Sparrow DB et al. (1998), Thylacine 1 is expressed segmentally within the...

XB-ART-14753

Development 1998 Jun 01;12511:2041-51. doi: 10.1242/dev.125.11.2041.

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Thylacine 1 is expressed segmentally within the paraxial mesoderm of the Xenopus embryo and interacts with the Notch pathway.

Sparrow DB , Jen WC , Kotecha S , Towers N , Kintner C , Mohun TJ .

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The presomitic mesoderm of vertebrates undergoes a process of segmentation in which cell-cell interactions mediated by the Notch family of receptors and their associated ligands are involved. The vertebrate homologues of Drosophila &Dgr ; are expressed in a dynamic, segmental pattern within the presomitic mesoderm, and alterations in the function of these genes leads to a perturbed pattern of somite segmentation. In this study we have characterised Thylacine 1 which encodes a basic helix-loop-helix class transcription activator. Expression of Thylacine is restricted to the presomitic mesoderm, localising to the anterior half of several somitomeres in register with domains of X-Delta-2 expression. Ectopic expression of Thylacine in embryos causes segmentation defects similar to those seen in embryos in which Notch signalling is altered, and these embryos also show severe disruption in the expression patterns of the marker genes X-Delta-2 and X-ESR5 within the presomitic mesoderm. Finally, the expression of Thylacine is altered in embryos when Notch signalling is perturbed. These observations suggest strongly that Thylacine 1 has a role in the segmentation pathway of the Xenopus embryo, by interacting with the Notch signalling pathway.

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Species referenced: Xenopus
Genes referenced: ank1 dlc dll1 esr-5 max mespa mt-tr neurog2 notch1 trna tubb2b

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	Fig. 1. Sequence of the Xenopus Thylacine 1 and Thylacine 2 genes. (A) Xenopus Thylacine 1 cDNA sequence and deduced amino acid sequence. The amino acids in bold represent the basic helix-loop-helix (bHLH) domain. (B) Comparison of the amino acid sequences of Thylacine 1 and Thylacine 2. Dots indicate amino acid identity, dashes represent gaps introduced to maximise the alignment and black shading indicates the bHLH domain. (C) Comparison of Thylacine 1 and Thylacine 2 bHLH domains with other members of the bHLH family. Black shading indicates amino acid identity with Thylacine 1. Sequences were aligned using the MegAlign program (DNASTAR Inc.). GenBank accession numbers for the Thylacine 1 and Thylacine 2 nucleotide sequences are Y14446 and Y14447 respectively.
	Fig. 2. Expression of Thylacine 1. The distribution of Thylacine 1 was analysed by RNase protection assay. (A) Distribution in Xenopus embryos. Lane 1, undigested probes; lane 2, tRNA control; lanes 3- 11, unfertilised egg, stages 8, 10, 12.5, 14, 17, 20, 23, 34 embryo RNA respectively. Two embryo equivalents of RNA (10 mg) was used in each case. (B) Distribution in adult frog tissues. Lane 1, undigested probes; lane 2, tRNA control; lanes 3-10, intestine, stomach, gall bladder, spleen, skeletal muscle, lung, liver, stage 20 RNA respectively. 5 mg of total RNA was used in each assay. As an internal control a probe for XMax2, which is expressed at constant levels throughout early development and in adult tissues (Tonissen and Krieg, 1994), was included in the Thylacine 1 assay.
	Fig. 3. Localisation of Thylacine 1 transcripts in Xenopus embryos. In this and other figures, embryos and sections are oriented with anterior to the left. Embryos were stained in whole mount for the expression of Thylacine 1 at neurula stages (stage 17: A,B; stage 19: C,D), tailbud (stage 22/23: E,F; stage 26: G; stage 28: H) and swimming tadpole (stage 35: I). Dorsal views are shown in A,C and E, and lateral views in B,D,F,G,H and I.
	Fig. 4. Comparison of Thylacine, X-Delta-2 and X-ESR5 expression patterns. Tailbud embryos (stage 27) were stained by whole-mount in situ hybridisation for Thylacine 1 (A,B), X-Delta-2 (C,D) or XESR5 (E,F) expression. Lateral views are shown in A,C and E, and sections of the same embryo along the longitudinal axis are shown in B,D and F respectively, viewed from a dorsal aspect. Expression is shown in dark blue and nuclei are counter-stained in red with Fuelgen. Brackets are used to indicate the relative positions of somitomeres (numbered) and mature somites (labelled in lower case letters). The forming somite (undergoing rotation) is designated somitomere 1. In all cases, sections from 10-20 embryos were examined to confirm the precise positioning of the transcripts relative to the somitomeres.
	Fig. 5. Mapping the transcriptional activation domain of Thylacine 1. Yeast strain Y190 was transformed with a yeast expression vector encoding the indicated Gal 4-Thylacine 1 chimeras. Amino acids contained in each mutant are indicated on the left. lacZ activity was determined by liquid culture assay. Values are expressed as a percentage of full length Thylacine 1 activity observed for each mutant and are means ± standard errors of at least three experiments.
	Fig. 6. Expression of Thylacine 1 alters the pattern of segmentation in Xenopus embryos. Embryos were injected on one side, at the twocell stage, with (A) nlacZ RNA or (B,C) a mixture of Thylacine 1 and nlacZ RNAs. At early tadpole stages, embryos were fixed and stained in whole-mount with X-gal, which stains the nuclei blue, and with the 12/101 antibody using HRP immunohistochemistry, which stains the muscle cells brown. Representative longitudinal sections are shown with the uninjected side oriented to the top in each case. (A) Embryo injected with just the nlacZ tracer. (B) Embryo expressing Thylacine 1 RNA with a ‘mild’ phenotype. (C) Embryo expressing Thylacine 1 with a ‘strong’ phenotype.
	Fig. 7. Ectopic Thylacine expression does not affect neurogenesis. Embryos injected with (A) nlacZ, (B) a mixture of nlacZ and Thylacine 1 or (C) a mixture of nlacZ and XNGNR1 RNAs were fixed at early neurula stages and stained for N-tubulin expression using whole-mount in situ hybridisation (dark blue-purple). To determine the injected side, nlacZ expression was detected by X-gal staining. In all cases dorsal views are shown with the uninjected side of the embryo oriented to the top of the panel.
	Fig. 8. Thylacine expression alters the expression of segmental markers of the paraxial mesoderm. Embryos injected with (A,B,E,F) nlacZ, or (C,D,G,H) a mixture of nlacZ and Thylacine 1 RNAs were fixed at early neurula stages and stained for X-Delta-2 (A-D) or XESR5 (E-H) expression using whole-mount in situ hybridisation (dark blue-purple). To determine the injected side, nlacZ expression was detected by X-gal staining. In each pair of panels lateral views of the uninjected and injected sides of the same embryo are shown.
	Fig. 9. Thylacine 1 expression is altered by expression of XSu( H)DBM and X-Su(H)/Ank. Embryos injected with (A,D) nlacZ, (B,E) a mixture of nlacZ and X-Su(H)DBM, or (C,F) a mixture of nlacZ and X-Su(H)/Ank RNAs were fixed at early neurula stages and stained for either X-Delta-2 (A-C) or Thylacine 1 (D-F) expression using whole-mount in situ hybridisation (dark blue-purple). To determine the injected side, nlacZ expression was detected by X-gal staining. In all cases dorsal views are shown with the injected side of the embryo oriented to the top of the panel.