Multiphoton excitation fluorescence imaging generates an optical section of sample by restricting fluorophore excitation to the plane of focus. High photon densities, achieved only in the focal volume of the objective, are sufficient to excite the fluorescent probe molecules by density-dependent, multiphoton excitation processes. We present comparisons of confocal with multiphoton excitation imaging of identical optical sections within a sample. These side-by-side comparisons of imaging modes demonstrate a significant advantage of multiphoton imaging; data can be obtained from deeper within biological specimens. Observations on a variety of biological samples showed that in all cases there was at least a twofold improvement in the imaging penetration depth obtained with multiphoton excitation relative to confocal imaging. The more pronounced degradation in image contrast deep within a confocally imaged sample is primarily due to scattered emission photons, which reduce the signal and increase the local background as measurements of point spread functions indicated that resolution does not significantly change with increasing depth for either mode of microscopy. Multiphoton imaging does not suffer from degradation of signal-to-background to nearly the same extent as confocal imaging because this method is insensitive to scatter of the emitted signal. Direct detection of emitted photons using an external photodetector mounted close to the objective (possible only in a multiphoton imaging system) improves system sensitivity and the utilization of scattered emission photons for imaging. We demonstrate that this technique provides yet further improvements in the capability of multiphoton excitation imaging to produce good quality images from deeper within tissue relative to confocal imaging.

Autosomal dominant mutations in the bHLH transcription factor TWIST1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome (SCS). The molecular mechanism underlying these phenotypes is poorly understood. We show that the ectopic expression of the related bHLH factor Hand2 phenocopies Twist1 loss-of-function phenotypes in the limb, and that they display a gene dosage-dependent antagonistic interaction. Twist1 and Hand2 dimerization partner choice can be modulated by PKA and protein phosphatase 2A-regulated phosphorylation of conserved helix I residues. Interestingly, multiple TWIST1 mutations associated with SCS alter PKA-mediated Twist1 phosphorylation, suggesting that misregulation of Twist1 dimerization via either stoichiometric or posttranslational mechanisms underlies SCS phenotypes.Studies of developing vertebrate limbs have yielded many insights into the process of embryonic pattern formation. Prominent among these are the identification of a growing catalog of transcription factors that orchestrate limb patterning. While the genetic and biochemical interactions of these transcription factors are clearly important for integrating patterning information, these interactions are poorly understood. Twist1 and Hand2 are basic helix-loop-helix (bHLH) transcription factors within the Twist family, and are attractive candidates for investigating such interactions. Each is required for distinct yet subtly related aspects of limb development, and biochemical studies have revealed a complex regulation of their protein-protein interactions 1-3 .Early limb bud expression of Twist1 is observed primarily in the peripheral mesenchyme, and Twist1 is required for maintenance of the overlying apical ectodermal ridge (AER) 4-7 . Twist1 haploinsufficiency in mice and humans is associated with a range of limb abnormalities. Twist1 heterozygous null mice display a partially penetrant preaxial polydactyly 8,9 . Human Correspondence should be addressed to A.B.F. tfirulli@iupui.edu (317) 278-5814 and E.L. elaufer@columbia.edu (212) Here we investigate the biochemical and genetic interactions between Twist1 and Hand2 both in vitro and during limb development. We show that PKA and B56δ-containing PP2A can regulate Twist1 and Hand2 phosphorylation at the conserved helix I residues, that hypophosphorylation and phosphorylation mimics of these residues alter bHLH dimerization affinities, and that a population of TWIST1 mutations that cause SCS in humans exhibit disregulation of this phosphoregulatory circuit. We also show that ectopic Hand2 expression phenocopies multiple SCS-like limb phenotypes, that the appropriate genetic dosage of Hand2 and Twist1 is critical for proper limb development, and that these interactions require the phosphoregulated helix I residues. These findings support a mechanism where the Twist family dimerization partner choices are modulated by both the relative levels of gene expression and the phosphorylation state of key helix I residues, thereby dictating changes i...

Violet-blue light is toxic to mammalian cells, and this toxicity has been linked with cellular production of H 2 O 2 . In this report, we show that violet-blue light, as well as UVA,

SUMMARY Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.