Abstract
BackgroundMouse limb bud is a prime model to study the regulatory interactions that control vertebrate organogenesis. Major aspects of limb bud development are controlled by feedback loops that define a self-regulatory signalling system. The SHH/GREM1/AER-FGF feedback loop forms the core of this signalling system that operates between the posterior mesenchymal organiser and the ectodermal signalling centre. The BMP antagonist Gremlin1 (GREM1) is a critical node in this system, whose dynamic expression is controlled by BMP, SHH, and FGF signalling and key to normal progression of limb bud development. Previous analysis identified a distant cis-regulatory landscape within the neighbouring Formin1 (Fmn1) locus that is required for Grem1 expression, reminiscent of the genomic landscapes controlling HoxD and Shh expression in limb buds.ResultsThree highly conserved regions (HMCO1-3) were identified within the previously defined critical genomic region and tested for their ability to regulate Grem1 expression in mouse limb buds. Using a combination of BAC and conventional transgenic approaches, a 9 kb region located ~70 kb downstream of the Grem1 transcription unit was identified. This region, termed Grem1 Regulatory Sequence 1 (GRS1), is able to recapitulate major aspects of Grem1 expression, as it drives expression of a LacZ reporter into the posterior and, to a lesser extent, in the distal-anterior mesenchyme. Crossing the GRS1 transgene into embryos with alterations in the SHH and BMP pathways established that GRS1 depends on SHH and is modulated by BMP signalling, i.e. integrates inputs from these pathways. Chromatin immunoprecipitation revealed interaction of endogenous GLI3 proteins with the core cis-regulatory elements in the GRS1 region. As GLI3 is a mediator of SHH signal transduction, these results indicated that SHH directly controls Grem1 expression through the GRS1 region. Finally, all cis-regulatory regions within the Grem1 genomic landscape locate to the DNAse I hypersensitive sites identified in this genomic region by the ENCODE consortium.ConclusionsThis study establishes that distant cis-regulatory regions scattered through a larger genomic landscape control the highly dynamic expression of Grem1, which is key to normal progression of mouse limb bud development.
Highlights
Mouse limb bud is a prime model to study the regulatory interactions that control vertebrate organogenesis
Analysis of ShhΔ/Δ GRS1tg10/+ embryos revealed the complete absence of ß-galactosidase activity in Shh-deficient forelimb buds (Figure 6A). This contrasts with the endogenous Grem1 expression, which is activated but not maintained in Shh-deficient limb buds [11,14]. These results show that activation of the Grem1 Regulatory Sequence 1 (GRS1) region depends on Sonic Hedgehog (SHH) signalling, which indicated that it could participate in ectopic Grem1 activation due to anterior ectopic SHH signalling in mouse [14] and chicken limb buds [12]
Its activity is first apparent when the SHH/ GREM1/apical ectodermal ridge (AER)-Fibroblast Growth Factor (FGF) feedback is already established, and the expected termination does not occur, as ß-galactosidase activity remains in the posterior mesenchyme after the endogenous Grem1 transcripts have been down-regulated (Figure 4B, D). These results indicate that FGF-mediated termination of Grem1 expression and the underlying self-regulatory feedback signalling system [40] does not occur by FGF signal transduction impacting the GRS1 region
Summary
Mouse limb bud is a prime model to study the regulatory interactions that control vertebrate organogenesis. Vertebrate limb bud development is controlled by interactions between two main signalling centres, the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) located in the posterior limb mesenchyme. ZPA cells produce the Sonic Hedgehog (SHH) signal, which together with GREMLIN1-mediated antagonism of Bone Morphogenetic Proteins (BMPs) in the posterior-distal limb bud mesenchyme propagates Fibroblast Growth Factor (FGF) signalling in the AER [11,12,13,14]. This SHH/GREM1/AER-FGF feedback-signalling loop promotes distal progression of limb bud outgrowth and formation of the autopod which gives rise to carpals and digit rays These include HOX proteins, the bHLH transcription factor HAND2, and ETS transcription factors, providing a glimpse of the complex transcriptional regulation of Shh in the posterior limb bud mesenchyme [20,21,22]
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