Biomedical subjects

Huai-Jen Tsai

Publications and source records attributed to Huai-Jen Tsai.

At least 19 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

Egr1 gene knockdown affects embryonic ocular development in zebrafish.

PURPOSE: To identify the changes in zebrafish embryonic ocular development after early growth response factor 1 (Egr1) gene knockdown by Egr1-specific translation inhibitor, morpholino oligonucleotides (MO). METHODS: Two kinds of Egr1-MO were microinjected separately with various dosages into one to four celled zebrafish embryos to find an optimal dose generating an acceptable mortality rate and high frequency of specific phenotype. Chordin-MO served as the positive control; a 5 mismatch MO of Egr1-MO1 and a nonspecific MO served as negative controls. We graded the Egr1 morphants according to their gross abnormalities, and measured their ocular dimensions accordingly. Western blot analysis and synthetic Egr1 mRNA rescue experiments confirmed whether the deformities were caused by Egr1 gene knockdown. Histological examination and three kinds of immunohistochemical staining were applied to identify glutamate receptor one expression in retinal ganglion cells and amacrine cells, to recognize acetylated alpha-tubulin expression which indicated axonogenesis, and to label photoreceptor cells with zpr-1 antibody. RESULTS: After microinjection of 8 ng Egr1-MO1 or 2 ng Egr1-MO2, 81.8% and 97.3% of larvae at 72 h postfertilization had specific defects, respectively. The gross phenotype included string-like heart, flat head, and deformed tail. The more severely deformed larvae had smaller eyes and pupils. Co-injection of 8 ng Egr1-MO1 and supplementary 12 pg synthetic Egr1 mRNA reduced the gross abnormality rate from 84.4% to 29.7%, and decreased the severity of deformities. Egr1 protein appeared in the wildtype and rescued morphants, but was lacking in the Egr1 morphants with specific deformities. Lenses of Egr1 morphants were smaller and had some residual nucleated lens fiber cells. Morphants' retinal cells arranged disorderly and compactly with thin plexiform layers. Immunohistochemical studies showed that morphants had a markedly decreased number of mature retinal ganglion cells, amacrine cells, and photoreceptor cells. Retinal axonogenesis was prominently reduced in morphants. CONCLUSIONS: The Egr1 gene plays an important role in zebrafish embryonic oculogenesis. Ocular structures including lens and retina were primitive and lacked appropriate differentiation. Such arrested retinal and lenticular development in Egr1 morphants resulted in microphthalmos.

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PubMed2006

TEL-AML1 transgenic zebrafish model of precursor B cell acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a clonal disease that evolves through the accrual of genetic rearrangements and/or mutations within the dominant clone. The TEL-AML1 (ETV6-RUNX1) fusion in precursor-B (pre-B) ALL is the most common genetic rearrangement in childhood cancer; however, the cellular origin and the molecular pathogenesis of TEL-AML1-induced leukemia have not been identified. To study the origin of TEL-AML1-induced ALL, we generated transgenic zebrafish expressing TEL-AML1 either ubiquitously or in lymphoid progenitors. TEL-AML1 expression in all lineages, but not lymphoid-restricted expression, led to progenitor cell expansion that evolved into oligoclonal B-lineage ALL in 3% of the transgenic zebrafish. This leukemia was transplantable to conditioned wild-type recipients. We demonstrate that TEL-AML1 induces a B cell differentiation arrest, and that leukemia development is associated with loss of TEL expression and elevated Bcl2/Bax ratio. The TEL-AML1 transgenic zebrafish models human pre-B ALL, identifies the molecular pathways associated with leukemia development, and serves as the foundation for subsequent genetic screens to identify modifiers and leukemia therapeutic targets.

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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

Semaphorin3D regulates invasion of cardiac neural crest cells into the primary heart field.

The primary heart field in all vertebrates is thought to be derived exclusively from lateral plate mesoderm (LPM), which gives rise to a cardiac tube shortly after gastrulation. The heart tube then begins looping and additional cells are added from other embryonic regions, including the secondary heart field, cardiac neural crest and the proepicardial organ. Here we show in zebrafish that neural crest cells invade and contribute cardiac myosin light chain2 (cmlc2)-positive cardiomyocytes to the primary heart field. Knockdown of semaphorin3D, which is expressed in the neural crest but apparently not in LPM, reduces the size of the primary heart field and the number of cardiomyocytes in the primary heart field by 20% before formation of the primary heart tube. Sema3D morphants have subsequent complex congenital heart defects, including hypertrophic cardiomyocytes, decreased ventricular size and defects in trabeculation and in atrioventricular (AV) valve development. Neuropilin1A, a semaphorin receptor, is expressed in LPM but apparently not in the neural crest, and nrp1A morphants have cardiac development defects. We propose that a population of sema3D-dependent neural crest cells follow a novel migratory pathway, perhaps toward nrp1A-expressing LPM, and serve as an important early source of cardiomyocytes in the primary heart field.

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PubMed2006

The embryonic vertebrate heart tube is a dynamic suction pump.

The embryonic vertebrate heart begins pumping blood long before the development of discernable chambers and valves. At these early stages, the heart tube has been described as a peristaltic pump. Recent advances in confocal laser scanning microscopy and four-dimensional visualization have warranted another look at early cardiac structure and function. We examined the movement of cells in the embryonic zebrafish heart tube and the flow of blood through the heart and obtained results that contradict peristalsis as a pumping mechanism in the embryonic heart. We propose a more likely explanation of early cardiac dynamics in which the pumping action results from suction due to elastic wave propagation in the heart tube.

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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

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.

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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.

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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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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.

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PubMed2005

Conditional expression of a myocardium-specific transgene in zebrafish transgenic lines.

To develop the first heart-specific tetracycline (Tet)-On system in zebrafish, we constructed plasmids in which the cardiac myosin light chain 2 promoter of zebrafish was used to drive the reverse Tet-controlled transactivator (rtTA) and the green fluorescent protein (GFP) reporter gene was preceded by an rtTA-responsive element. In the zebrafish fibroblast cell-line, rtTA-M2, one of rtTA's derivatives, demonstrated the highest increase in luciferase activity upon doxycycline (Dox) induction. We then generated two germ lines of transgenic zebrafish: line T03 was derived from microinjection of a plasmid containing rtTA-M2 and a plasmid containing a responsive reporter gene, whereas line T21 was derived from microinjection of a single dual plasmid. Results showed that line T21 was superior to line T03 in terms of greater GFP intensity after induction and with of minimal leakiness before induction. The photographic images of induced GFP in the heart of F2 larvae showed that the fluorescent level of GFP was dose-responsive. The level of GFP expressed in the F3 3 days postfertilization larvae that were treated with Dox for 1 hr decreased gradually after the withdrawal of the inducer; and the fluorescent signal disappeared after 5 days. The GFP induction and reduction were also tightly controlled by Dox in the F3 adult fish from line T21. This Tet-On system developed in zebrafish shows much promise for the study of the gene function in a specific tissue at the later developmental stage.

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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.

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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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PubMed2004

Higher harmonic generation microscopy for developmental biology.

Optical higher harmonic generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to its interacted matters due to an energy-conservation characteristic, providing the "noninvasiveness" nature desirable for biological studies. Combined with its nonlinearity, higher harmonic generation microscopy provides excellent three-dimensional (3D) sectioning capability, offering new insights into the studies of embryonic morphological changes and complex developmental processes. By choosing a laser working in the biological penetration window, here we present a noninvasive in vivo light microscopy with sub-micron 3D resolution and millimeter penetration, utilizing endogenous higher harmonic generation signals in live specimens. Noninvasive imaging was performed in live zebrafish (Danio rerio) embryos. The complex developmental processes within > 1-mm-thick zebrafish embryos can be observed in vivo without any treatment. No optical damage was found even with high illumination after long-term observations and the examined embryos all developed normally at least to the larval stage. The excellent 3D resolution of the demonstrated technology allows us to capture the subtle developmental information on the cellular or sub-cellular levels occurring deep inside the live embryos and larvae. This technique can not only provide in vivo observation of the cytoarchitecture dynamics during embryogenesis with submicron resolution and millimeter penetration depth, but would also make strong impact in developmental and structural biology studies.

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PubMed2003

Na,K-ATPase is essential for embryonic heart development in the zebrafish.

Na,K-ATPase is an essential gene maintaining electrochemical gradients across the plasma membrane. Although previous studies have intensively focused on the role of Na,K-ATPase in regulating cardiac function in the adults, little is known about the requirement for Na,K-ATPase during embryonic heart development. Here, we report the identification of a zebrafish mutant, heart and mind, which exhibits multiple cardiac defects, including the primitive heart tube extension abnormality, aberrant cardiomyocyte differentiation, and reduced heart rate and contractility. Molecular cloning reveals that the heart and mind lesion resides in the alpha1B1 isoform of Na,K-ATPase. Blocking Na,K-ATPase alpha1B1 activity by pharmacological means or by morpholino antisense oligonucleotides phenocopies the patterning and functional defects of heart and mind mutant hearts, suggesting crucial roles for Na,K-ATPase alpha1B1 in embryonic zebrafish hearts. In addition to alpha1B1, the Na,K-ATPase alpha2 isoform is required for embryonic cardiac patterning. Although the alpha1B1 and alpha2 isoforms share high degrees of similarities in their coding sequences, they have distinct roles in patterning zebrafish hearts. The phenotypes of heart and mind mutants can be rescued by supplementing alpha1B1, but not alpha2, mRNA to the mutant embryos, demonstrating that alpha1B1 and alpha2 are not functionally equivalent. Furthermore, instead of interfering with primitive heart tube formation or cardiac chamber differentiation, blocking the translation of Na,K-ATPase alpha2 isoform leads to cardiac laterality defects.

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PubMed2003

Transgenic zebrafish with fluorescent germ cell: a useful tool to visualize germ cell proliferation and juvenile hermaphroditism in vivo.

Juvenile zebrafish are hermaphroditic; undifferentiated gonads first develop into ovary-like tissues, which then either become ovaries and produce oocytes (female) or degenerate and develop into testes (male). In order to fully capture the dynamic processes of germ cells' proliferation and juvenile hermaphroditism in zebrafish, we established transgenic lines TG(beta-actin:EGFP), harboring an enhanced green fluorescent protein (EGFP) gene driven by a medaka beta-actin promoter. In TG(beta-actin:EGFP), proliferating germ cells and female gonads strongly expressed EGFP, but fluorescence was only dimly detected in male gonads. Based on the fluorescent (+) or nonfluorescent (-) appearance of germ cells seen in living animals, three distinct groups were evident among TG(beta-actin:EGFP). Transgenics in ++ group (44%) were females, had fluorescent germ cells as juveniles, and female gonads continuously fluoresced throughout sexual maturation. Transgenics in +- (23%) and -- (33%) groups were males. Fluorescent germ cells were transiently detected in +- transgenics from 14 to 34 days postfertilization (dpf), but were not detected in -- transgenics throughout their life span. Histological analyses showed that 26-dpf-old transgenics in ++, +-, and -- groups all developed ovary-like tissues: Germ cells in -- group juveniles arrested at the gonocyte stage and accumulated low quantities of EGFP, while those in ++ group juveniles highly proliferated into diplotene to perinucleolar stages and accumulated high quantities of EGFP. In +- group juveniles, degenerating oocytes, gonocytes, and spermatogonia were coexistent in transiently fluorescent gonads. Therefore, the fluorescent appearance of gonads in this study was synchronous with the differentiation of ovary-like tissues. Thus, TG(beta-actin:EGFP) can be used to visualize germ cells' proliferation and juvenile hermaphroditism in living zebrafish for the first time.

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PubMed2003

Activation of Notch signaling pathway precedes heart regeneration in zebrafish.

Several vertebrates display the ability to regenerate parts of their body after amputation. During this process, differentiated cells reenter the cell cycle and proliferate to generate a mass of undifferentiated cells. Repatterning mechanisms act on these cells to eventually shape a regenerated tissue or organ that replaces the amputated one. Experiments with regenerating limbs/fins in newts and zebrafish have shown that members of the Msx family of homeodomain-containing transcription factors play key roles during blastema formation and patterning. Here we show that adult zebrafish have a remarkable capacity to regenerate the heart in a process that involves up-regulation of msxB and msxC genes. We present evidence indicating that heart regeneration involves the execution of a specific genetic program, rather than redeployment of a cardiac development program. Preceding Msx activation, there is a marked increase in the expression of notch1b and deltaC, which we show are also up-regulated during fin regeneration. These data suggest a role for the Notch pathway in the activation of the regenerative response. Taken together, our results underscore the use of zebrafish as a model for investigating the process of regeneration in particular and the biology of stem cells in general. Advances in these fields will undoubtedly aid in the implementation of strategies for regenerative medicine.

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