Transcription Factor Lbx1 Expression in Mouse Embryonic Stem Cell-Derived Phenotypes

Transcription factor Lbx1 is known to play a role in the migration of muscle progenitor cells in limb buds and also in neuronal determination processes. In addition, involvement of Lbx1 in cardiac neural crest-related cardiogenesis was postulated. Here, we used mouse embryonic stem (ES) cells which have the capacity to develop into cells of all three primary germ layers. During in vitro differentiation, ES cells recapitulate cellular developmental processes and gene expression patterns of early embryogenesis. Transcript analysis revealed a significant upregulation of Lbx1 at the progenitor cell stage. Immunofluorescence staining confirmed the expression of Lbx1 in skeletal muscle cell progenitors and GABAergic neurons. To verify the presence of Lbx1 in cardiac cells, triple immunocytochemistry of ES cell-derived cardiomyocytes and a quantification assay were performed at different developmental stages. Colabeling of Lbx1 and cardiac specific markers troponin T, α-actinin, GATA4, and Nkx2.5 suggested a potential role in early myocardial development.


Introduction
Lbx1 is a member of the Ladybird-like homeobox gene family that encodes a homeodomain transcription factor. This mouse counterpart of Drosophila m. ladybird gene was first discovered by Jagla et al. [1]. In vertebrates, expression of Lbx1 has been described in the CNS and in migrating muscle precursor cells. During early mouse embryonic development, the presence or absence of Lbx1 distinguishes two major neuronal classes generated in the dorsal spinal cord [2]. Specifically, Lbx1 is essential for determining a somatosensory instead of a viscerosensory fate in relay neurons in the hindbrain [3]. At later stages of mouse neurogenesis, expression of Lbx1 defines a basal GABAergic differentiation state for dorsal horn neurons [4].
Furthermore, it was found that Lbx1 plays an important role in the migration of hypaxial muscle precursor cells during development. It was suggested by Brohmann et al. [5] that Lbx1 controls the expression of genes that are essential for the recognition or interpretation of cues that guide migrating muscle precursors and maintain their migratory potential. Watanabe et al. [6] detected Lbx1 in activated but not quiescent satellite cells of adult mice. They suggested that Lbx1 plays important roles in the differentiation and maintenance of satellite cells of mature myofibers.
In addition to its relevance in neuronal [7] and muscle cell [8] development in Drosophila ladybird genes have also been reported to be expressed in a specific subset of cardioblasts, required for the diversification of heart precursor cells [9]. Until today, there are only few data about the involvement of Lbx1 in mouse cardiogenesis. Inactivation of Lbx1 in mice mainly resulted in defects in heart looping and increased cell proliferation leading to myocardial hyperplasia [10]. Obviously there are striking morphological and functional differences between the tubular Drosophila heart and the four-chambered mammalian heart. However, the specification of cardiac primordia in both Drosophila 2 Stem Cells International and vertebrate embryos is under the control of conserved core cardiac transcription factors encoding, for example, Nkx2.5/Tinman, GATA/Pannier, Mef2, and Hand family members [11].
Murine embryonic stem (ES) cells are characterized by the capacity to differentiate into virtually any cell type of an organism, including neurons, skeletal, and cardiac muscle cells [12]. In vitro differentiation of mouse ES cells into cardiomyocytes recapitulates the programmed expression of cardiac genes observed in the mouse embryo in a timecontrolled manner [13]. During ES cell differentiation, cardiac-specific genes are up-or downregulated dependent on extracellular signals and cell-cell interactions, thus providing an excellent model system to study early embryonic development at the cellular level.
Therefore, the aim of this study was the identification of Lbx1 expression at the transcript and protein level in ES cellderived neurons and muscle cell progenitors to ensure the presence of Lbx1 in our in vitro model system. Specifically, we investigated whether Lbx1 is also expressed in ES cellderived cardiac myocytes. The presence of Lbx1 was clearly demonstrated in a small subpopulation of ES cell-derived cardiomyocytes by immunocytochemistry. [14] were cultured on a mitotically inactivated embryonic fibroblast feeder layer in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Karlsruhe, Germany) and supplements as described [15]. The two-step differentiation protocol included (i) the formation of embryoid bodies (EBs) and (ii) after EB plating on adhesive substrate (0.1% gelatin) the expansion of multilineage progenitor cells and spontaneous differentiation to form differentiated phenotypes. In short, ES cells (n = 600 cells/20 μL drop) were plated on the lids of Petri dishes (∅ 10 cm), cultured as "hanging drops" for 2 days and on bacteriological plates (∅ 6 cm) in suspension for additional 3 days to form EBs (five days in total = 5 d). The differentiation medium consisted of Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% FCS (selected batches), Lglutamine/penicillin/streptomycin (1 : 100 of stock solution), nonessential amino acids (1 : 100 of stock solution, all from Invitrogen), and α-monothioglycerol (final concentration 450 μM; Sigma-Aldrich, Taufkirchen, Germany). At day 5, EBs were plated in IMDM with the supplements mentioned above and cultivated for further two (= 5 + 2 d) up to 35 days (= 5 + 35 d) onto gelatin-coated 6 cm culture dishes for RT-PCR analysis and cardiomyocyte isolation (30 EBs). 30 EBs were plated onto gelatin-coated 6 cm culture dishes containing cover slips for immunocytochemistry, and 100 EBs onto 10 cm culture plates for protein isolation. Cultures were maintained in a 37 • C/5% CO 2 incubator. Although this differentiation approach is preferentially used to obtain cardiomyocytes and skeletal muscle cell progenitor cells, a small population of neuronal cells can be generated, too.
The PCR reactions were performed on a 25 μL reaction volumes. One-third of each PCR reaction was electrophoretically separated on 2% agarose gels containing GelRed nucleic Acid Gel Stain (Biotium, Hayward, USA). Gels were illuminated with UV light, and the GelRed fluorescence signals were analyzed by the TINA2.08e software (Raytest Isotopenmeßgeräte, Straubenhardt, Germany). Data of the target genes were plotted as percentage changes in relation to the expression of the housekeeping gene Gapdh. Gels of ten independent experiments were analyzed. For statistical evaluation, data were compared using analysis of variance (ANOVA).

Immunofluorescence Analysis.
Indirect immunofluorescence (IF) analysis of EB outgrowths was carried out at days 5 + 11 for neuronal phenotypes and 5 + 14 for skeletal muscle cell progenitors. To quantify the amount of Lbx1-positive cardiac cells in EB outgrowths at various differentiation stages, beating cardiac clusters were isolated as described [15] at days 5 + 4, 5 + 7, 5 + 9, 5 + 11, and 5+15. Clusters were replated onto gelatin-coated cover slips (∅10 mm) in wells of 4-well plates and allowed to attach overnight. Cells were rinsed with PBS and fixed with PBS containing 4% paraformaldehyde at RT for 20 min. After washing, cells were permeabilized with 0.1% Triton in PBS at RT for 10 min. Preparations were incubated with 1% bovine serum albumine in PBS for 1 h followed by incubation with the primary antibodies at RT in a humidified chamber for 1 h. The following antibodies were applied: rabbit anti mouse Lbx1 The quantitative estimation based on the evaluation of approximately 1000 cardiac cells (n = 10 experiments) positively stained for troponin T at the given differentiation stages. Lbx1-immunopositive cells displaying a strong fluorescence signal coexpressing troponin T (Lbx1+, troponin T+) and cells positive for troponin only T (Lbx1−, troponin T+) were counted. Lbx1 expressing cells not costained by troponin T antibody in close proximity to cardiac clusters were not counted. Most likely, these cells did not rearrange the sarcomeric apparatus properly after dissociation and replating.

RT-PCR Analysis of Lbx1
Expression. Embryonic mouse heart and brain tissues as well as ES cells and differentiated progeny of 2 to 5+35 days of cultivation were analyzed by RT-PCR. Heart and brain tissue from E12.5 revealed transcript levels with slightly higher levels in brain tissue (Figure 1(a)). Transcript levels of Lbx1 were low in undifferentiated ES cells (8.2%) but showed a significant transient upregulation at the progenitor cell stage from 5 + 4 d (55.9%) to 5 + 14 d (72.3%; Figure 1(b)). Maximum level of 86.7% was reached at day 5+11. At the terminal differentiation stage 5+30 d, Lbx1 was significantly downregulated to 30.3%.

Detection of Lbx1 by Western Blot Analysis.
To confirm the presence of Lbx1 at the protein level, heart and brain tissues from different developmental stages were analyzed. Lbx1 was moderately detectable in comparative brain tissues (Figure 2(a)). Lbx1 could also be detected in E12. 5  was expressed at all stages of differentiation at moderate levels ( Figure 2(b)).

Immunocytochemical Analysis of Lbx1 in Differentiated
ES Cell Progeny. Immunocytochemical analysis was first performed with skeletal muscle progenitors and neuronal cells to confirm the involvement of Lbx1 in early developmental processes in ES cell-derived cultures in vitro. Costaining with sarcomeric α-actinin revealed a small number of Lbx1positive skeletal muscle cell progenitors (Figure 3(a)). Lbx1 immunoreactivity was also detected in numerous β III tubulin-positive neuronal cells (Figure 3(b) and at higher magnification Figure 3(c)). To identify the neuronal subtype, costaining with anti-GABA antibody was performed. As shown in Figures 3(d ) and 3(d ), Lbx1 was expressed in the nuclei of GABA-positive neuronal cells. Immunocytochemical analysis of Lbx1 in undifferentiated ES cells revealed its absence (not shown).

Lbx1 Is Colocated in ES Cell-Derived Cardiomyocytes.
In order to discover whether Lbx1 could also be detected in ES cell-derived cardiomyocytes, triple immunofluorescence staining including sarcomeric α-actinin and early cardiac transcription factors was performed in isolated beating clusters. Lbx1 was found in a small subpopulation of cardiomyocytes in coexpression with GATA4 (Figure 4(a )) and Nkx2.5 (Figure 4(b )).
To further investigate the amount of Lbx1-expressing cardiomyocytes, a quantification assay was performed at different developmental stages. Immunocytochemistry revealed that about 10% of troponin T-positive cardiomyocytes stained positive for Lbx1 as early as day 5 + 4 ( Figure 5(a)). This amount slightly changed at days 5 + 7 (7.8%) and 5 + 9 (8.2%) to 10.9% at 5 + 11 d and 9.6% at day 5+15, but changes were not significant. Figures 5(b)-5(d) showed representative images of immunofluorescence staining of isolated beating cardiomyocytes partially coexpressing Lbx1 at given time points.
Because there was no Lbx1-specific purification or enrichment procedure included, approximately less than five percent of ES cell-derived cells expressed Lbx1. Expression was restricted to skeletal myocyte progenitors, cardiomyocytes, and neuronal cells.

Discussion
Analysis of Lbx1 at the transcript and protein level in ES cell-derived progeny revealed the expression in neurons as well as skeletal muscle progenitors and in a small subpopulation of cardiomyocytes. To our knowledge, this is the first time that a coexpression of Lbx1 and several cardiacspecific markers could be demonstrated in ES cell-derived cardiomyocytes.
RT-PCR analysis showed an expression signal in embryonic heart tissue. While Schäfer et al. [10] detected Lbx1-LacZ-positive cells in embryonic mouse hearts but failed to detect Lbx1 mRNA, Chao et al. [17] found moderate expression levels of Lbx1 gene in porcine hearts. Lbx1 was significantly upregulated at the progenitor cell stage around day 5 + 11 of ES cell differentiation. Western blot analysis of embryonic hearts tissue samples and several ES cell differentiation stages confirmed the presence of Lbx1 protein in our model system. Because there are progenitors from several lineages including the mesodermal as well as ectodermal lineage present at this time of ES cell differentiation [18], double immunocytochemistry using cell type-specific antibodies was performed to assign Lbx1 signal to specific phenotypes. As expected, Lbx1 immunoreactivity could be detected in skeletal muscle cell precursors and GABAergic neurons, reflecting the tissue expression pattern in mice as described previously [4,19].
Costaining of Lbx1 with cardiac transcription factors (GATA4, Nkx2.5) and the continuous expression in a subpopulation of troponin T-positive cardiomyocytes suggested a potential role in myocardial differentiation and function. During mouse cardiogenesis at E11.0, single Lbx1-LacZpositive cells were detected in the myocardium of the left ventricle [10]. The authors stated that this small population of Lbx1-LacZ-positive cells might originate from the neural tube migrating to the caudal branchial arch and the truncus arteriosus between E9.0 and E9.5 or reflect a de novo Lbx1 expression in the myocardium. Our data provide first evidence for an expression of Lbx1 in murine cardiomyocytes independent from the cardiac neural crest system.
Long-term expression of the Lbx1 homolog Ladybird was also found in Drosophila [20]. The authors performed a muscle and heart-targeted genome-wide transcriptional profiling and a chromatin-immunoprecipitation-(ChIP-)on-chip search for direct Ladybird targets. They concluded that Ladybird contributes to specifying the identity of cardiac precursors, regulates genes required for the acquisition of cell-type specific properties (e.g. motility, shape, and size),  and might be involved in the regulation of genes required for terminal differentiation and the functional properties of cardiac cells [20]. A similar approach in mice would help to discover the entire function of Lbx1 in vertebrate heart development.
Taken together, our findings clearly demonstrated the expression of Lbx1 in embryonic stem cell-derived cardiomyocytes, thus providing a model system for the identification of Lbx1 target genes and signaling pathways involved in early heart failure caused by Lbx1 inactivation.