Quantitative imaging of the Dorsal nuclear gradient reveals limitations to threshold-dependent patterning in Drosophila

The NF-κB-related transcription factor, Dorsal, forms a nuclear concentration gradient in the early Drosophila embryo, patterning the dorsal-ventral (DV) axis to specify mesoderm, neurogenic ectoderm, and dorsal ectoderm cell fates. The concentration of nuclear Dorsal is thought to determine these patterning events; however, the levels of nuclear Dorsal have not been quantified previously. Furthermore, existing models of Dorsal-dependent germ layer specification and patterning consider steady-state levels of Dorsal relative to target gene expression patterns, yet both Dorsal gradient formation and gene expression are dynamic. We devised a quantitative imaging method to measure the Dorsal nuclear gradient while simultaneously examining Dorsal target gene expression along the DV axis. Unlike observations from other insects such as Tribolium, we find the Dorsal gradient maintains a constant bell-shaped distribution during embryogenesis. We also find that some classical Dorsal target genes are located outside the region of graded Dorsal nuclear localization, raising the question of whether these genes are direct Dorsal targets. Additionally, we show that Dorsal levels change in time during embryogenesis such that a steady state is not reached. These results suggest that the multiple gene expression outputs observed along the DV axis do not simply reflect a steady-state Dorsal nuclear gradient. Instead, we propose that the Dorsal gradient supplies positional information throughout nuclear cycles 10-14, providing additional evidence for the idea that compensatory combinatorial interactions between Dorsal and other factors effect differential gene expression along the DV axis.

[1]  P. Simpson,et al.  Maternal-Zygotic Gene Interactions during Formation of the Dorsoventral Pattern in Drosophila Embryos. , 1983, Genetics.

[2]  R. Steward,et al.  The dorsal protein is distributed in a gradient in early drosophila embryos , 1988, Cell.

[3]  M. Levine,et al.  The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila , 1989, Cell.

[4]  C. Nüsslein-Volhard,et al.  A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo , 1989, Cell.

[5]  M. Levine,et al.  The dorsal morphogen is a sequence-specific DNA-binding protein that interacts with a long-range repression element in drosophila , 1991, Cell.

[6]  M. Levine,et al.  dorsal-twist interactions establish snail expression in the presumptive mesoderm of the Drosophila embryo. , 1992, Genes & development.

[7]  Michael Levine,et al.  Binding affinities and cooperative interactions with bHLH activators delimit threshold responses to the dorsal gradient morphogen , 1993, Cell.

[8]  M. Levine,et al.  Interactions between dorsal and helix-loop-helix proteins initiate the differentiation of the embryonic mesoderm and neuroectoderm in Drosophila. , 1993, Genes & development.

[9]  S. Roth,et al.  The role of brinker in mediating the graded response to Dpp in early Drosophila embryos. , 1999, Development.

[10]  S. Roth,et al.  The maternal NF-kappaB/dorsal gradient of Tribolium castaneum: dynamics of early dorsoventral patterning in a short-germ beetle. , 2000, Development.

[11]  C. Doe,et al.  Convergence of dorsal, dpp, and egfr signaling pathways subdivides the drosophila neuroectoderm into three dorsal-ventral columns. , 2000, Developmental biology.

[12]  Michael Levine,et al.  Linear signaling in the Toll-Dorsal pathway of Drosophila: activated Pelle kinase specifies all threshold outputs of gene expression while the bHLH protein Twist specifies a subset. , 2002, Development.

[13]  Michael Levine,et al.  Whole-Genome Analysis of Dorsal-Ventral Patterning in the Drosophila Embryo , 2002, Cell.

[14]  M. Levine,et al.  Ventral dominance governs sequential patterns of gene expression across the dorsal-ventral axis of the neuroectoderm in the Drosophila embryo. , 2003, Developmental biology.

[15]  William McGinnis,et al.  Multiplex Detection of RNA Expression in Drosophila Embryos , 2004, Science.

[16]  M. Levine,et al.  Immunity regulatory DNAs share common organizational features in Drosophila. , 2004, Molecular cell.

[17]  M. Levine,et al.  Localized repressors delineate the neurogenic ectoderm in the early Drosophila embryo. , 2005, Developmental biology.

[18]  S. Roth,et al.  Dorsoventral Axis Formation in the Drosophila Embryo—Shaping and Transducing a Morphogen Gradient , 2005, Current Biology.

[19]  Dmitri Papatsenko,et al.  Quantitative analysis of binding motifs mediating diverse spatial readouts of the Dorsal gradient in the Drosophila embryo. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Charless C. Fowlkes,et al.  Three-dimensional morphology and gene expression in the Drosophila blastoderm at cellular resolution I: data acquisition pipeline , 2006, Genome Biology.

[21]  M. Levine,et al.  Computational Models for Neurogenic Gene Expression in the Drosophila Embryo , 2006, Current Biology.

[22]  James Briscoe,et al.  The interpretation of morphogen gradients , 2006, Development.

[23]  Ethan Bier,et al.  Threshold-Dependent BMP-Mediated Repression: A Model for a Conserved Mechanism That Patterns the Neuroectoderm , 2006, PLoS biology.

[24]  S. Bergmann,et al.  Pre-Steady-State Decoding of the Bicoid Morphogen Gradient , 2007, PLoS biology.

[25]  J. Lippincott-Schwartz,et al.  Nucleocytoplasmic shuttling mediates the dynamic maintenance of nuclear Dorsal levels during Drosophila embryogenesis , 2007, Development.

[26]  W. Bialek,et al.  Stability and Nuclear Dynamics of the Bicoid Morphogen Gradient , 2007, Cell.

[27]  Manolis Kellis,et al.  Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo. , 2007, Genes & development.

[28]  D. W. Knowles,et al.  Transcription Factors Bind Thousands of Active and Inactive Regions in the Drosophila Blastoderm , 2008, PLoS biology.

[29]  S. Roth,et al.  Self-regulatory circuits in dorsoventral axis formation of the short-germ beetle Tribolium castaneum. , 2008, Developmental cell.

[30]  D. Papatsenko,et al.  How the Dorsal gradient works: Insights from postgenome technologies , 2008, Proceedings of the National Academy of Sciences.

[31]  A. Tsirigos,et al.  Anterior-posterior positional information in the absence of a strong Bicoid gradient , 2009, Proceedings of the National Academy of Sciences.

[32]  A. Stathopoulos,et al.  Design flexibility in cis-regulatory control of gene expression: synthetic and comparative evidence. , 2009, Developmental biology.