Twist1 Controls a Cell-Specification Switch Governing Cell Fate Decisions within the Cardiac Neural Crest

<div><p>Neural crest cells are multipotent progenitor cells that can generate both ectodermal cell types, such as neurons, and mesodermal cell types, such as smooth muscle. The mechanisms controlling this cell fate choice are not known. The basic Helix-loop-Helix (bHLH) transcription factor Twist1 is expressed throughout the migratory and post-migratory cardiac neural crest. <i>Twist1</i> ablation or mutation of the Twist-box causes differentiation of ectopic neuronal cells, which molecularly resemble sympathetic ganglia, in the cardiac outflow tract. Twist1 interacts with the pro-neural factor Sox10 via its Twist-box domain and binds to the <i>Phox2b</i> promoter to repress transcriptional activity. Mesodermal cardiac neural crest <i>trans</i>-differentiation into ectodermal sympathetic ganglia-like neurons is dependent upon Phox2b function. Ectopic Twist1 expression in neural crest precursors disrupts sympathetic neurogenesis. These data demonstrate that Twist1 functions in post-migratory neural crest cells to repress pro-neural factors and thereby regulate cell fate determination between ectodermal and mesodermal lineages.</p> </div

Ectopic sympathetic-like neurons in <i>Twist1</i>;<i>Wnt1-Cre</i> CKO APCs are not <i>bona fide</i> sympathetic neurons.

<p>A–H) At E11.5, NCC-specific <i>Hand2</i> ablation causes reduced <i>DBH</i> expression (compare inset 1 in A and C), and loss of <i>Hand1</i> expression (compare inset 1 in E and G). However, in E11.5 <i>Twist1^fx/−</i>;<i>Hand2^fx/−</i>;<i>Wnt1-Cre(+)</i> mutants, robust ectopic sympathetic-like neurons persist and express comparatively high levels of <i>DBH</i> (inset D2; n = 4) and <i>Hand1</i> (inset H2; n = 4). I, J) A Hand2-dependent <i>cis</i>-regulatory element proximal to <i>Hand1</i> is sufficient to drive reporter transgene (<i>Hand1^SG+hsp68-lacZ</i>) expression in developing SGNs at E11.5 (inset 1 in I and J). However, reporter transgene expression is not detectable in the ectopic neurons in the <i>Twist1^fx/−</i>;<i>Wnt1-Cre(+)</i> CKO APCs (J2), indicating that this tissue-specific <i>cis</i>-regulatory element is not active in these cells (n = 4).</p

The Twist-box domain of Twist1 is required for molecular interaction with Sox10 and for Twist1 binding to the <i>Phox2b</i> promoter.

<p>A) Co-immunoprecipitation assays (n = 3) using epitope-tagged variants of Twist1 and Sox10 show that Twist1 molecularly interacts with Sox10. This interaction is diminished in the Twist1 S192P mutant (asterisk), but not the Twist1 T125;S127A mutant. B) Densitometry analyses quantitate these results. C) Bioinformatic analyses uncovered four evolutionarily conserved HMG Box binding sites (highlighted in blue) and two conserved E-Box binding sites (highlighted in yellow) within in the 827 bps 5′ to the <i>Phox2b</i> transcription start site. Nucleotides that diverge from established consensus sequences are highlighted in red. D) Transactivation assays of the <i>Phox2b</i> promoter show that Sox10 upregulates gene expression from the <i>Phox2b</i> promoter 48.9+/−2.2-fold. Transcriptional upregulation is significantly reduced to 26.3+/−1.9-fold (p-value = 0.0003) when Sox10 is co-transfected with Twist1. When Sox10 is co-transfected with the Twist1 S192P mutant, Twist1 repression of Sox10 activity on the <i>Phox2b</i> promoter is lost (49.3+/−3.9-fold; p-value = 0.92). E–I) Both (E) radiolabeled oligonucleotides containing HMG Box or E-Box consensus sites (bold) and (F) reticulocyte lysates programmed with Myc+Sox10 and/or Myc+Twist1 were used to confirm that (G) Sox10 directly binds to <i>cis</i>-elements within the <i>Phox2b</i> promoter (asterisks), and that Twist1 has no appreciable effect upon Sox10 DNA binding. An open circle denotes a complex formed between HMG 3 and Twist1. H) A Western blot for α-Myc verifies that the Myc-tagged Twist1 variants transcribed <i>in vitro</i> were synthesized in equivalent amounts. I) EMSAs employing these lysates revealed that E-Box 1 is bound robustly by wild-type Twist1 (open circle), weakly by Twist1 S192P, and not at all by Twist1 R116W, Twist1 R118H, or Twist1 R120A. Mutation of the E-Box, E-Box 1(Mut), completely ablated Twist1 binding. Underlined nucleotides in oligo sequences indicate those that diverge from established consensus sequences. Mutated nucleotides are in lower case. Arrowheads in EMSAs denote non-specific binding. NS, non-specific unlabeled competitor; Mut, mutant unlabeled competitor; S, specific unlabeled competitor.</p

Abnormal NCCs in <i>Twist1</i>;<i>Wnt1-Cre</i> CKOs express neuronal, not mesenchymal, markers.

<p>A, B) Alcian blue staining to visualize ECM reveals that the cNCC-derived nodules (B, arrowheads) in E11.5 <i>Twist1^fx/−</i>;<i>Wnt1-Cre(+)</i> mutants are devoid of ECM. C, D) <i>Sox9</i>, a transcriptional regulator of ECM components, is also absent from the nodules (D, arrowheads). E–H) Cell aggregates express the neuronal precursor marker <i>Sox10</i> (F, arrowheads) and neuronal marker Tubb3 (H, arrowheads). For all figures: LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Scale bars = 100 µm.</p

Ectopic sympathetic-like neurons are detectable in <i>Twist1</i>;<i>Hand1^eGFPCre</i> CKO APCs.

<p>E11.5 <i>Twist1^fx/−</i>;<i>Hand1^Cre</i> mutant aggregates (arrowheads) express TH (B; n = 2) and <i>Ascl1</i> (D; n = 4), whereas these factors are not detectable in <i>Control</i> APCs (A and C, respectively).</p

Sympathetic neurogenesis is disrupted by Twist1 mis-expression.

<p>In E12.5 Control embryos, the thoracic sympathetic chain ganglia (arrowheads) express TH (A), Tubb3 (D), <i>Phox2b</i> (J), and <i>Hand2</i> (M). <i>Sox10</i>-expressing support cells surround the thoracic sympathetic chain ganglia (G, arrowhead). In thoracic sympathetic chain ganglia in E12.5 <i>CAGCCAT-Twist1(+)</i>;<i>Wnt1-Cre(+)</i> transgenic embryos, expression of TH, Tubb3, <i>Phox2b</i>, and <i>Hand2</i> is either absent (B, E, K, and N), or markedly reduced (C, F, L, and O). In the latter instance, TH (green) and Twist1 (red) expression is largely mutually exclusive (n = 3). These hypoplastic thoracic sympathetic chain ganglia are either entirely positive for <i>Sox10</i> (H, arrowhead) or display a markedly reduced core of <i>Sox10</i>-negative cells (I, arrowhead).</p

Ectopic neurogenesis in <i>Twist1^CC</i> mutants is Phox2b-dependent.

<p>A) All <i>Twist1^CC/CC</i> homozygous OFTs examined contain robust aggregates of <i>Ascl1</i>-positive cells. B) Concurrent <i>Phox2b^lacz/+</i> haploinsufficiency tended to reduce the size of these aggregates (arrowhead). C) No <i>Ascl1</i>-positive cells were evident in <i>Twist1^CC/CC</i>;<i>Phox2b^lacz/lacZ</i> OFTs. D) Morphometric quantification of these analyses validated these results.</p

Ectopic neurons in <i>Twist1</i>;<i>Wnt1-Cre</i> CKO APCs express sympathetic neuron markers.

<p><i>In situ</i> hybridization shows that, in <i>Control</i> E11.5 embryos, <i>Hand1</i> (A) and <i>Hand2</i> (C) expression is detectable throughout the cNCC-derived APCs, where as expression of other components of the sympathetic neurogenesis pathway, including <i>Phox2b</i> (E), <i>Ascl1</i> (G), <i>Gata3</i> (I), TH (K, as assessed via immunohistochemistry), and <i>DBH</i> (M) are not detectable in this tissue. E11.5 <i>Twist1^fx/−</i>;<i>Wnt1-Cre(+)</i> mutants, aggregates (arrowheads) express the transcription factors <i>Hand1</i> (B), <i>Hand2</i> (D), <i>Phox2b</i> (F), <i>Ascl1</i> (H), and <i>Gata3</i> (J), and the norepinephrine biosynthetic enzymes TH (L), and <i>DBH</i> (N). (n = 3).</p

The Twist-box domain of Twist1 is required for repression of ectopic neurogenesis.

<p>Analyses of <i>Twist1^CC</i> mutant embryos that harbor this Twist box mutation, reveal that <i>Twist1^CC/+</i> heterozygous OFTs contain dispersed or relatively small aggregates of <i>Phox2b</i>-positive cells (B) and rare TH-positive cells (E, arrowhead) <i>Twist1^CC/CC</i> embryos contain robust ectopic <i>Phox2b</i>-positive ganglia (C) and a greater number of TH-positive neurons (F, arrowhead). Neither <i>Phox2b</i>- nor TH-positive cells are detectable in <i>Twist1^+/+</i> APCs (A and D, respectively).</p

Twist1 promotes tumor progression <i>in vivo</i>.

<p>Transgene expression was driven by the MMTV-Cre. While MMTV-Cre; <i>Twist1</i> mice (TWIST1; n = 71) do not develop skin lesions, MMTV-Cre;<i>K-rasG12D</i> animals (RAS; n = 89) spontaneously develop anal and oral papillomas. In MMTV-Cre;<i>K-rasG12D</i>;<i>Twist1</i> mice (RAS+TWIST1; n = 19) papillomas evolve into squamous carcinomas. (A) HPS staining of skins either from <i>K-rasG12D</i> or <i>K-rasG12D</i>;<i>Twist1</i> transgenic mice. Transgene expression is induced by the MMTV-Cre. (B) Kaplan-Meier survival curves of transgenic mice. Survival corresponds to the end point of the experiment, the tumor burden requiring euthanasia of the animal. Open circles indicate mice censored from the dataset.</p