David Ish-Horowicz

Molecular mechanisms regulating segmentation and growth in Drosophila and vertebrate embryos

Many mRNA transcripts are localised to specific cytoplasmic subregions in order to help target proteins to their sites of action. We have used in vivo injection assays to show the generality of minus-end-directed RNA transport along microtubules (Bullock and Ish-Horowicz, Nature 2001, 414: 611-616; Hughes et al., Curr Biol 2004, 14: 1950-1956), and to define Egalitarian and Bicaudal-D as novel components of the dynein motor complex transporting RNAs and probably other cargoes (Bullock and Ish-Horowicz, 2001). We have defined RNA transcript signals (localisation elements) that direct cargo recognition by the transport machinery, and also studied the kinetics of RNA transport in real time, leading us to propose that dynein motor activity is modulated by the nature of its cargo (Bullock et al., 2003).

Current potential projects include:

• identification and functional analysis of factors that bind selectively to localisation elements.
• establishing an in vitro system of transcript transport whose cargo specificity mimics that seen in vivo.
• screening by RNA-interference for factors require for RNA transport.

Regulation of cyclic gene expression and axial growth during vertebrate segmentation
Serial production of somites in vertebrate embryos is driven by the segmentation clock, a molecular oscillator that drives cyclic transcription in the unsegmented presomitic mesoderm [PSM] (Palmeirim et al., Cell 1997; 91: 639-648). We have shown that the clock drives cyclic initiation of transcription, and defined a small promoter region (clock element) that can drive pulses of transcription in the PSM of transgenic mice (Morales et al., Developmental Cell 2002; 3: 63-74).

Possible projects include:

• Comparing clock elements from different cycling genes to find conserved regulatory sites through which trans-acting regulatory factors may act. Testing the in vivo significance of sites and factors in transgenic mice, with an emphasis on negatively-acting factors that drive cyclic repression.
• Altering the period of the segmentation clock in order to identify rate-limiting components of the clock that must be part of its machinery. These experiments test our view that cyclic Notch activity underlies the segmentation clock.
• Visualising endogenous targets of cycling transcriptional regulators.

Identification and characterisation of axial stem cells in vertebrate embryos
The anteroposterior axis extends posteriorly via a "growth zone" that has been proposed to include a population of axial stem cells that lie in or near the tailbud (Davis and Kirschner, Development 2000, 127: 255-267; Cambray and Wilson, Development 2002, 129: 4855-4866). We are using transcriptional profiling of single cells on microarrays to identify and characterise these stem cells and their progeny. These experiments will address mechanisms for specifying and maintaining a stem cell population, and for coupling axis extension to axial patterning.