First published online April 3, 2008
Stem Cells
Vol. 26 No.
6
June 2008, pp.
1490
-1495
doi:10.1634/stemcells.2007-1099; www.StemCells.com
© 2008 AlphaMed Press
The Neuronal Differentiation Potential of Ldb1-Null Mutant Embryonic Stem Cells Is Dependent on Extrinsic Influences
Minyoung Hwanga,
Marat Gorivodskyb,
Minjung Kimc,
Heiner Westphalb,
Dongho Geuma
aGraduate School of Medicine, Korea University, Seoul, Korea;
Laboratories of bMammalian Genes and Development and
cMolecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA
Key Words. Ldb1 • Embryonic stem cells • Neuronal differentiation • Embryoid body • Five-stage method • Adherent monolayer culture method
Correspondence:
Correspondence: Heiner Westphal, M.D., Ph.D., Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2790, USA. Telephone: 301-496-1855; Fax: 301-402-0543; e-mail: hw{at}helix.nih.gov; or Dongho Geum, Ph.D., Graduate School of Medicine, Brain Korea 21, Korea University, Seoul 136-705, South Korea. Telephone: 82-2-920-6091; Fax: 82-2-929-5696; e-mail: geumd{at}korea.ac.kr
Received on January 8, 2008;
accepted for publication on March 19, 2008.
First published online in STEM CELLS EXPRESS April 3, 2008.
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ABSTRACT
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LIM-domain binding protein 1 (Ldb1) is a multiadaptor protein that mediates the action of transcription factors, including LIM-homeodomain proteins. To elucidate the functional role of Ldb1 in the neuronal differentiation of embryonic stem (ES) cells, we have generated Ldb1-null mutant (Ldb1–/–) ES cells and examined neuronal differentiation potentials in vitro using two different neuronal differentiation protocols. When subjected to a five-stage protocol that recapitulates in vivo conditions of neuronal differentiation, wild-type ES cells differentiated into a wide spectrum of neuronal cell types. However, Ldb1–/– ES cells did not differentiate into neuronal cells; instead, they differentiated into sarcomeric 
-actinin-positive muscle cells. In contrast, when an adherent monolayer culture procedure (which is based on the default mechanism of neural induction and eliminates environmental influences) was applied, both wild-type and Ldb1–/– ES cells differentiated into MAP2-positive mature neurons. Comparison of the results obtained when two different neuronal differentiation protocols were used suggests that Ldb1–/– ES cells have an innate potential to differentiate into neuronal cells, but this potential can be inhibited by environmental influences.
Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION
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The LIM-domain-binding protein 1 (Ldb1) is a transcription cofactor, and it was originally isolated by its ability to bind directly to LIM-homeodomain (LIM-HD) transcription factors and LIM-only proteins (LMOs) [1, 2]. Ldb1 contains many conserved functional domains for binding proteins, and a large number of Ldb1 binding partners have been identified [3]. Among these binding partners, LIM-HD proteins make complexes by binding to the LIM-interacting domain (LID) of Ldb1. These complexes play essential roles in the development and differentiation of the central nervous system by controlling the formation of organizing centers and regulating the specification of developing neurons [4]. Other Ldb1 binding partners, LMOs, contain two tandem LIM domains and also bind to the LID of Ldb1 [5–8]. LMOs regulate the transcriptional activity of Ldb1-LIM-HD complexes by competing with LIM-HD proteins for binding to the LID of the Ldb1 protein, and Ldb1-LMO complexes play essential roles in hematopoietic and nervous system development [9–12]. Ldb1-null mutant mice show multifarious defects, including the truncation of anterior brain structures and posterior duplication [13]. Truncation of the forebrain may be caused by the malformation or malfunction of organizers. In the mouse, forebrain formation requires signals from two different organizers, such as the anterior primitive streak/node and the anterior visceral endoderm (AVE). The anterior primitive streak/node is located at the midline of the embryo and secretes bone morphogenetic protein (BMP) inhibitors [14]. The embryonic ectoderm is under the control of the BMP signal transduction pathway that inhibits proneural genes, which allows the embryonic ectoderm to form epidermis. If the BMP signal transduction pathway is blocked by BMP inhibitors secreted from the organizer, proneural factors induce the embryonic ectoderm to form neural ectoderm [15]. Therefore, the establishment of neuronal identity from the uncommitted embryonic ectoderm occurs by default (i.e., the state is autonomously achieved after the removal of inhibitory signals). Neuronal identity can be directly acquired from embryonic stem (ES) cells through default mechanisms [16]. The AVE is a secondary organizer and is required for forebrain development [17, 18]. Wnt signal transduction pathway is important for the formation of the anterior-posterior axis, and Wnt inhibitors, such as Dkk1 and Cerberus, secreted in the AVE, play an essential role in forebrain formation [19–24]. The truncation of anterior brain structures of Ldb1-null mutant mice may reflect the malfunction of a diverse array of LIM-HD transcription factors whose functions are largely dependent on Ldb1. However, it is not yet clear whether the loss of Ldb1 directly causes the truncation of forebrain structures. In the current study, we have generated Ldb1–/– ES cells and addressed the primary function of Ldb1 in neuronal differentiation in vitro by comparing two different neuronal differentiation methods: a five-stage protocol developed by Lee et al. [25] and an adherent monolayer culture protocol developed by Ying et al. [26]. The potential of Ldb1–/– ES cells to differentiate into forebrain neurons was also examined to evaluate the primary action of Ldb1 in forebrain formation.
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MATERIALS AND METHODS
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Generation of Ldb1–/– ES Cells
Ldb1–/– ES cells were generated by consecutive cre recombination and homologous recombination. For cre recombination, a single loxP and floxed neomycin were inserted in the promoter region and into intron 9 of the Ldb1 gene, respectively. The CMV-cre vector was transfected into ES cells, and Ldb1 coding sequences (exons 1–9) were deleted by cre recombination in one allele. Homologous recombination to delete exons 3–9 of Ldb1 in another allele was performed as described by Mukhopadhyay et al. without any modifications [13]. Targeted disruption of the Ldb1 gene in both alleles via cre and homologous recombination was confirmed by Southern blot analyses (details given in Fig. 1B). The absence of Ldb1 expression in the Ldb1–/– ES cells was confirmed by Northern blot analyses (details given in Fig. 1C).
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Figure 1. Gene targeting in both alleles of Ldb1 locus. (A): Partial restriction map of the WT Ldb1 locus, the targeting vectors, and disrupted Ldb1 alleles. Ldb1 coding sequences (exons 1–9) were deleted by CMV-cre in one allele, and the neomycin-resistance gene replaced the deleted Ldb1 coding sequences (exons 1–9) following homologous recombination in another allele. (B): Southern blot analysis of targeted embryonic stem (ES) cells. Upper panel shows the Southern blot analysis after cre recombination. The HIII-digested ES cell DNA was used for Southern blot analysis with a 3' probe. The probe detected a 6.1-kb WT and an 11.2-kb mutant fragment ( /+). Lower panel shows the Southern blot analysis after homologous recombination. The EcoRI-digested ES cell DNA was used for Southern blot analysis with a 3' probe. The probe detected a 4-kb cre recombinant fragment and a 5-kb homologous recombinant fragment (/–). (C): Northern blot analysis of Ldb1 expression. Total RNA isolated from R1 and DH3 ES cells were used as WT controls (lanes R1, H). Ldb1 was not detected in any of the targeted ES cell clones; the IIA4 targeted ES cell line was used for additional experiments. Abbreviations: B, BamHI; HIII, HindIII; K, KpnI; kb, kilobases; R1, ECOR1; WT, wild-type.
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Maintenance and Differentiation of ES Cells
Wild-type and Ldb1–/– R1 ES cells were maintained in an undifferentiated state on mouse embryonic fibroblast feeder cells with leukemia inhibitory factor (1,000 U/ml; Chemicon, Temecula, CA, http://www.chemicon.com) in ES cell medium consisting of Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum, 100 mM minimal essential medium nonessential amino acids, and 0.55 mM 2-mercaptoethanol. The five-stage method for the differentiation of ES cells into neuronal cells was carried out as previously described, with the minor modification that embryoid bodies (EBs) were formed by hanging drops of 2 x 103 cells instead of suspension culture [25]. The adherent monolayer culture method was performed as previously described without any modifications [26].
Whole-Mount Immunohistochemistry
Whole-mount immunohistochemistry was performed as previously described, with modifications [27]. EBs were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBT) for 3 hours, washed with PBT, and then incubated for 2 hours in 0.9% saline at room temperature. EBs were serially dehydrated from 50% up to 80% methanol and bleached with 3% peroxide in a 80% methanol/20% dimethyl sulfoxide (DMSO) solution. After washing with PBS/0.1% Tween 20 for 3 hours, EBs were permeabilized with 0.5% Triton X-100 and 5% DMSO in blocking solution with 5% skim milk. EBs were incubated with primary antibodies for 2 days at room temperature. A list of the primary antibodies used is given in supplemental online Table 1. After washing with PBT/0/1% Triton X-100 at least five times for 1 hour each time, EBs were incubated overnight at 4°C with secondary horseradish peroxidase-conjugated antibodies (1:200; Dako, Glostrup, Denmark, http://www.dako.com). Signals were visualized by a color reaction with DAB for 10–30 minutes and cleared in 50% glycerol, and pictures were taken in 80% glycerol using a Carl Zeiss (Jena, Germany, http://www.zeiss.com) imaging system.
Immunohistochemistry
Cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 20 minutes at room temperature and permeabilized with 0.3% Triton X-100 in blocking solution for 45 minutes. Cells were incubated at 4°C overnight with the primary antibodies shown in supplemental online Table 1. After washing with PBS containing 0.1% bovine serum albumin, cells were incubated at room temperature for 1 hour with fluorescently labeled secondary antibodies. After a secondary wash, the cells were mounted with Vectashield (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) containing 4,6-diamidino-2-phenylindole for nuclear staining. Specimens were examined on a Carl Zeiss LSM 510 confocal imaging system.
Real-Time Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated using a TRIzol Plus RNA Purification System (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer's guidance. One microgram of the RNA template was reverse-transcribed by 200 units of Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, http://www.promega.com) according to the manufacturer's instructions. Subsequently, a 2-µl aliquot of each room-temperature sample was subjected to real-time polymerase chain reaction (PCR) in a 20-µl reaction mixture containing 4 mM MgCl2, 10 pmol of upstream and downstream primers, and 2 µl of 10 x LightCycler FastStart DNA Master SYBR Green 1 (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). Data were analyzed with LightCycler software, version 3.5. Primer sequences for real-time reverse transcription (RT)-PCR are summarized in supplemental online Table 2. Each RT-PCR experiment was repeated five times, and data were statistically evaluated with Student's t test. A probability level of p < .05 was taken as statistically significant.
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RESULTS
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Generation and Differentiation of Ldb1–/– ES Cells
Null mutant ES cells for both alleles of the Ldb1 locus were generated by consecutive cre and homologous recombination and confirmed by Southern blot hybridization analysis (Fig. 1A, 1B). Northern blot hybridization analysis revealed that the established Ldb1–/– ES cell lines were devoid of Ldb1 expression (Fig. 1C). Ldb1–/– ES cells were aggregated to form EBs to monitor the development of three early germ layers. Both wild-type and Ldb1–/– ES cells were capable of forming EBs; however, their potentials to differentiate into three germ cell layers were quite different (Fig. 2). Whole-mount immunohistochemistry and RT-PCR analyses showed that the number of neural ectodermal cells expressing nestin and sox1 were remarkably decreased in the Ldb1–/– EBs (Fig. 2A, 2B). However, foxa (hnf3β)- and T (brachyury)-positive mesodermal cells and endodermal cells expressing sox17 were highly increased in the Ldb1–/– EBs (Fig. 2A, 2B). These results suggest that Ldb1 may function in a positive manner to induce neural ectoderm, but it inhibits the development of the early epiblast into mesodermal and endodermal lineages.
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Figure 2. Differentiation potentials of Ldb1–/– embryonic stem cells into three germ cell layers. (A): Whole-mount immunohistochemical analysis of three germ cell layer markers in WT and Ldb1–/– EBs. Fewer nestin- and sox1-positive cells and more foxa (hnf3β)-, T (brachyury)- and sox17-positive cells were present in Ldb1–/– EBs. (B): Reverse transcription-polymerase chain reaction analysis of three germ cell layer markers in WT and Ldb1–/– EBs. GAPDH was used as an internal control. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild-type.
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Differentiation of Ldb1–/– ES Cells into Neuronal Cells by Two Different Neuronal Differentiation Methods
Ldb1–/– ES cells were induced to differentiate into neuronal cells by the well-established five-stage method to perform a more profound evaluation of the functional role of Ldb1 in neuronal differentiation. Figure 3A represents the five stages of neuronal differentiation. ES cells were maintained on the feeder cells at stage 1 and aggregated to EBs to mimic in vivo conditions for three-germ-layer development at stage 2. EBs were then plated on gelatin-coated plates with insulin/transferrin/selenium/fibronectin medium to promote growth of the neuronal progenitors. At stage 4, clump cells, including muscle cells that can contract, were removed and then plated on the ornithine/fibronectin-coated plates with fibroblast growth factor 2 (FGF2) to amplify the neural progenitors. With the removal of FGF2, the neuronal progenitors underwent postmitotic differentiation at stage 5. Wild-type ES cells differentiated into neurogenin2-, RbpJ/K1-, and ATOH1 (math1)-positive neural progenitors and various neurotransmitter-specific neurons that are positive for tyrosine hydroxylase, serotonin, GAD2, GluR1, adrenaline, and calbindin (Fig. 3B). Wild-type ES cells also differentiated into a wide variety of neurons that were positive to pax5, pax6, nkx2.2, nkx2.5, en1, islet1, lhx1, lhx2, lhx3, and otx2 (Fig. 3B). These results suggest that the five-stage method is not selective for neuronal cell types but just leaves the cells to randomly differentiate. Surprisingly, the Ldb1–/– cells were unable to differentiate into neuronal cells using the same five-stage method. Instead, they uniformly differentiated into sarcomeric -actinin-positive muscle cells (Fig. 4).
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Figure 3. Wild-type embryonic stem (ES) cells differentiated into a wide spectrum of neuronal cell types by a five-step method. (A): Wild-type ES cells progressively differentiated into neurons. Representative pictures show the morphology of the cells present at each of the five stages. (B): Wild-type ES cells differentiated into TH+, 5-HT+, GAD2+, GluR1+, adrenaline+, calbindin+, Pax5+, Pax6+, Nkx2.2+, Nkx2.5+, En1+, Islet1+, Lhx1+, Lhx2+, Lhx3+, Neurogenin2+, Otx1+, RbpJ/K+, and ATOH1+ neurons. Abbreviations: 5-HT, serotonin; TH, tyrosine hydroxylase.
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Figure 4. WT and Ldb1–/– embryonic stem (ES) cells have different neuronal differentiation potentials by a five-stage method. Only WT, but not Ldb1–/–, ES cells differentiate into neurons. Left panel shows the differentiation of WT ES cells into MAP2+ and tubulin β3+ neurons. Right panel shows the differentiation of Ldb1–/– ES cells into sarcomeric -actinin+ muscle cells. Abbreviation: WT, wild-type.
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To investigate whether Ldb1 plays a direct role in neuronal differentiation, we subjected Ldb1–/– ES cells to another differentiation method, an adherent monolayer culture procedure that is based on the default mechanism of neural induction. ES cells were plated at a very low density (104 cells per 60-mm dish) to eliminate the effects of extrinsic influences. Six days after differentiation, neural rosettes appeared, and neuronal processes could be observed 9 days after differentiation in both wild-type and Ldb1–/– cells (Fig. 5A). Neuron-specific MAP2 was detected by immunocytochemical analysis in both wild-type and Ldb1–/– cells 12 days after differentiation (Fig. 5A). Quantitative real-time RT-PCR analysis revealed no significant differences in the expression of MAP2 between wild-type (100.5 ± 2.6; n= 5) and Ldb1–/– (98.3 ± 6.5; n= 5) cells (Fig. 5B). These results suggest that Ldb1–/– ES cells have the same potential to differentiate into mature neurons as wild-type ES cells.
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Figure 5. Both WT and Ldb1–/– embryonic stem (ES) cells differentiate into neurons by an adherent monolayer culture method. (A): Representative pictures show the progressive differentiation of WT and Ldb1–/– ES cells into neurons. (B): Expression of MAP2 12 days after differentiation. Left panel shows the immunocytochemical analysis of nestin protein in both WT and Ldb1–/– cells. Upper right panel shows the relative transcription levels determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis, normalized with GAPDH and expressed as mean ± SEM. Lower right panel shows a representative picture for the RT-PCR product of MAP2 and GAPDH. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild-type.
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The Role of Ldb1 in Wnt Signal Transduction Pathway-Dependent Forebrain Neuron Specification
During early development, the Wnt signaling pathway determines the posterior portion of the embryo, and inhibition of Wnt signaling is required for the specification of forebrain neurons. Since our previous study showed that the forebrain is truncated in Ldb1 knockout mice [13], we examined the expression of WNT inhibitors and forebrain neuronal cell markers in Ldb1–/– differentiated neuronal cells. The expression of Wnt inhibitors, such as Dkk1 (98.5 ± 4.6; n= 5) and Cerberus (110.5 ± 18.2; n= 5), was not changed significantly in Ldb1–/– cells, and the levels of telencephalic neuronal cell markers, such as Foxg1 (97.8 ± 6.1; n= 5) and Gsh2 (94.8 ± 18.7; n= 5), were consistently the same as those in control cells (Fig. 6). These results suggest that truncation of the forebrain in Ldb1-null mutant mice is an indirect effect of Ldb1 elimination, and Ldb1–/– cells have the innate potential to differentiate into forebrain neurons.
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Figure 6. Ldb1–/– embryonic stem cells have the potential to differentiate into forebrain neurons by an adherent monolayer culture method. The expression of Dkk1 and Cer. (Wnt inhibitors) and the expression of Foxg1 and Gsh2 (forebrain neuronal cell markers) show no significant differences between WT and Ldb1–/– cells. (A): Relative transcription levels of Dkk1, Cer., Foxg1, and Gsh2 determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis, normalized with glyceraldehyde-3-phosphate dehydrogenase, and expressed as mean ± SEM. (B): Representative picture for the RT-PCR product of Dkk1, Cer., Foxg1, and Gsh2. Abbreviations: Cer., Cerberus; WT, wild-type.
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DISCUSSION
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One of the noticeable findings of our study is the difference in the potential of Ldb1–/– ES cells to differentiate into neuronal cells depending on whether the five-stage method or adherent monolayer culture method is used for differentiation (Figs. 4, 5). The fundamental difference between these two methods is whether environmental influences are present or not. The five-stage method mimics in vivo conditions of early embryonic development; all cells are under the control of organic relationships, including cell-cell communication or signals from organizing centers [25]. In contrast, the adherent monolayer culture method is based on the default mechanism of neural induction, which eliminates the influence of signals from neighboring cells [26]. These differences afford us crucial insights into the function of Ldb1 in neuronal differentiation. As shown in Figure 2A, only a smaller number of Ldb1–/– ES cells differentiated into neural precursors, and they could not be differentiated into mature neurons by the five-stage method. These results are consistent with our previous study that the anterior brain is truncated in Ldb1 knockout mice [13]. These data suggest that Ldb1 plays an important functional role in forebrain formation in vivo and in neural differentiation in vitro. Nevertheless, it is still unclear whether these phenotypes are due to direct functions of Ldb1. In contrast, data from the adherent monolayer culture method, which demonstrates the differentiation of Ldb1–/– ES cells into neuronal cells under environmental-effect-free conditions, suggest that indirect functions of Ldb1 are responsible for these phenotypes. Therefore, missing anterior brain structures in Ldb1 knockout mice and the inability of Ldb1–/– ES cells to differentiate into neuronal cells by a five-stage method might be mediated by the secondary indirect effects of Ldb1 elimination. Wnt is one of the primary candidates for these phenotypes, because the Wnt signal transduction pathway determines the anterior-posterior axis, and inhibition of Wnt signaling is required for forebrain development and neural differentiation of ES cells [19–24, 28]. Our previous study showed the curtailed expression of Wnt inhibitors in the head organizer of Ldb1 knockout mice, which may lead to truncation of the forebrain structures [13]. However, according to the current adherent monolayer culture method, expression levels of the Wnt inhibitors Dkk1 and Cerberus were not altered, and levels of forebrain neural markers Foxg1 and Gsh2 in the Ldb1–/– cells were the same as those in wild-type cells. Taking all the data into account, it seems likely that Ldb1 may be involved in the formation of head organizers or specification of cells that might produce Wnt inhibitors, instead of directly involved in the formation of forebrain structures or specification of forebrain neurons in the early brain development.
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CONCLUSION
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The differentiation potentials of Ldb1–/– ES cells into neuronal cells are greatly dependent on in vitro culture conditions and the missing of forebrain structures in Ldb1 null mutant mouse may be caused by the indirect function of Ldb1.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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This work was supported by the Korea Research Foundation, funded by the Korean government (Ministry of Education & Human Resources Development) (Grant KRF-2006-R0606941-C00222), and by the Korea Science and Engineering Foundation (KOSEF), funded by the Korean government (MOST) (Grant M10641040004-07N4104-00410).
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FOOTNOTES
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Author contributions: M.H.: conception and design, collection and/or assembly of data, data analysis and interpretation; M.G.: provision of study material or patient, collection and assembly of data; M.K.: provision of study material or patient; H.W.: conception and design, data analysis and interpretation, final approval of manuscript; D.G.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript.
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Abstract
Introduction
