Temporal Regulation of the Muscle Gene Cascade by Macho1 and Tbx6 Transcription Factors in Ciona Intestinalis

For over a century, muscle formation in the ascidian embryo has been representative of \u27mosaic\u27 development. The molecular basis of muscle-fate predetermination has been partly elucidated with the discovery of Macho1, a maternal zinc-finger transcription factor necessary and sufficient for primary muscle development, and of its transcriptional intermediaries Tbx6b and Tbx6c. However, the molecular mechanisms by which the maternal information is decoded by cis-regulatory modules (CRMs) associated with muscle transcription factor and structural genes, and the ways by which a seamless transition from maternal to zygotic transcription is ensured, are still mostly unclear. By combining misexpression assays with CRM analyses, we have identified the mechanisms through which Ciona Macho1 (Ci-Macho1) initiates expression of Ci-Tbx6b and Ci-Tbx6c, and we have unveiled the cross-regulatory interactions between the latter transcription factors. Knowledge acquired from the analysis of the Ci-Tbx6b CRM facilitated both the identification of a related CRM in the Ci-Tbx6c locus and the characterization of two CRMs associated with the structural muscle gene fibrillar collagen 1 (CiFCol1). We use these representative examples to reconstruct how compact CRMs orchestrate the muscle developmental program from pre-localized ooplasmic determinants to differentiated larval muscle in ascidian embryos

Sequential activation of Brachyury target genes in the developing notochord of <i>C. intestinalis</i>.

<p>(A–O) WMISH of <i>C. intestinalis</i> embryos at the 110-cell (A,F,K), gastrula (B,G,L), neural plate (C,H,M), neurula (D,I,N), and initial tailbud (E,J,O) stage. Embryos in (A,F,K) are photographed with anterior up, all other embryos are oriented with anterior to the left. Scale bar: approximately 100 µm. Red arrowheads indicate the regions containing stained notochord cells. (P) Schematic representation of the developmental time-courses of Ci-Bra (red horizontal bar) and its target genes (colored bars), plotted against a time-table denoting notochord morphogenetic events and the embryonic stages for <i>Ciona</i> at 18°C <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001697#pbio.1001697-Hotta4" target="_blank">[32]</a>. Early-onset genes are depicted as pink bars, middle-onset genes as orange bars, and the two late-onset genes are indicated by blue bars. All bars are dashed on their right side because the expression of the genes that they represent is yet to be determined at the larval stage. hpf, hours post-fertilization.</p

Effects of the removal of functional Ci-Bra binding sites on the developmental onset of a multiple-site notochord CRM.

<p>(A–E) Low-magnification microphotographs of <i>Ciona</i> embryos carrying either the wild-type <i>Ci-lamc1>LacZ</i> notochord CRM transgene (A,D) or a mutated version of this CRM lacking its distal-most Ci-Bra binding site (<i>Ci-lamc1-T1M>LacZ</i>; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001697#pbio-1001697-g007" target="_blank">Figure 7F</a>) or its proximal Ci-Bra binding site (<i>Ci-lamc1-T4M>LacZ</i>; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001697#pbio-1001697-g007" target="_blank">Figure 7F</a>), fixed at the early gastrula (A–C) and mid-gastrula (D,E) stages, and stained with X-Gal. Insets in the low right corners show high-magnifications view of representative stained embryos. The inset in the top right corner of (D) shows an embryos carrying the <i>434-bp Ci-Bra>LacZ</i> transgene, which was used to label the notochord lineage at this stage for comparison. Red and white arrowheads: notochord staining, or lack thereof, respectively; orange: muscle staining. (F) Graph showing the percentage of embryos showing notochord staining as a fraction of the total number of stained embryos scored. Blue bars, embryos at the early gastrula stage; brown, embryos at mid-gastrula; green, embryos at the late tailbud stage (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001697#pbio.1001697.s006" target="_blank">Figure S6</a>). The number of embryos scored for each construct was: WT, early gastrula, <i>n</i> = 646; mid-gastrula, <i>n</i> = 672; mid-tailbud, <i>n</i> = 502. T1M, early gastrula, <i>n</i> = 705; mid-gastrula, <i>n</i> = 994; mid-tailbud, <i>n</i> = 449. T4M, early gastrula, <i>n</i> = 756; mid-gastrula, <i>n</i> = 700; mid-tailbud, <i>n</i> = 431. Statistically significant <i>p</i>-values are indicated by asterisks.</p

Developmental onsets of activity of notochord CRMs representative of the multiple- and single-site Ci-Bra targets, and of two notochord CRMs lacking Ci-Bra binding sites.

<p>Time-course experiments were carried out by WMISH on transgenic embryos (A–F) carrying the notochord CRMs indicated on the top right corner of (A, C, E), fixed at the 110-cell (A,C,E) and early gastrula stage (B,D,F). Insets in the bottom right corners display high-magnification views of representative stained embryos. The inset in the top right corner of (F) shows an embryo carrying the −0.6 kb <i>Ci-Noto1>LacZ</i> transgene stained with X-Gal. (G) Graph of a representative time-course experiment, reporting the number of transgenic embryos showing <i>LacZ</i> expression in notochord precursors, for the constructs and stages detailed in the panel. <i>n</i>, number of scored embryos showing hybridization signal. (H) Graph of time-course experiments for the notochord CRMs associated with the late-onset genes <i>Ci-ACL</i> and <i>Ci-β4GalT</i>, as determined by X-Gal staining. Results of three representative experiments were averaged. The number of embryos scored for the <i>Ci-ACL</i> CRM was: neural plate, <i>n</i> = 327; neurula, <i>n</i> = 419; initial tailbud, <i>n</i> = 389; early tailbud, <i>n</i> = 237. The number of embryos scored for the <i>Ci-β4GalT</i> CRM was: neural plate, <i>n</i> = 342; neurula, <i>n</i> = 402; initial tailbud, <i>n</i> = 348; early tailbud, <i>n</i> = 220.</p

Different structural features of notochord CRMs associated with early, middle, and late-onset Ci-Bra targets.

<p>Summary of the developmental onsets of different Ci-Bra targets plotted against the developmental stages indicated at the bottom. Red bars symbolize minimal notochord CRMs; yellow vertical bars indicate functional Ci-Bra binding sites; a blue and a grey square represent binding sites for transcription factors other than Ci-Bra. CRM structures and sizes are approximate, for simplicity. (Left) The notochord CRMs associated with the early-onset genes <i>Ci-thbs3</i>, <i>Ci-FCol1</i>, <i>Ci-ERM</i>, and <i>Ci-lamc1</i> are controlled by Ci-Bra through multiple functional binding sites. (Center) The notochord CRMs associated with the middle-onset genes <i>Ci-Noto1</i>, <i>Ci-Noto8</i>, <i>Ci-Noto4</i>, <i>Ci-Noto9</i>, and <i>Ci-ABCC10</i> are controlled by Ci-Bra through one main binding site. (Right) The minimal notochord CRMs associated with the late-onset genes <i>Ci-ACL</i> and <i>Ci-β4GalT</i> are controlled indirectly by Ci-Bra via a relay mechanism.</p

Notochord CRMs controlled by Ci-Bra through two cooperative binding sites.

<p>Identification of the minimal sequences necessary for notochord activity of the <i>Ci-FCol1</i> and <i>Ci-Noto5</i> notochord CRMs, through sequence-unbiased truncation analyses and site-directed mutations. All schematics are as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001697#pbio-1001697-g002" target="_blank">Figure 2</a>. (A) Sequence-unbiased truncation and site-directed mutation analyses of the <i>Ci-FCol1</i> notochord CRM. (B–F) Low-magnification group microphotographs of embryos electroporated in parallel with either the wild-type 65-bp <i>Ci-FCol1</i> minimal notochord CRM (B) or with mutated versions of this construct carrying individual mutations in each Ci-Bra binding site, as shown at the bottom of each panel (C–E) or a double mutation in the two proximal Ci-Bra binding sites (F). (G) Quantification of the activity of the constructs shown in (B–F) in different tissues, as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001697#pbio-1001697-g002" target="_blank">Figure 2</a>. (H) Sequence-unbiased truncation and site-directed mutation analyses of the <i>Ci-Noto5</i> notochord CRM. (I–M) Low-magnification group microphotographs of embryos electroporated in parallel with either the wild-type 83-bp <i>Ci-Noto5</i> minimal notochord CRM (I) or with mutated versions of this construct carrying the individual mutations in each Ci-Bra binding site shown at the bottom of each panel (J–L) or a double mutation in the two proximal Ci-Bra binding sites (M). (N) Quantification of the activity of the constructs shown in (I–M) in different tissues, plotted as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001697#pbio-1001697-g002" target="_blank">Figure 2</a>. Insets show higher magnifications of representative stained embryos.</p

Comparison of functional Ci-Bra binding sites with the consensus binding site identified for other Brachyury proteins.

<p>Bases that diverge from the consensus are indicated in lower case. The core TNNCAC sequence is underlined.</p><p>N, any base; R, A or G; W, A or T; Y, C or T.</p

Occurrence and functional requirement of different core T-box binding sites found within notochord CRMs.

a<p>Representation in plain font.</p>b<p>Requirement in bold font.</p><p>EMSA, electrophoretic mobility shift assay.</p

Notochord CRMs controlled by Ci-Bra through single binding sites.

<p>(A,H,M) Identification of the minimal sequences necessary for notochord activity of the <i>Ci-Noto1</i>, <i>Ci-Noto9</i>, and <i>Ci-Noto4</i> notochord CRMs, through sequence-unbiased truncation analyses and site-directed mutations. All schematics are as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001697#pbio-1001697-g002" target="_blank">Figure 2</a>. In the 1.1-kb <i>Ci-Noto1</i> construct, only the binding sites that were analyzed are depicted. (B) Control late tailbud embryo carrying the 170-bp minimal <i>Ci-Noto1</i> notochord CRM. (C) Embryo from the same clutch as (B), co-electroporated with the 170-bp construct and the <i>Ci-FoxA-a>Bra</i> construct; misplaced notochord, endoderm, and CNS cells are indicated by an aqua arrowhead. (D) Control late tailbud embryo carrying the 170-bp minimal <i>Ci-Noto1</i> notochord CRM in which the Ci-Bra site shown in (A) has been mutated as shown at the bottom of the panel. (E) Embryo from the same clutch as (D) co-electroporated with the minimal CRM and the <i>Ci-FoxA-a>Bra</i> construct. (F) Control embryo carrying a larger 1.1-kb <i>Ci-Noto1</i> CRM truncation. (G) Embryo electroporated in parallel with the embryo in (F), carrying the 1.1-kb <i>Ci-Noto1</i> CRM truncation with only the Ci-Bra binding site mutated, as shown at the bottom of (A). Note that in the 1.1-kb construct, only the binding sites that were subjected to mutation analysis are depicted. (I,J) Low-magnification group microphotographs of embryos electroporated in parallel with either the 248-bp <i>Ci-Noto9</i> minimal notochord CRM (I) or with a version of this construct carrying a mutation in the Ci-Bra binding site (J). (K,L) Individual embryos selected from the experiments shown in (I,J). The mutation of the Ci-Bra binding site is shown at the bottom of (L). (M) Sequence-unbiased truncation analysis of the <i>Ci-Noto4</i> notochord CRM. Additional constructs used for the truncation and mutation analysis are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001697#pbio.1001697.s004" target="_blank">Figure S4</a>. In the 0.88-kb construct, only the sites that were analyzed are depicted. (N) Embryo electroporated with the 0.88-kb <i>Ci-Noto4</i> CRM fragment shown in (M). (O) Embryo from the same clutch as (N) co-electroporated with the construct in (N) and the <i>Ci-FoxA-a>Bra</i> construct. (P) Embryo electroporated with the 0.88-kb <i>Ci-Noto4</i> CRM carrying the mutation of a single Ci-Bra binding site, shown at the bottom. (Q) Embryo co-electroporated with the construct in (P) and the <i>Ci-FoxA-a>Bra</i> construct. Representative cells of stained tissues are indicated by arrowheads, which are color-coded as follows: red, notochord; blue, CNS; green, epidermis and tail epidermal neurons; purple, mesenchyme; orange, muscle; yellow, endoderm. Green arrowheads are used also to indicate papillary neurons in (F,G).</p