A 37 kb region upstream of brachyury comprising a notochord enhancer is essential for notochord and tail development.

Dennis Schifferl,Manuela Scholze-Wittler,Jesse V. Veenvliet,Lars Wittler,Frederic Koch,Bernhard G. Herrmann

doi:10.1242/dev.200059

Abstract

ABSTRACTThe node-streak border region comprising notochord progenitor cells (NPCs) at the posterior node and neuro-mesodermal progenitor cells (NMPs) in the adjacent epiblast is the prime organizing center for axial elongation in mouse embryos. The T-box transcription factor brachyury (T) is essential for both formation of the notochord and maintenance of NMPs, and thus is a key regulator of trunk and tail development. The T promoter controlling T expression in NMPs and nascent mesoderm has been characterized in detail; however, control elements for T expression in the notochord have not been identified yet. We have generated a series of deletion alleles by CRISPR/Cas9 genome editing in mESCs, and analyzed their effects in mutant mouse embryos. We identified a 37 kb region upstream of T that is essential for notochord function and tailbud outgrowth. Within that region, we discovered a T-binding enhancer required for notochord cell specification and differentiation. Our data reveal a complex regulatory landscape controlling cell type-specific expression and function of T in NMP/nascent mesoderm and node/notochord, allowing proper trunk and tail development.

Highlights

The mammalian embryo is generated in three consecutive phases, starting with head formation from the epiblast, continued by trunk development from the primitive streak acting as growth zone for posterior elongation, and tail development from the tailbud
A T deletion termed TCD spanning from −62 kb upstream to 10 kb downstream of T, including the T transcription unit, resulted in axial truncation and absence of the trunk notochord in homozygous embryos, as expected from the analysis of the original T mutant (Fig. 1D,D′, Chesley, 1935)
The axial truncation phenotype is demonstrated by the depletion of Neuro-mesodermal progenitors (NMPs) and differentiation of their descendants into neural tissue at the expense of paraxial mesoderm (Koch et al, 2017), as visualized by an expansion of the neural plate identified by Sox2 protein, and lack ofsomitic mesoderm posterior of the forelimb buds (Fig. 1E-E′′)

Summary

Introduction

The mammalian embryo is generated in three consecutive phases, starting with head formation from the epiblast, continued by trunk development from the primitive streak acting as growth zone for posterior elongation, and tail development from the tailbud. Immunofluorescent staining showed the expected expression of T in the notochord, NMPs and nascent mesoderm of wild-type embryos (Fig. 1B-C′′, Wymeersch et al, 2016). The data show that the 37 kb upstream region contains control elements essential for tailbud formation, tail outgrowth, and proper neural tube and somite differentiation, whereas T expression from the streak promoter is sufficient to support trunk formation from NMPs. The trunk phenotype can be attributed to the lack of signaling inputs from the missing notochord (Chiang et al, 1996).

Results

Conclusion