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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 vivodetection 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.

The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells fromDrosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, we show that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation were normal inhrT morphant embryos, subsequently the hearts failed to undergo looping, and late onset defects in chamber morphology and gene expression were observed. In particular, we found that the loss of hrT function led to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, we show that overexpression of hrT causes a significant downregulation of tbx5, indicating that one key role ofhrT is to regulate the levels of tbx5. Secondly, we found that HrT is required to inhibit the expression of the blood lineage markersgata1 and gata2 in the most posterior lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis.

The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells from Drosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, we show that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation were normal in hrT morphant embryos, subsequently the hearts failed to undergo looping, and late onset defects in chamber morphology and gene expression were observed. In particular, we found that the loss of hrT function led to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, we show that overexpression of hrT causes a significant downregulation of tbx5, indicating that one key role of hrT is to regulate the levels of tbx5. Secondly, we found that HrT is required to inhibit the expression of the blood lineage markers gata1 and gata2 in the most posterior lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis.

Pituitary gland development serves as an excellent model system in which to study the emergence of distinct cell types from a common primordium in mammalian organogenesis. We have investigated the role of the morphogen Sonic hedgehog (SHH) in outgrowth and differentiation of the pituitary gland using loss-and gain-of-function studies in transgenic mice. Shh is expressed throughout the ventral diencephalon and the oral ectoderm, but its expression is subsequently absent from the nascent Rathke’s pouch as soon as it becomes morphologically visible, creating a Shh boundary within the oral epithelium. We used oral ectoderm/Rathke’s pouch-specific 5′ regulatory sequences (Pitx1HS) from the bicoid related pituitary homeobox gene (Pitx1) to target overexpression of the Hedgehog inhibitor Hip (Huntingtin interacting protein) to block Hedgehog signaling, finding that SHH is required for proliferation of the pituitary gland. In addition, we provide evidence that Hedgehog signaling, acting at the Shh boundary within the oral ectoderm, may exert a role in differentiation of ventral cell types (gonadotropes and thyrotropes) by inducing Bmp2 expression in Rathke’s pouch, which subsequently regulates expression of ventral transcription factors, particularly Gata2. Furthermore, our data suggest that Hedgehog signaling, together with FGF8/10 signaling, synergizes to regulate expression of the LIM homeobox gene Lhx3, which has been proved to be essential for initial pituitary gland formation. Thus, SHH appears to exert effects on both proliferation and cell-type determination in pituitary gland development.