Regulatory Analysis of the Mouse Hoxb3 Gene: Multiple Elements Work in Concert to Direct Temporal and Spatial Patterns of Expression

Abstract

The expression pattern of the mouse Hoxb3 gene is exceptionally complex and dynamic compared with that of other members of the Hoxb cluster. There are multiple types of transcripts for Hoxb3 gene, and the anterior boundaries of its expression vary at different stages of development. Two enhancers flanking Hoxb3 on the 3' and 5' sides regulate Hoxb2 and Hoxb4, respectively, and these control regions define the two ends of a 28-kb interval in and around the Hoxb3 locus. To assay the regulatory potential of DNA fragments in this interval we have used transgenic analysis with a lacZ reporter gene to locate cis-elements for directing the dynamic patterns of Hoxb3 expression. Our detailed analysis has identified four new and widely spaced cis-acting regulatory regions that can together account for major aspects of the Hoxb3 expression pattern. Elements Ib, IIIa, and IVb control gene expression in neural and mesodermal tissues; element Va controls mesoderm-specific gene expression. The most anterior neural expression domain of Hoxb3 is controlled by an r5 enhancer (element IVa); element IIIa directs reporter expression in the anterior spinal cord and hindbrain up to r6, and the region A enhancer (in element I) mediates posterior neural expression. Hence, the regulation of segmental expression of Hoxb3 in the hindbrain is different from that of Hoxa3, as two separate enhancer elements contribute to expression in r5 and r6. The mesoderm-specific element (Va) directs reporter expression to prevertebra C1 at 12.5 dpc, which is the anterior limit of paraxial mesoderm expression for Hoxb3. When tested in combinations, these cis-elements appear to work as modules in an additive manner to recapitulate the major endogenous expression patterns of Hoxb3 during embryogenesis. Together our study shows that multiple control elements direct reporter gene expression in diverse tissue-, temporal-, and spatially restricted subset of the endogenous Hoxb3 expression domains and work in concert to control the neural and mesodermal patterns of expression.

Notch activity permits retinal cells to progress through multiple progenitor states and acquire a stem cell property

“…All tissue sections (20 m) were prepared after embedding in OCT. For X-Gal staining, retinal tissue was fixed with 1% PFA/0.5% glutaraldehyde (52). Retinae were stained as whole mounts overnight at 37°C.…”

The significance of Hox gene collinearity

“…Krumlauf and colleagues proposed that cluster organization is maintained because each Hox gene must remain associated with its enhancer elements, and these may be distributed widely up the distinct anterior expression boundaries of Hoxb4 and Hoxb3 (Kwan et al, 2001). Further examples of shared enhancers are a somitic enhancer located between Hoxb5 and Hoxb4 (Sharpe et al, 1998), a neural enhancer between Hoxb6 and Hoxb5 (Sharpe et al, 1998), and a neural/mesoderm enhancer between Hoxd11 and Hoxd10 (Zakany et al, 1997).…”

Notch 1 inhibits photoreceptor production in the developing mammalian retina

“…Retinae were then stained as whole mounts for 1-4 hours at room temperature (Fekete and Cepko, 1993). For Xgal staining, retinal tissue was harvested and fixed with a 1% PFA/0.5% glutaraldehyde mix as described (Kwan et al, 2001). Retinae were stained as whole mounts overnight at 37°C.…”