Positioning a multifunctional basic helix-loop-helix transcription factor within the Ciona notochord gene regulatory network

Abstract

In a multitude of organisms, transcription factors of the basic helix-loop-helix (bHLH) family control the expression of genes required for organ development and tissue differentiation. The functions of different bHLH transcription factors in the specification of nervous system and paraxial mesoderm have been widely investigated in various model systems. Conversely, the knowledge of the role of these regulators in the development of the axial mesoderm, the embryonic territory that gives rise to the notochord, and the identities of their target genes, remain still fragmentary. Here we investigated the transcriptional regulation and target genes of Bhlh-tun1, a bHLH transcription factor expressed in the developing Ciona notochord as well as in additional embryonic territories that contribute to the formation of both larval and adult structures. We describe its possible role in notochord formation, its relationship with the key notochord transcription factor Brachyury, and suggest molecular mechanisms through which Bhlh-tun1 controls the spatial and temporal expression of its effectors.

Keywords: Brachyury; Ciona; Enhancer; Gene regulatory network; Notochord; bHLH.

Figure 1.. The Ci-Bra-dependent expression of Bhlh-tun1 is recapitulated by its cis-regulatory region.

(A,B) Whole-mount in situ hybridizations (WMISH) of late tailbud II embryos (stages according to Hotta et al., 2007) with a Bhlh-tun1 digoxygenin-labeled antisense RNA probe. (A) Control embryo. (B) Transgenic embryo carrying the Ci-FoxA.a>Ci-Bra construct (abbreviated as FoxA.a>Bra). (C,D) Whole-mount late tailbud II embryos stained to detect beta-galactosidase activity after having been electroporated with a 1.7-kb genomic fragment from the 5’-upstream region of Bhlh-tun1, either alone (C), or in combination with the FoxA.a>Bra construct (D). (E,F) WMISH of early tailbud I embryos with the same probe used in (A,B). (E) Control embryo. (F) Mutant embryo, presumably with Ci-Bra^−/− genotype, displaying a shorter tail and malformed notochord compared to stage-matched controls. These embryos lack expression of Bhlh-tun1 in the notochord (white arrowhead), yet they retain expression of this gene in its additional expression domains (arrowheads). Arrowheads are color-coded as follows: red, notochord; blue, CNS; green, epidermis; purple, mesenchyme; grey, cells that are misplaced to the ventral region of the embryo as a consequence of the ectopic expression of Ci-Bra in CNS and endoderm precursors and its over-expression in notochord cells. White, notochord cells showing no expression of Bhlh-tun1. All embryos are oriented with anterior to the left and dorsal up. Scale bars indicate approximately 100 micrometers in A and 50 micrometers in E. Ctrl, control wild-type embryo.

Figure 2.. Identification and characterization of an epidermis CRM and a notochord CRM from the Bhlh-tun1 genomic locus.

Schematic representation of the Bhlh-tun1 locus and its genomic surroundings, which include the flanking genes KH.C7.157 (Emc; light blue) and KH.C7.805 (lilac). Exons are indicated as blue boxes, introns are shown as lines between them. Two enhancer regions are depicted as compact bars, colored in green (epidermis) and red (notochord), respectively. Oblique lines indicated intervening genomic regions devoid of gene models that have not been depicted. Black bent arrows indicate the approximate transcription start sites. Within the red bar that represents the 1.7-kb CRM identified directly upstream of the Bhlh-tun1 coding region, the 144-bp notochord CRM is indicated, and is then magnified and annotated with putative binding sites for Ci-FoxA.a (blue ovals, consensus sequence shown on the left side of the figure) and for Ci-Bra (yellow vertical bars). The specific sequences of these sites are indicated below the 144-bp CRM depiction (blue letters for Ci-FoxA.a binding sites, black for Ci-Bra), and mutant base pairs are indicated by lower-case letters. All fragments were cloned upstream of the Ci-FoxA.a basal promoter (pink box) driving the LacZ reporter gene (blue bar, annotated). Plus or minus signs in the ‘Notochord Activity’ column indicate the presence or absence of notochord staining related to each construct. Two plus signs indicate a qualitatively stronger notochord staining in >50% of the electroporated embryos. (A-C) Mid-tailbud embryos electroporated at the 1-cell stage with either the 995-bp genomic fragment (green box) (A), or the Bhlh-tun1 1.7-kb CRM (red box) (B) or the empty pFBΔSP6 vector (C), fixed and stained with X-Gal. The image in Panel A is a composite of 3 microphotographs of the same embryo, photographed in different planes of focus, z-stacked using the ImageJ software (www.imagej.net). (D-G) Low-magnification microphotographs of representative transgenic embryos selected from larger batches to display the staining produced by the 58-bp minimal notochord enhancer fragment (D) and two of its mutant forms (E,F), alongside the 49-bp truncation that lacks notochord activity (G). Arrowheads are color-coded as in Fig. 1; orange arrowheads indicate muscle staining. SV, sensory vesicle; VG, visceral ganglion.

Figure 3.. Effects of the overexpression of Bhlh-tun1 on notochord and body plan formation.

(A,B) Confocal microphotographs of Ciona embryos carrying the Ci-Bra>GFP neutral notochord marker, either alone (A) or in combination with the Ci-Bra>Bhlh-tun1 construct (abbreviated as Bra> Bhlh-tun1) (B). Insets show close-ups of the regions boxed in white in each panel. The percentage of embryos displaying the notochord phenotype is reported in the top right corner of Panel B. Both embryos were stained with rhodamine-phalloidin as previously described (José-Edwards et al., 2013). (C) Overlap of bright-field and fluorescent confocal microphotographs of an embryo carrying the FoxA.a>Bhlh-tun1 construct, representative of the phenotype induced by this transgene. All 40 notochord cells are fluorescent, and the panel shows a collapsed z-stack of their images acquired serially from the whole-mount embryo. The frequency of embryos displaying this phenotype is indicated in the bottom left corner. Inset shows the notochord cells in dark field (see also Supplemental Movie 1). The area boxed in red in (C) and in the inset is shown at higher magnification in (C’) to highlight the changes in cell shape induced by the overexpression of Bhlh-tun1. Scale bar in C’: 10 micrometers.

Figure 4.. Expression studies of putative Bhlh-tun1 target genes.

(A-P) WMISH of Ciona embryos with digoxigenin-labeled antisense RNA probes for genes identified in the microarray screen. The EST clones used to generate each probe are reported in the top right corner of each panel. Most embryos are at tailbud stages (approximately ranging from mid-neurula to mid-tailbud II; Hotta et al., 2007); (B) dorsal view of mid-neurula embryo; (C) slightly lateral view of a mid-neurula embryo. Arrowheads are color-coded as in Fig. 1. (Q,R) Pie graphs showing the distribution within different expression territories of the genes identified as putative Bhlh-tun1 targets (Q) and the gene ontologies available for the 16 of the 21 Bhlh-tun1 targets expressed in notochord cells. Genes expressed in epidermis are framed in green (N-P). Current gene models are indicated in the bottom left corner of each panel; gene names, whenever available, are reported on the bottom right corner of each panel. TXF, transcription factor; ECM, extracellular matrix.

Figure 5.. Up-regulation of Claudin16/17/19 in embryos over-expressing Bhlhtun1.

(A-D) WMISH of control (A,C) and transgenic (B,D) Ciona embryos, performed using the digoxigenin-labeled antisense RNA probes synthesized using as templates the EST clones reported in the bottom right corner of panels A and C. The current gene name is indicated on the left side of each row. Gene models are indicated in the left bottom corner of panels A and C. Arrowheads are color-coded as in Fig. 1; yellow arrowheads indicate endodermal cells. (E) Graph showing the results of qRT-PCR experiments aimed at validating the over-expression detected by WMISH, performed at the same stage (stage 19/early tailbud stage 1; see Methods). Details of the experimental results are listed in Table S1, oligonucleotide sequences can be found in Table S2. p, p-value; ctrl, control (wild-type) embryos. (F) Late-tailbud II embryo electroporated at the 1-cell stage with a 300-bp fragment identified in the 5’-flanking region of Claudin16/17/19, containing a cluster of three E-boxes. The embryo was stained using X-Gal for about 4 hrs at 37°C. Scale bars indicate approximately 100 micrometers in A and 50 micrometers in E.

Figure 6.. Validation of additional Bhlh-tun1 early and late target genes expressed in the developing notochord.

(A-D) WMISH of control (A,C) and transgenic (B,D) Ciona embryos, performed using the digoxigenin-labeled antisense RNA probes synthesized using as templates the EST clones reported in the bottom right corner of panels A and C. The current gene name is indicated on the left side of each row. Gene models are indicated in the left bottom corner of panels A and C. Arrowheads are color-coded as in Fig. 1; yellow arrowheads indicate endodermal cells. The light pink arrowhead in C indicates weak notochord staining (see also Fig. 4D). (E,F) Graphs showing the results of qRT-PCR experiments aimed at validating the over-expression detected by WMISH, performed at the same stage as panels A-D (stage 19/early tailbud stage I; panel E), or at the stage 23/late tailbud I (F). p, p-value; ctrl, control (wild-type) embryos. Details of the experimental results are listed in Table S1, oligonucleotide sequences can be found in Table S2.

Figure 7.. Genes down-regulated by the over-expression of Bhlh-tun1.

(A-D) WMISH of control (A,C) and transgenic (B,D) Ciona embryos, performed using the digoxigenin-labeled antisense RNA probes synthesized using as templates the EST clones reported in the bottom right corner of panels A and C. The current gene name is indicated on the left side of each row. Gene models are indicated in the left bottom corner of panels A and C. Arrowheads are color-coded as in Fig. 1.

Figure 8.. Position of Bhlh-tun1 and a few of its notochord target genes within the Ci-Bra-downstream gene regulatory network.

Left side: main stages of Ciona development that precede lumen formation (tubulogenesis) are shown for reference. Right side: a few of the main developmental milestones that punctuate notochord morphogenesis are listed. Hpf: hours post-fertilization. Central panel: a simplified view of the branch of the Ciona notochord GRN controlled by Bhlh-tun1. Additional transcription factors that compose the Ciona notochord GRN can be found elsewhere (Imai et al., 2006; Kugler et al., 2008; José-Edwards et al., 2011). Names of transcription factors are colored in blue (Bhlh-tun1 in violet), structural genes or enzymes in black. A Myb-like transcription factor that activates expression of B4GalT is colored in grey (Katikala et al., 2013 and our unpublished results). Arrows symbolize activation of gene expression. Dashed lines with flat ends indicate down-regulation of gene expression, the extent of which remains to be determined. HD, homeodomain transcription factor that contributes to activate expression of Bhlh-tun1, presumed on the basis of the notochord CRM analysis (Fig. 2).