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Islet-1 is a LIM-homeodomain transcription factor that has been defined to label cardiac progenitor cells of the second heart field. Here we provide the first analysis of the expression pattern of Xenopus islet-1 (Xisl-1) in the context of cardiovascular development. During early stages of heart development Xisl-1 is co-expressed with Nkx2.5 in the cardiac crescent in Xenopus supporting the notion of an initially single heart field. At subsequent stages of cardiogenesis the expression domains of Xisl-1 and Nkx2.5 become more distinct with Xisl-1 being detected more anterior to Nkx2.5, however both factors continue to be co-expressed in the dorsal mesocardium and pericardial roof of the linear heart tube. The presence of a cardiac Xisl-1 progenitor pool in an amphibian whose heart lacks an anatomically separated rightventricle is intriguing. Functional analyses show that Xisl-1 is required for normal heart development. Inhibition of Xisl-1 results in defects in heart morphogenesis and in the downregulation of early cardiac markers implicating a role for Xisl-1 in cardiac specification. Additionally, Xisl-1 loss-of-function affects the expression of several vascular markers demonstrating the involvement of Xisl-1 in vasculogenesis.
Fig. 1. Xisl-1 protein domains and temporal expression pattern. (A) The Xisl-1 ORF codes for a 354 amino acid (aa) protein, which contains two LIM protein–protein interaction domains and a homeobox DNA binding domain as indicated. A comparison of the Islet-1 amino acid sequence between different species demonstrates a high evolutionary conservation. The Isl-1 protein of frog, fish, chick, mouse and human show a 96–98% overall homology. The overall homology to the Drosophila Islet-1 protein is only 55%, however the LIM domains and the homeodomain are more conserved with 69–89% homology. The functional domains of vertebrate Isl-1 are 95% to 100% homologous. (B) RT-PCR analysis of the temporal expression pattern of Xisl-1. Xisl-1 transcripts are present maternally at low levels. During late gastrula stages (st.12.5) Xisl-1 is significantly upregulated and continues to be expressed at similar levels at least until tailbud stages.
Fig. 2. Spatial expression pattern of Xisl-1. (A) Xisl-1 is expressed in the anteriormost region of the embryo (shown from an anterior view, dorsal is up) including the cement gland anlage (asterisk) and the cardiac crescent (arrow). (B) During early neurula stages Xisl-1 expression extends dorsally adjacent to the neural folds (cephalic placodal area) (arrows). (C) Xisl-1 mRNA additionally marks the profundal–trigeminal placodal area. (D) A parasagittal section (anterior is to the left, dorsal is up) shows the expression of Xisl-1 in the anteriormesendoderm (arrow). (E) At neural tube stages Xisl-1 transcripts are present in the cardiac crescent (arrow) and are also detected in the stomodeum abutting the cement gland on the dorsal side (arrowhead). Xisl-1 expression is maintained in the cephalic placodes and their associated ganglia at least until st. 36 (E–K and data not shown). (F) Xisl-1 is expressed in the heart region (arrow). Punctate expression is seen in precursor cells of the developing vascular system (arrowheads). (G) Xisl-1 transcripts are abundant in the foregutendoderm and pharyngeal mesoderm and expression in the heart region has become restricted to an anterior domain (arrow); arrowhead points to pineal gland. (H) Co-staining for Xisl-1 (light blue) and Nkx2.5 (dark blue) shows that Xisl-1 is expressed more anteriorly compared to Nkx2.5. (I–K) Expression of Xisl-1 in the developing heart (asterisk) decreases and becomes restricted to a dorsolateral domain. aa: aortic arches; IFT: inflow tract; lpm: lateral plate mesoderm; np: neural plate; pPrV: profundal–trigeminal placodal area; gPr: profundal ganglion; gV: trigeminal ganglion; gVII: ganglia of facial nerve VII.
Fig. 3. Characterization of the spatial relationship between the Xisl-1 and Nkx2.5 expression domains. Single and double whole mount in situ hybridizations were performed to depict the expression domains of Xisl-1 and Nkx2.5 during early heart development in more detail. Pictures A, F, K, P show detection of Xisl-1 transcripts in light blue. Pictures B, G, L, Q show detection of Nkx2.5 transcripts in red. Pictures C, H, M, R show staining for both transcripts with the overlap of expression exhibiting a darker, rather purple staining. The asterisk behind a letter denotes images of identical embryos. Other embryos were stage matched. Pictures E, J, O depict schematics of the relative expression domains of Xisl-1 (blue dots) and Nkx2.5 (red lines) from embryos shown in pictures D, I, and N. (A–D) At neurula stages, both Xisl-1 and Nkx2.5 demarcate the cardiac crescent, which lies ventrally to the cement gland (arrows in pictures C, D). All embryos are shown from an anterior view. Picture D shows an enlargement of the cardiac crescent region of the embryo depicted in picture C. (F–I) At early tailbud stages, Xisl-1 and Nkx2.5 expression still overlaps largely in the heart region (arrow in picture H). (I) A ventral view of the heart region of an embryo to better demonstrate the overlapping expression of Xisl-1 and Nkx2.5 (arrow) during early tailbud stages. Xisl-1 only (blue) is also strongly expressed in the stomodeal region abutting the cement gland. (K–N) At stage 30 when the linear heart tube begins to form, Xisl-1 transcripts become restricted to a more anterior domain of the heart region (arrowhead) and the overlapping domain with Nkx2.5 decreases (black arrow). The white arrow points to the domain in which only Nkx2.5 is expressed. Picture N is a ventral view of the same embryo shown in picture M; head is to the left. The dotted lines highlight the Xisl-1 (white lines) and Nkx2.5 (black lines) expression domains. The ventral view of a stage matched embryo depicted in picture T shows a single staining for Xisl-1 in dark blue, in which the gap of Xisl-1 expression (asterisk) marks the region that expresses Nkx2.5 as seen in picture N. The arrow points to Xisl-1 expression in the posterior domain of the heart. (P–S) At progressive stages Xisl-1 transcripts are detected in the most anterior part of the heart region and its cardiac expression can only be documented in transverse sections. (U–X) Anterior to posterior series of transverse sections of a st. 28 embryo stained for Xisl-1 (purple) and Nkx2.5 (red). In the most anterior section (U) Xisl-1 transcripts are localized in two circular domains in the mesodermal layer (arrows) and in the pharyngeal endoderm (asterisk). No Nkx2.5 staining is detectable. (V) Along the A–P axis, expression of Xisl-1 becomes crescent-shaped in the mesoderm. (W) The section reveals an extensive overlap of Xisl-1 and Nkx2.5 in the cardiogenic mesoderm (arrows). (X) Further posterior, Xisl-1 becomes restricted to the dorsolateral domain (black arrows). In the ventralmost expression domain only Nkx2.5 transcripts are detected (white arrow). (Y–Z3) Transverse sections through the linear heart tube of st.30 embryos are shown. (Y, Y1) Nkx2.5 expression (red) throughout the heart tube (myocardium, mesocardium and pericardial roof) is depicted. A subsection of the mesocardium and pericardial roof is shown as an enlargement in picture Y1. (Y2, Y3) Nkx2.5 (red) and Xisl-1 (blue) expression is seen in the linear heart tube of a st.30 embryo. Xisl-1 transcripts are absent from the myocardium but are detected in the endocardium and in the foregutendoderm (asterisk). The Xisl-1 and Nkx2.5 expression domains overlap in the mesocardium and pericardial roof (see enlargement Y3). (Z, Z1) TnIc (red) is expressed in differentiated cardiomyocytes of the myocardium. Note the absence of TnIc transcripts in the mesocardium and pericardial roof. The enlargement shows a subsection of these domains (Z1). (Z2, Z3) TnIc (red) and Xisl-1 (blue) expression in the linear heart tube of a st.30 embryo. Note the complementary expression domains of TnIc and Xisl-1 from the myocardium, mesocardium, and pericardial roof, respectively. cg: cement gland; ec: endocardium; mc: myocardium; me: mesocardium; nf: neural fold; np: neural plate; pc: pericardial roof.
Fig. 6. Xisl-1 is required for cardiac marker gene expression. 10 ng islMO were injected unilaterally into the dorsal marginal zone of 8-cell stage embryos. At st. 28 these embryos were analysed for cardiac gene expression by whole mount in situ hybridization. Xisl-1 loss-of-function results in a reduction or even loss of expression of the early cardiac gene Tbx20, as well as of the differentiation marker genes Troponin Ic (TnIc) and cardiac-α-actin on the injected side (arrows). The specificity of the morpholino effect was verified by coinjection of RNA transcribed from the δisl-1 deletion construct, whose translation is not inhibited by the islMO. Coinjection of δisl-1 RNA and islMO partially restored the expression of the investigated marker genes.
Fig. 5. Xisl-1 is required for normal heart development. (A) At early tadpole stages gross morphological heart defects are clearly seen in whole embryos that have been injected with 20 ng islMO (arrow); dotted line demarcates the heart in the WT embryo. Embryos exhibit an enlarged pericardial cavity and in the most severe cases the heart is reduced to a remnant of beating tissue. About 80% of the islMO injected embryos have heart defects (upper bar graph). These defects were further qualified (lower bar graph). (B) Hearts were dissected from st. 42/43 embryos that have been injected with either islMO or control MO (CMO) to better visualize the morphological defects. In addition to the obvious phenotype of an unlooped heart tube, there are cases when the heart does undergo looping however it is smaller compared to a normal heart. Smaller hearts were measured as indicated in the cartoon and the sizes are presented in μm total length in the bar graph. (C) At earlier stages a heart phenotype in whole embryos injected with islMO is visible by the reduced staining for TnIc. Vibratome sections show that the heart tube is reduced in size and fails to loop as a result of Xisl-1 loss-of-function. Dotted lines depict section plane. a: atria; oft: outflow tract; v: ventricle.
