic microRNA represses zebrafish myf5 promoter activity through silencing dickkopf-3 gene

A strong, negative cis-element located at the first intron +502/+835 (I300) of zebrafish myf5 has been reported. To elucidate the molecular mechanism underlying this repression network, we microinjected zebrafish single-cell embryos with I300 RNA, resulting in the dramatic reduction of luciferase activity driven by the myf5 promoter. Within this I300 segment, we identified an intronic microRNA (miR-In300) located at +609/+632 and found that it was more highly expressed in the older mature somites than those newly formed, which negatively correlated with the distribution of zebrafish myf5 transcripts. We proved that miR-In300 suppressed the transcription of myf5 through abolishing myf5 promoter activity, and we subsequently identified the long isoform of the Dickkopf-3 gene (dkk3) as the target gene of miR-In300. We further found that injection of the dkk3-morpholinos (MOs) resulted in downregulation of myf5 transcripts in somites, whereas co-injection of myf5 mRNA with dkk3-MO1 enabled rescue of the defects induced by dkk3-MO1 alone. Finally, injection of miR-In300-MO enhanced both myf5 transcripts in somites and the level of Dkk3 protein in zebrafish embryos. Based on these findings, we concluded that miR-In300 binds to its target gene dkk3, which inhibits the translation of dkk3 mRNA and, in turn, suppresses zebrafish myf5 promoter activity.

miR-1 and miR-206 target different genes to have opposing roles during angiogenesis in zebrafish embryos

As miR-1 and miR-206 share identical seed sequences, they are commonly speculated to target the same gene. Here, we identify an mRNA encoding seryl-tRNA synthetase (SARS), which is targeted by miR-1, but refractory to miR-206. SARS is increased in miR-1-knockdown embryos, but it remains unchanged in the miR-206 knockdown. Either miR-1 knockdown or sars overexpression results in a failure to develop some blood vessels and a decrease in vascular endothelial growth factor Aa (VegfAa) expression. In contrast, sars knockdown leads to an increase of VegfAa expression and abnormal branching of vessels, similar to the phenotypes of vegfaa-overexpressed embryos, suggesting that miR-1 induces angiogenesis by repressing SARS. Unlike the few endothelial cells observed in the miR-1-knockdown embryos, knockdown of miR-206 leads to abnormal branching of vessels accompanied by an increase in endothelial cells and VegfAa. Therefore, we propose that miR-1 and miR-206 target different genes and thus have opposing roles during embryonic angiogenesis in zebrafish.

Transgenic zebrafish model to study translational control mediated by upstream open reading frame of human chop gene

Upstream open reading frame (uORF)-mediated translational inhibition is important in controlling key regulatory genes expression. However, understanding the underlying molecular mechanism of such uORF-mediated control system in vivo is challenging in the absence of an animal model. Therefore, we generated a zebrafish transgenic line, termed huORFZ, harboring a construct in which the uORF sequence from human CCAAT/enhancer-binding protein homologous protein gene (huORFchop) is added to the leader of GFP and is driven by a cytomegalovirus promoter. The translation of transgenic huORFchop-gfp mRNA was absolutely inhibited by the huORFchop cassette in huORFZ embryos during normal conditions, but the downstream GFP was only apparent when the huORFZ embryos were treated with endoplasmic reticulum (ER) stresses. Interestingly, the number and location of GFP-responsive embryonic cells were dependent on the developmental stage and type of ER stresses encountered. These results indicate that the translation of the huORFchop-tag downstream reporter gene is controlled in the huORFZ line. Moreover, using cell sorting and microarray analysis of huORFZ embryos, we identified such putative factors as Nrg/ErbB, PI3K and hsp90, which are involved in huORFchop-mediated translational control under heat-shock stress. Therefore, using the huORFZ embryos allows us to study the regulatory network involved in human uORFchop-mediated translational inhibition.

MicroRNA-3906 Regulates Fast Muscle Differentiation through Modulating the Target Gene homer-1b in Zebrafish Embryos

A microRNA, termed miR-In300 or miR-3906, suppresses the transcription of myf5 through silencing dickkopf-related protein 3 (dkk3r/dkk3a) during early development when myf5 is highly transcribed, but not at late stages when myf5 transcription is reduced. Moreover, after 24 hpf, when muscle cells are starting to differentiate, Dkk3a could not be detected in muscle tissue at 20 hpf. To explain these reversals, we collected embryos at 32 hpf, performed assays, and identified homer-1b, which regulates calcium release from sarcoplasmic reticulum, as the target gene of miR-3906. We further found that either miR-3906 knockdown or homer-1b overexpression increased expressions of fmhc4 and atp2a1 of calcium-dependent fast muscle fibrils, but not slow muscle fibrils, and caused a severe disruption of sarcomeric actin and Z-disc structure. Additionally, compared to control embryos, the intracellular calcium concentration ([Ca2+]i) of these treated embryos was increased as high as 83.9–97.3% in fast muscle. In contrast, either miR-3906 overexpression or homer-1b knockdown caused decreases of [Ca2+]i and, correspondingly, defective phenotypes in fast muscle. These defects could be rescued by inducing homer-1b expression at later stage. These results indicate that miR-3906 controls [Ca2+]i homeostasis in fast muscle through fine tuning homer-1b expression during differentiation to maintain normal muscle development.