A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies long-range ventral forebrain enhancers.
The secreted protein sonic hedgehog (Shh) plays an integral role in forming the ventral midline of the vertebrate central nervous system (CNS). In the absence of Shh function, ventral midline development is perturbed resulting in holoprosencephaly (HPE), a structural malformation of the brain, as well as in neuronal patterning and path finding defects along the length of the anteroposterior neuraxis. Central to the understanding of ventral neural tube development is how Shh transcription is regulated in the CNS. To address this issue, we devised an enhancer trap assay to systematically screen 1 Mb of DNA surrounding the Shh locus for the ability to target reporter gene expression to sites of Shh transcription in transgenic mouse embryos. This analysis uncovered six enhancers distributed over 400 kb, the combined activity of which covered all sites of Shh expression in the mouse embryonic CNS from the ventral forebrain to the posterior extent of the spinal cord. To evaluate the relative contribution of these enhancers to the overall pattern of Shh expression, individual elements were deleted in the context of a transgenic Bac reporter assay. Redundant mechanisms were found to control Shh-like reporter activity in the ventral spinal cord, hindbrain and regions of the telencephalon, whereas unique elements regulated Shh-like expression in the ventral midbrain, the majority of the ventral diencephalon and parts of the telencephalon. Three ventral forebrain enhancers locate on the distal side of translocation breakpoints that occurred upstream of Shh in human cases of HPE, suggesting that displacement of these regulatory elements from the Shh promoter is a likely cause of HPE in these individuals.
Jeong
Yongsu
Y
Department of Genetics, University of Pennsylvania School of Medicine, Clinical Research Building, Room 470, 415 Curie Boulevard, Philadelphia, PA 19104, USA.
El-Jaick
Kenia
K
Roessler
Erich
E
Muenke
Maximilian
M
Epstein
Douglas J
DJ
D000818:Animals; D001001:Anura; D001483:Base Sequence; D002490:Central Nervous System / Q000033:anatomy & histology / Q000196:embryology / Q000378:metabolism; D002645:Chickens; D017124:Conserved Sequence; D004742:Enhancer Elements, Genetic; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D053823:Hedgehog Proteins; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016548:Prosencephalon / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D023721:Tetraodontiformes; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D053823:Hedgehog Proteins; C465570:Shh protein, mouse; D015534:Trans-Activators