Identification of a conserved cis‐regulatory element controlling mid‐diencephalic expression of mouse Six3

Abstract

The sine oculis homeobox protein Six3 plays pivotal roles in the development of the brain and craniofacial structures. In humans, SIX3 haploinsufficiency results in holoprosencephaly, a defect in anterior midline formation. Although much is known about the evolutionarily conserved functions of Six3, the regulatory mechanism responsible for the expression pattern of Six3 remains relatively unexplored. To understand how the transcription of Six3 is controlled during embryogenesis, we screened ∼300 kb of genomic DNA encompassing the Six3 locus for cis-acting regulatory elements capable of directing reporter gene expression to sites of Six3 transcription in transgenic mouse embryos. We identified a novel enhancer element, whose activity recapitulates endogenous Six3 expression in the ventral midbrain, pretectum, and thalamus. Cross-species comparisons revealed that this Six3 brain enhancer is functionally conserved in other vertebrates. We also showed that normal Six3 transcription in the ventral midbrain and pretectum is dependent on Ascl1, a basic helix-loop-helix proneural factor. Moreover, loss of Ascl1 resulted in downregulation of the Six3 brain enhancer activity, emphasizing its unique role in regulating Six3 expression in the developing brain.

Common genetic causes of holoprosencephaly are limited to a small set of evolutionarily conserved driver genes of midline development coordinated by TGF-β, hedgehog, and FGF signaling

“…However, almost every case has the potential to fit an autosomal driver (novel or de novo) with common modifier model that can include ultraconserved noncoding regions. Interestingly, many of the most ultraconserved regions examined occur near known HPE genes, such as SIX3 (Beccari, Conte, Cisneros, & Bovolenta, ; (Beccari et al., ; Lee et al., ) and FGF8 (Beermann et al, ; Komisarczuk et al, ).…”

“…Interestingly, many of the most ultraconserved regions examined occur near known HPE genes, such as SIX3 (Beccari, Conte, Cisneros, & Bovolenta, 2012;(Beccari et al, 2015;Lee et al, 2017) and FGF8 (Beermann et al, 2006;Komisarczuk et al, 2009).…”

“…HPE is best understood as a conserved sequence of developmental steps that begin in the early gastrulating embryo (Arnold & Robertson, 2009). Driver genes are inextricably linked to this developmental sequence, beginning with TGF-signals around the node (Barratt, Glanville-Jones, & Arkell, 2014;Beddington & Robertson, 1999;Houtmeyers et al, 2016;Powers et al, 2010;Robertson, 2014;Warr et al, 2008), the establishment of the primitive streak and its midline progenitors (Herion, Salbaum, & Kappen, 2014), the precise positioning of the prechordal plate beneath the midline neuro-ectoderm (England, Blanchard, Mahadevan, & Adams, 2006), the activation of midline hedgehog signals (Geng et al, 2008;Jeong et al, 2008;Ohkubo, Chiang, & Rubenstein, 2002;Okada, Okumura, Motoyama, & Ogawa, 2008), and/or cooperating with them in the forebrain (Carlin et al, 2012;Gestri et al, 2005;Geng, Acosta, Lagutin, Gil, & Oliver, 2016;Lavado, Lagutin, & Oliver, 2008;Lee et al, 2017;Storm et al, 2006).…”

Specification of neurotransmitter identity by Tal1 in thalamic nuclei

“…We previously identified and characterized two enhancers that regulate Six3 transcription in the developing forebrain (Lee et al, 2013;Lee et al, 2017). We previously identified and characterized two enhancers that regulate Six3 transcription in the developing forebrain (Lee et al, 2013;Lee et al, 2017).…”

“…Generation of a Transgenic Mouse Line Driving Cre-mediated Recombination in the Rostral Thalamic Domain To generate a system for conditional gene targeting within the developing thalamus, we chose a regulatory element to direct Cre expression in transgenic mice. We previously identified and characterized two enhancers that regulate Six3 transcription in the developing forebrain (Lee et al, 2013;Lee et al, 2017). Among these, Six3 mid-diencephalic enhancer (named CNS7) was sufficient to direct the transcription of a reporter gene to the rostral thalamus in a pattern fitting with the spatial and temporal profiles of the endogenous Six3 gene (Lee et al, 2017).…”