Daniel P. Szeto

Pitx2 regulates lung asymmetry, cardiac positioning and pituitary and tooth morphogenesis

Pitx1 (refs 1,2,3) and Pitx2 (refs 4, 5) are highly homologous, bicoid-related transcription factors. Pitx2 was initially identified as the gene responsible for the human Rieger syndrome, an autosomal dominant condition that causes developmental abnormalities. Pitx2 is asymmetrically expressed in the left lateral-plate mesoderm, and mutant mice with laterality defects show altered patterns of Pitx2 expression that correlate with changes in the visceral symmetry (situs). Ectopic expression of Pitx2 in the right lateral-plate mesoderm alters looping of the heart and gut and reverses body rotation in chick and Xenopus embryos. Here we describe the phenotype of Pitx2 gene-deleted mice, characterized by defective body-wall closure, right pulmonary isomerism, altered cardiac position, arrest in turning and, subsequently, a block in the determination and proliferation events of anterior pituitary gland and tooth organogenesis. Thus, Pitx2 is a transcription factor that encodes ‘leftness’ of the lung.

Signal-specific co-activator domain requirements for Pit-1 activation

POU-domain proteins, such as the pituitary-specific factor Pit-1, are members of the homeodomain family of proteins which are important in development and homeostasis, acting constitutively or in response to signal-transduction pathways to either repress or activate the expression of specific genes. Here we show that whereas homeodomain-containing repressors such as Rpx seem to recruit only a co-repressor complex, the activity of Pit-1 (ref. 3) is determined by a regulated balance between a co-repressor complex that contains N-CoR/SMRT,, mSin3A/B and histone deacetylases and a co-activator complex that includes the CREB-binding protein (CBP) and p/CAF. Activation of Pit-1 by cyclic AMP or growth factors depends on distinct amino- and carboxy-terminal domains of CBP, respectively. Furthermore, thehistone acetyltransferase functions of CBP, or p/CAF are required for Pit-1 function that is stimulated by cyclic AMP or growth factors, respectively. These data show that there is a switch in specific requirements for histone acetyltransferases and CBP domains in mediating the effects of different signal-transduction pathways on specific DNA-bound transcription factors.

Reciprocal Interactions of Pit1 and GATA2 Mediate Signaling Gradient–Induced Determination of Pituitary Cell Types

The mechanisms by which transient gradients of signaling molecules lead to emergence of specific cell types remain a central question in mammalian organogenesis. Here, we demonstrate that the appearance of four ventral pituitary cell types is mediated via the reciprocal interactions of two transcription factors, Pit1 and GATA2, which are epistatic to the remainder of the cell type-specific transcription programs and serve as the molecular memory of the transient signaling events. Unexpectedly, this program includes a DNA binding-independent function of Pit1, suppressing the ventral GATA2-dependent gonadotrope program by inhibiting GATA2 binding to gonadotrope- but not thyrotrope-specific genes, indicating that both DNA binding-dependent and -independent actions of abundant determining factors contribute to generate distinct cell phenotypes.

Biocompatibility and Biodistribution of Surface-Enhanced Raman Scattering Nanoprobes in Zebrafish Embryos: In vivo and Multiplex Imaging

Nanoparticles are increasingly being used to investigate biological processes in various animal models due to their versatile chemical, unique optical, and multifunctional properties. In this report we address the biocompatibility and biodistribution of nanoparticle sensors used for Raman chemical imaging in live zebrafish (Danio rerio) embryos. Surface-enhanced Raman scattering (SERS) nanoprobes (NPs) comprising gold nanoparticles (AuNPs) as enhancing substrate and nonfluorescent Raman labels were synthesized and microinjected into zebrafish embryos at the one-cell stage. Raman mapping was performed to assess their distribution in various cell-types and tissues of developing embryo at five different stages between 6 and 96 hpf (hours post-fertilization). Biocompatibility and toxicity studies indicate that the NPs are not toxic and the embryos were found to exhibit normal morphological and gene expression in addition to the proper form and function of major organs such as the heart and vasculature (of 7 day old NPs injected zebrafish embryos). A multiplex in vivo detection protocol was developed by SERS imaging to demonstrate that multiple labels can be detected by Raman mapping in undifferentiated cells as they develop into distinct cell- and tissue-types. The present work is the first to report on multiplex Raman imaging of zebrafish embryos with potential implications in tracking tissue development and biological processes at single molecule sensitivity using appropriate target molecules in vivo.

Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP.

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP‐labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual‐channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual‐channel, three‐dimensional (3D) and four‐dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer‐assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos. Developmental Dynamics 232:359–368, 2005.

Combinatorial gene regulation by Bmp and Wnt in zebrafish posterior mesoderm formation

Combinatorial signaling is an important mechanism that allows the embryo to utilize overlapping signaling pathways to specify different territories. In zebrafish, the Wnt and Bmp pathways interact to regulate the formation of the posterior body. In order to understand how this works mechanistically, we have identified tbx6 as a posterior mesodermal gene activated by both of these signaling pathways. We isolated a genomic fragment from the tbx6 gene that recapitulates the endogenous tbx6 expression, and used this to ask how the Bmp and Wnt signaling pathways combine to regulate gene expression. We find that the tbx6 promoter utilizes distinct domains to integrate the signaling inputs from each pathway, including multiple Tcf/LEF sites and a novel Bmp-response element. Surprisingly, we found that overexpression of either signaling pathway can activate the tbx6 promoter and the endogenous gene, whereas inputs from both pathways are required for the normal pattern of expression. These results demonstrate that both Bmp and Wnt are present at submaximal levels, which allows the pathways to function combinatorially. We present a model in which overlapping Wnt and Bmp signals in the ventrolateral region activate the expression of tbx6 and other posterior mesodermal genes, leading to the formation of posterior structures.

Disruption of Zebrafish Cyclin G-Associated Kinase (GAK) Function Impairs the Expression of Notch-Dependent Genes During Neurogenesis and Causes Defects in Neuronal Development.

BackgroundThe J-domain-containing protein auxilin, a critical regulator in clathrin-mediated transport, has been implicated in Drosophila Notch signaling. To ask if this role of auxilin is conserved and whether auxilin has additional roles in development, we have investigated the functions of auxilin orthologs in zebrafish.ResultsLike mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK), differing in their domain structures and expression patterns. Both zebrafish auxilin and GAK can functionally substitute for the Drosophila auxilin, suggesting that they have overlapping molecular functions. Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene. Furthermore, inhibition of GAK function caused an elevated level of apoptosis in neural tissues, resulting in severe degeneration of neural structures.ConclusionIn support of the notion that endocytosis plays important roles in Notch signaling, inhibition of zebrafish GAK function affects embryonic neuronal cell specification and Her4 expression. In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

In vivo imaging and quantitative analysis of zebrafish embryos by digital holographic microscopy

Digital holographic microscopy (DHM) has been applied extensively to in vitro studies of different living cells. In this paper, we present a novel application of an off-axis DHM system to in vivo study of the development of zebrafish embryos. Even with low magnification microscope objectives, the morphological structures and individual cell types inside developing zebrafish embryos can be clearly observed from reconstructed amplitude images. We further study the dynamic process of blood flow in zebrafish embryos. A calibration routine and post-processing procedures are developed to quantify physiological parameters at different developmental stages. We measure quantitatively the blood flow as well as the heart rate to study the effects of elevated D-glucose (abnormal condition) on circulatory and cardiovascular systems of zebrafish embryos. To enhance our ability to use DHM as a quantitative tool for potential high throughput screening application, the calibration and post-processing algorithms are incorporated into an automated processing software. Our results show that DHM is an excellent non-invasive imaging technique for visualizing the cellular dynamics of organogenesis of zebrafish embryos in vivo.

Zebrafish eve1 regulates the lateral and ventral fates of mesodermal progenitor cells at the onset of gastrulation

Zebrafish eve1, a member of the even-skipped related gene family, is expressed initially in the animal pole of late blastula embryo and subsequently restricted to the ventral mesoderm of the gastrula embryo under the signaling control of bone morphogenetic protein (Bmp). Overexpression of eve1 in embryos results in similar ventralized phenotypes to that seen in embryos overexpressing Bmp, suggesting that Eve1 acts downstream of the Bmp signaling pathway to regulate the fate of mesodermal progenitor cells (MPCs). How eve1 functions in the normal development of MPCs is unknown. Using overexpression of a chimeric protein of Eve1 fused to the Gal4 activation domain and gene-knockdown approaches, we investigated the role of eve1 in MPC development of zebrafish embryos at early gastrulation. We find that Eve1 functions as a transcriptional repressor and is required for normal MPC development. The role of eve1 in MPCs requires the redundant and cooperative functions of Bmp-activated downstream homeobox genes, ved, vent and vox. Inhibition of eve1, ved, vent and vox in double and triple combinations results in dorsalized phenotypes. Furthermore, specific inhibition of eve1 and ved causes the expression of an ectopic patch of the brachyury ortholog no tail and leads to the formation of an ectopic tail. Our data show that Eve1 functions together with Ved, Vent and Vox in a transcriptional network to prevent the spread of anti-Bmp gene activity from the dorsal side, leading to the establishment of the Bmp gradient activity along the dorsoventral axis to induce distinct transcriptional outputs in MPCs to maintain the lateral and ventral MPC fates during gastrulation.

Analysis of the structure and expression of the chicken gene encoding a homolog of the human RREB-1 transcription factor

Ras proteins are involved in a number of signal transduction pathways including the mitogen-activated kinase cascade. Activated MAPKs translocate to the nucleus and phosphorylate transcription factors such as c-myc, TCF and AP-1. Recently, a Ras-responsive element binding transcription factor, RREB-1, was cloned from a human medullary thyroid carcinoma cell line. RREB-1 is a zinc finger protein that binds to a Ras-responsive element in the promoter of the human calcitonin gene. We report the cloning of the chicken homologue to human RREB-1. Amino-acid alignment demonstrates that chicken and human RREB-1 are 53% identical and 69% similar. Genomic southern analysis indicates that chicken rreb-1 is a single-copy gene in the chicken genome. We demonstrate that chicken and human rreb-1 display the same tissue distribution, being expressed in all tissues examined except the brain. Interestingly, chicken RREB-1 has an extended N-terminus and contains 16 zinc fingers of the TFIIIA subclass, in comparison to human RREB-1 which was reported to contain only four zinc fingers. The size discrepancy between the two predicted gene products is further discussed. An unusual structural feature of RREB-1 is the widely spaced arrangement of the zinc fingers.