Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers

Abstract

Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network.

Establishing neural crest identity: a gene regulatory recipe

“…Moreover, Pax3/7 and Msx1 are also required for the expression of Ets1 (Barembaum and Bronner, 2013), highlighting their importance during the early steps of neural crest specification. Similarly, Pax3/7 has also been shown to cooperate with Zic1 to drive neural crest specification in frog embryos (Bae et al, 2014;Plouhinec et al, 2014). In X. laevis, for example, Zic1 together with Pax3/7 (Milet et al, 2013) bind directly to the Snai1 and Snai2 promoters (Plouhinec et al, 2014).…”

“…Similarly, Pax3/7 has also been shown to cooperate with Zic1 to drive neural crest specification in frog embryos (Bae et al, 2014;Plouhinec et al, 2014). In X. laevis, for example, Zic1 together with Pax3/7 (Milet et al, 2013) bind directly to the Snai1 and Snai2 promoters (Plouhinec et al, 2014). They also directly regulate FoxD3 expression in the chick trunk neural crest (Simoes-Costa et al, 2014).…”

Ror2 signaling is required for local upregulation of GDF6 and activation of BMP signaling at the neural plate border

“…The combined activity of these major signaling pathways induces the expression of transcription factors including Gbx2, Dlx, Msx1 and Msx2, Pax3 and Pax7, Ap2alpha (also known as Tfap2a), and Zic1 (Brewer et al, 2004;Khadka et al, 2006;Li et al, 2009;Luo et al, 2003Luo et al, , 2001Monsoro-Burq et al, 2005;Plouhinec et al, 2014;Woda et al, 2003) in an area between neural and non-neural ectoderm. These factors cooperatively define the neural plate border (NPB) and interact to induce expression of neural crest specific genes.…”

PFKFB4 controls embryonic patterning via Akt signalling independently of glycolysis

“…During vertebrate gastrulation and neurulation, the dorsal ectoderm is patterned into several cell fates: neural plate (NP), non-neural ectoderm (NNE), neural crest (NC) and preplacodal ectoderm (P), a prerequisite for the differentiation of the central and peripheral nervous systems and surrounding tissues. Focusing on genes enriched dorsally during neurulation 9,12 , we found that : non-neural ectoderm (NNE), neural plate (NP), neural border (NB), preplacodal ectoderm (P) and blastopore (Blastp). In late gastrulas, pfkfb4 is expressed broadly dorsally, encompassing sox2 pan-neural expression.…”