In a sister study to that of Bmp2
by K. Chandler et al
, the role of distant cis
-regulatory elements in Bmp4
expression was addressed with the same initial approach used for Bmp2
]. The 2 BAC clones used for this study collectively spanned about 400 kb, centered on the Bmp4
gene. Separately, they contained a reporter gene in place of the Bmp4
coding sequence, along with roughly 230 kb of flanking sequence. Many domains of endogenous Bmp4
expression were recapitulated in transgenic mouse embryos generated with the pair of BAC constructs; however, there was a conspicuous absence of reporter expression in the extra-embryonic ectoderm, eye, trachea, and anterior limb bud. Lack of expression in these tissues was not likely due to positional effects because it was consistent across multiple independent lines.
As with Bmp2, the BACs each drove reporter expression in a mostly distinct subset of tissues where Bmp4 is typically expressed. For example, the 5’ BAC line drove expression in the mesoderm, tooth bud dermal papilla, kidney, pelage hair follicle placode, mammary glands, forebrain, apical ectodermal ridge, thymus, gut, inner ear, zone of polarizing activity, bladder, ventral pawpads, and heart outflow tract (). In contrast, the 3’ BAC drove expression in the roof palate mesenchyme, ventral ribs, vertebral column, proximal limb mesenchyme, umbilical artery, dura mater, dorsal aorta, pulmonary arteries, and craniofacial mesenchyme. Interestingly, expression in osteoblasts was driven by the 5’, but not the 3’ BAC. This situation mirrors that which was seen for Bmp2. Because the two BACs shared a 60 kb region of overlap extending out to 30 kb on either side of the Bmp4 transcription start site, regulatory elements located within this region were present in both BACs. Accordingly, there were several sites where reporter expression was consistently observed in lines harboring both constructs. These included the whisker hair shafts, dorsal root ganglia, digit tips, and genital tubercle. An important conclusion from these observations was that while several cis-regulatory elements are close to Bmp4, most are located more than 30 kb away.
5.1 Bmp4 expression in mesoderm is controlled by an ancient 5’ enhancer
To pinpoint the location of enhancers within the large areas defined by BAC transgenes, K. Chandler et al searched for sequences with high evolutionary conservation. They improved the specificity of their search by focusing on sequences that aligned not only across mammalian species but also with fish. Three such sequences were discovered, termed Evolutionarily-Conserved Regions (ECRs) 1, 2, and 3. These were located 101 and 46-kb upstream, and 80 kb downstream of the Bmp4 promoter, respectively. In addition to their nucleotide sequences, the spatial orientation of these elements relative to Bmp4 was conserved across species as well. This is true in spite of the fact that genes flanking the Bmp4 gene desert have undergone rearrangement between mammals and fish. Thus, ECRs 1, 2 and 3 are ancient vertebrate features.
To test the hypothesis that ECRs 1, 2, and 3 could function as developmental enhancers for Bmp4, K. Chandler et al took two complementary approaches. First, they deleted each ECR separately from the full-length BAC constructs via homologous recombination in bacteria. Remarkably, removal of ECR2 from the 5’ BAC resulted in a loss of reporter expression in the outflow tract of the heart, as well as mesoderm of the lateral plate and extra-embryonic tissues. In contrast, removal of ECR1 and ECR3 from the 5’ and 3’ full-length BACs, respectively, had no detectable effect on reporter gene expression during mouse development. ECR1 and ECR3 may be functionally redundant, either with each other or with enhancer elements elsewhere in the genome. This could explain why removal of either one fails to abrogate reporter expression. Alternatively, they may act at later stages of development. Regardless, ECR2 appears to be necessary for Bmp4 expression in several tissues during mouse development.
K. Chandler et al
also tested each ECR separately for ability to drive tissue-specific expression of a reporter during development. In agreement with their previous results, the ECR2-containing transgene drove reporter expression in the extra-embryonic mesoderm and lateral plate mesoderm. Likewise, the transgenes containing ECR1 and ECR3 could not drive reproducible expression in any tissue. These results indicate that ECR2 is a mesoderm-specific enhancer element. Accordingly, ECR2 contains putative bindings sites for several transcription factors that regulate embryonic mesoderm development, including Gata4, Cdx1, Nfe2l1(Tcf11), Zic3 and the Hand1/E47 dimer. Moreover, genetic studies suggest that Bmp4
is important for mesodermal development and function [44
]. The conservation of ECR2 in fish further suggests this is an ancient feature of vertebrate development.