Abstract
Split hand/foot malformation (SHFM) and SHFM combined with long-bone deficiency (SHFLD) are congenital dysgeneses of the limb. Although six different loci/mutations (SHFM1–SHFM6) have been found from studies on families with SHFM, the causes and associated pathogenic mechanisms for a large number of patients remain unidentified. On the basis of the identification of a duplicated gene region involving BHLHA9 in some affected families, BHLHA9 was identified as a novel SHFM/SHFLD-related gene. Although Bhlha9 is predicted to participate in limb development as a transcription factor, its precise function is unclear. Therefore, to study its physiological function, we generated a Bhlha9-knockout mouse and investigated gene expression and the associated phenotype in the limb bud. Bhlha9-knockout mice showed syndactyly and poliosis in the limb. Moreover, some apical ectodermal ridge (AER) formation related genes, including Trp63, exhibited an aberrant expression pattern in the limb bud of Bhlha9-knockout mice; TP63 (Trp63) was regulated by Bhlha9 on the basis of in vitro analysis. These observations suggest that Bhlha9 regulates AER formation during limb/finger development by regulating the expression of some AER-formation-related genes and abnormal expression of Bhlha9 leads to SHFM and SHFLD via dysregulation of AER formation and associated gene expression.
Keywords: Split hand/foot malformation, Split hand/ foot malformation combined with long-bone deficiency, Bhlha9
Introduction
Split hand/foot malformation (SHFM) is a form of limb dysgenesis. Its phenotype is extremely variable between and within families, ranging from ectrodactyly and syndactyly to monodactyly. Six loci/mutations (SHFM1– SHFM6) were found to be associated with human nonsyndromic SHFM [1]. Representatively, mutations in TP63 (Trp63), encoding a homologue of the tumor suppressor p53, were identified as a causative factor for SHFM4 [2, 3]. TP63 (Trp63) RNA is transcribed from two different transcription start sites, yielding both transactivating (TA-p63) and nontransactivating (ΔN-p63) isotypes. The dominant negative isotype ΔN-p63 is specifically expressed in the apical ectodermal ridge (AER) and is thought to function as a transcription factor that is involved in AER maintenance [4]. In fact, the forelimbs of Trp63-knockout mice are severely malformed, lacking the radius and autopod [5, 6]. However, the disease-associated genes and pathogenic mechanisms in a large number of nonsyndromic SHFM cases, including SHFM combined with long-bone deficiency (SHFLD), remain unidentified.
Recent reports have indicated that 17p13.3 duplications should be considered the commonest cause of SHFM/ SHFLD [7–14]. In particular, Klopocki et al. [9] scrutinized the minimal critical region (11.8 kb) by analyzing 17 families with 17p13.3 duplications, and identified BHLHA9 [which encodes basic helix–loop–helix (bHLH) family member A9] as a novel SHFM/SHFLD-related gene.
BHLHA9 encodes a member of the bHLH transcription factor family that is predicted to participate in limb development as a transcription factor. Knockdown of Bhlha9 in zebrafish results in severely truncated pectoral fins [9]. Furthermore, Bhlha9-knockout mice exhibit various degrees of syndactyly [15]. In addition, Malik et al. [16] demonstrated that three missense mutations affecting the highly conserved DNA-binding domain of BHLHA9 are associated with mesoaxial synostotic syndactyly, Malik–Percin type. From these studies, it appears that Bhlha9 is a key molecular player in limb and finger development. However, the precise function of Bhlha9 as a transcription factor is unknown, and its downstream genes have not been identified. To elucidate the physiological function of Bhlha9, we generated knockout mice and investigated their phenotypes and gene expression patterns in the limb bud.
Materials and Methods
Generation of Bhlha9 Knockout Mice
All animal experiments were performed according to protocols approved by the institutional animal care and use committees of the National Institute for Child Health and Development and Tokyo Medical and Dental University. A vector was constructed for replacement of the endogenous Bhlha9 locus with the Venus-Cre and PGK-Neo cassette via homologous recombination in embryonic stem cells (Fig. S1a). Through PCR, 5ʹ and 3ʹ sequences flanking the endogenous Bhlha9 locus were amplified from a C57BL/6 genomic bacterial artificial chromosome clone (BACPAC Resource Center). These homology arms were cloned into a vector harboring both a neomycin resistance cassette for positive selection and a diphtheria toxin A encoding gene for negative selection. The targeting vector was linearized and electroporated into TT2F embryonic stem cells. Recombinant embryonic stem cell clones were isolated after culturing in medium containing G418 antibiotic (Invitrogen) and were screened for proper integration by Southern blotting with use of a 5ʹ probe (Fig. S1b). The resulting chimeric offspring were crossed with C57BL/6 mice, and germline transmission was confirmed by PCR (Fig. S1c). The mouse genotypes were determined by PCR with use of tail DNA as the template and the following primers: T1, 5ʹ-TGAAGCTG TGAGACCAGGACA-3ʹ: T2, 5ʹ-GGCCGTGACACTCCA GGT-3ʹ; and T3, 5ʹ-GTTCTGCTGGTAGTGGTCGG-3ʹ.
In Situ Hybridization
In situ hybridization on whole mounts and sections was performed as previously described [17]. Briefly, tissues were embedded in optimal cutting temperature compound (Sakura Finetek) and ash-frozen in liquid nitrogen. Specimens were sectioned to 16μm with use of a CM3050 S cryostat (Leica) and hybridized with digoxigenin-labeled antisense RNA probes. Detailed descriptions of the RNA probes are available from the authors on request.
Histological Analysis
For histological analysis of the interdigit, the forelimbs were harvested from adult mice and fixed with 4% paraformaldehyde in phosphate-buffered saline at 4 °C overnight. Tissues were dehydrated, embedded in paraffin, and sectioned; these sections were stained with hematoxylin and eosin (Wako). Skeleton specimens from 3-month-old adult Bhlha9-knockout and wild-type littermate control mice were stained with Alcian blue/alizarin red as previously described [18]. The length of the limb skeleton was measured by analysis of an image obtained with an SZX16 stereoscopic microscope (Olympus).
RNA Isolation and Quantitative Real-Time PCR
Total RNA was isolated from limb buds and cells with use ISOGEN (Nippon Gene), and was reverse-transcribed with a ReverTraAce kit (Toyobo) according to the manufacturer’s instructions. Complementary DNA was used for quantitative real-time PCR (qPCR), and qPCR was performed with use of Thunderbird SYBR mix (Toyobo). β-Actin expression served as the control for messenger RNA expression. Changes in gene expression were quantified by the ΔΔCT method. The primer sequences for qPCR are described in Table S1.
Microarray Analysis
RNA samples were obtained from the whole limb bud of embryonic day 10.5 (E10.5) wild-type and Bhlha9-knockout embryos, as described already. Total RNA (200 ng) was reverse-transcribed and biotinylated with use of a GeneChip 3′ IVT Express kit (Affymetrix). Finally, the complementary RNA was hydrolyzed and hybridized to a GeneChip Mouse Genome 430 2.0 array (Affymetrix). The microarray data were summarized with use of the MAS 5.0 method. We applied the following thresholds for data analysis: signal intensity greater than 100, detection call present, and signal intensity upregulated or downregulated more than twofold compared with that of the control. Microarray data are shown in Table S2 and were deposited in the Gene Expression Omnibus repository under accession number GSE81223.
Plasmid constriction
To prepare pMIGR-3×FLAG-BHLHA9, complementary DNA encoding full-length human BHLHA9 was artificially constructed (GenScript) and cloned into pMIGR (Addgene). Each 4-kb upstream region of the TA-p63 and ΔN-p63 first protein coding exon was cloned from the A549 genome and subcloned into a promoterless pNL1.1 nanoluciferase vector to be used for luciferase assays. The plasmids were sequenced to verify the absence of mutations.
Cell culture and Luciferase assay
HeLa cells and PLAT-E cells were cultured in 10% fetal bovine serum–Dulbecco’s modified Eagle’s medium containing 1% penicillin–streptomycin (all from Sigma-Aldrich). PLAT-E cells were transfected with the pMIGR construct (as a control) or the pMIGR-3×FLAG-BHLHA9 construct with use of FugeneHD (Promega). Forty-eight hours later, the media were collected, filtered, and transferred to the HeLa stable cell line in a medium containing puromycin (1μg/ml). Control or 3×FLAG-BHLHA9-expressing HeLa stable cells were cotransfected with each TP63 promoter–nanoluciferase reporter gene construct and the pGL4.13 luciferase construct (for normalization; Promega) with use of Fugene HD (Promega). Cell extracts were prepared 48 h after transfection, and luciferase activity was measured with a Nano-Glo dual-luciferase reporter assay system (Promega).
Results
Bhlha9 expression in mouse limb buds
First, we assessed endogenous Bhlha9 expression in the limb buds of mouse embryos. Previous studies using a LacZ reporter system showed that Bhlha9 is expressed in the ectodermal region of the digit near the AER on embryonic days 10–15 [15]; however, a time-dependent change in endogenous Bhlha9 expression had not been demonstrated. Therefore, we investigated endogenous Bhlha9 expression in the limb bud using in situ hybridization with whole mounts and sections from different stages of embryonic development.
Endogenous Bhlha9 expression in the limb bud was clearly detected on both the dorsal side and the ventral side of the autopod surface, covering the progress zone near the AER, at E10.5 to embryonic day 11.5 (E11.5) (Fig. 1, panels a, b, d, e, g, h). In contrast, Bhlha9 expression in the limb bud was undetectable at embryonic day 12.5 (E12.5) in whole mounts, and was barely visible in tissue sections (Fig. 1, panels c, f, i). We were unable to perform this technique at later developmental stages (data not shown). This observation was confirmed with use of limb bud RNA and qPCR analysis (Fig. 1j). From these results, we estimated that Bhlha9 potentially functions during early embryonic stages (E10.5–E11.5) but not during later developmental stages.
Fig. 1. Bhlha9 is expressed in the limb ectoderm.
Bhlha9 is expressed on both the dorsal side and the ventral side of the autopod in wild-type mouse limb buds at embryonic day 10.5 (E10.5) and embryonic day (E11.5), as assessed by whole-mount in situ hybridization (a–f). Although Bhlha9 expression in the limb at embryonic day 12.5 (E12.5) was unclear from whole-mount in situ hybridization, it was detectable in the longitudinal section of wild-type forelimbs at each stage (g–i). Expression of Bhlha9 relative to β-Actin in E10.5–E12.5 wild-type or Bhlha9-knockout whole limb buds was assessed by quantitative real-time PCR (j) The relative ratios against E10.5 limb RNA (set to 1.0) are presented as the mean ± standard deviation (n = 4). Asterisks represent a statistically significant difference on the basis of Tukey’s test (one asterisk p < 0.05, two asterisks p < 0.01, three asterisks p < 0.001). D dorsal, V ventral. Scale bar 200 μm
Bhlha9-knockout mouse phenotype
To study the physiological function of Bhlha9, we generated the Bhlha9-knockout mouse and investigated its phenotype. Our Bhlha9-knockout mouse was constructed by replacement of endogenous Bhlha9 with Venus-Cre (Fig. S1a). Unfortunately, Venus-Cre protein expression in Bhlha9-knockout mouse limb bud was not detectable by general biological analyses, although genome mutation was confirmed by Southern blotting and PCR analysis (Fig. S1b, c).
As stated in a previous report, the Bhlha9-knockout mouse showed syndactyly in the forelimb bud (Fig. 2a) [15]. Furthermore, we also found the new phenotype that the Bhlha9-knockout mouse frequently showed poliosis in the hindlimb finger (Fig. 2b). Neither phenotype was observed in heterozygous mice. Next, we used the forelimb to investigate the nature of the syndactyly and the involvement of soft and/or skeletal tissues in it. We performed alizarin red and alcian blue staining of bone and cartilage respectively (Fig. 2c). We found no apparent skeletal abnormalities in the hand plates or arms; hence, the mechanism did not involve defects in skeletal patterning. To clarify the detailed phenotype of the Bhlha9-knockout mice, we investigated their interdigital webbing (Fig. 2d). The fused interdigital webbing in adult Bhlha9-knockout mice (3 months old) exhibited a large number of hematoxylin-positive cells by hematoxylin and eosin staining. This result shows a disruption of cell death in the Bhlha9-knockout mouse interdigital webbing tissue, as stated in previous reports [15].
Fig. 2. Phenotype of Bhlha9-knockout mice.
a The appearance of the forelimb in 3-month-old wild-type and Bhlha9-knockout mice. Various degrees of syndactyly were observed in the forelimbs of Bhlha9-knockout mice. A white arrow indicates interdigital webbing. Most Bhlha9-knockout mice (71.79%) had a webbed digit in the forelimb (n = 39). b The appearance of the hindlimb in 3-month-old wild-type and Bhlha9-knockout mice. All Bhlha9-knockout mice displayed poliosis in the hindlimbs (n = 17). c Skeletal staining of the forelimbs of 3-month-old wild-type and Bhlha9-knockout mice. Limbs were dissected from 3-month-old wild-type and Bhlha9-knockout mice and bone and cartridge were stained by alizarin red and alcian blue, respectively. In Bhlha9-knockout mice, no anomalies in humerus, radius, ulna, or forelimb phalange length were detected. d Hematoxylin and eosin staining of the interdigital webbing in adult Bhlha9-knockout mice. Longitudinal sections of interdigit 2–3 in 3-month-old adult Bhlha9-knockout mouse forelimbs were stained by hematoxylin and eosin. Staining revealed remaining cells and eosinophilic fibers in the interdigital region. The lower panel shows a higher magnification of the region marked in the upper panel. KO knockout, WT wild type. Scale bar a, b 1 mm, c, d 200 μm.
Gene expression in the Bhlha9-knockout mouse limb bud
The Bhlha9-knockout mouse exhibited abnormal limb development and a disruption of cell death in interdigital webbing tissue (Fig. 2) [15]; however, the molecular mechanism of this phenotype remains unknown. Hence, to investigate the physiological function of BHLHA9 as a transcription factor, we performed global gene expression analysis using limb bud RNA. Since Bhlha9 expression was predominant at the E10.5 embryonic stage (Fig. 1), we used E10.5 limb bud RNA for this experiment.
No apparent changes in the expression of major programmed-cell-death-related genes were found in Bhlha9-knockout mouse limb buds by microarray analysis. However, several genes related to limb development, including Trp63, were highly upregulated in the limb bud of Bhlha9-knockout mice (Fig. 3a, Table S2). Differential expression of selected genes was confirmed by qPCR analysis. It was evident that Trp63 and Fgf8, which is a gene downstream of Trp63 and a representative inhibitor of interdigital cell apoptosis, were upregulated in the limb bud of Bhlha9-knockout mice (Fig. 3b). In contrast, the expression of syndactyly-related genes was unaltered. In addition, Trp63 and Fgf8 overexpression in Bhlha9-knockout mouse limb buds was not confirmed at embryonic day 13.5 (E13.5) (Fig. S2). These results indicate that Bhlha9 transiently regulates the expression of Trp63 and its downstream genes during embryonic stages E10.5–E11.5, but not at later developmental stages.
Fig. 3. Trp63 and Fgf8 expression is upregulated in Bhlha9-knockout mouse limb buds.
a The GeneChip signal value of embryonic day 10.5 (E10.5) Bhlha9-knockout mouse limb bud RNA compared to E10.5 wild-type mouse limb bud RNA as a scatter plot (n = 1, R2 = 0.9717). The signal value of Trp63 (wild type 269, knockout 742) is shown by a red dot. Differentially expressed genes are shown in dark gray (fold change greater than 2). b Expression of various syndactyly-associated genes in Bhlha9-knockout mouse limbs. Expression of selected genes relative to β-Actin expression in E10.5 wild-type or Bhlha9-knockout mouse whole limb buds was assessed by quantitative real-time PCR. The relative ratios against E10.5 wild- type mouse limb RNA (set to 1.0) are presented as the mean ± standard deviation (n = 4). An asterisk represents statistical significance (p < 0.05), as assessed by Student’s t test. KO knockout, WT wild type
Bhlha9 regulates TP63 (Trp63) and Fgf8 expression
TP63 (Trp63) and Fgf8 are expressed in the AER, and serve as key molecular modulators of limb development [5, 6, 19, 20]. On the basis of gene expression analysis, abnormal expression of these genes was confirmed in E10.5 limb buds of Bhlha9-knockout mice. Thus, we investigated the expression patterns of Trp63 and Fgf8 in E10.5 Bhlha9-knockout mouse limb buds using in situ hybridization with whole mounts and sections.
Trp63 and Fgf8 were clearly detected in the AER of wild-type mouse limb buds (Fig. 4a, b). However, in the AER of Bhlha9-knockout mouse limb buds, the regions that expressed Trp63 and Fgf8 were expanded to the progress zone (Fig. 4c, d). The wild-type mouse AER also had a characteristic convex structure; however, this characteristic shape was not detected in the Bhlha9-knockout mouse AER (Fig. 4b, d).
Fig. 4. Bhlha9 contributes to apical ectodermal ridge (AER) formation.
Expression patterns of Trp63 and Fgf8 in embryonic day 10.5 wild-type and Bhlha9-knockout mouse limb buds. For confirmation of the real-time PCR results, wherein Trp63 and Fgf8 expression was increased in Bhlha9-knockout mouse limb buds, the expression pattern of each gene was detected by whole-mount in situ hybridization (a, b). Whole-mount in situ hybridization using embryo limb buds also showed the expansion of each gene’s expression region in the limb ectoderm (c, d). Arrows indicate the AER structure. D dorsal, KO knockout, V ventral, WT wild-type. Scale bar 200 μm
On the basis of in situ hybridization analysis using whole mounts and sections, we hypothesized that Bhlha9 regulates the expression of Trp63 in the progress zone. We validated this contention by in vitro analysis. TP63 (Trp63) RNA is transcribed from two different transcription start sites, yielding the TA-p63 and ΔN-p63 isotypes. Especially, the dominant-negative isoform (ΔN-p63) is found to be specifically expressed in the AER, and is thought to function as a transcription factor that is involved in AER maintenance [5]. First, we constructed TA-p63 and ΔN-p63 promoter driven luciferase reporter genes to investigate the effect of Bhlha9 on the transcription of these genes (Fig. 5a). We subcloned the 4-kb upstream regions of the TA-p63 and ΔN-p63 first protein coding exons, which include several E-box domains that are recognized by the bHLH transcription factor protein family. We also used the HeLa cell line for in vitro analysis and prepared control and 3×FLAG-BHLHA9-expressing HeLa stable cells using retrovirus systems (Fig. 5b), because these cells can be used to detect the expression of endogenous TP63 (data not shown). Luciferase assays using TA-p63 and ΔN-p63 promoter driven luciferase reporter genes resulted in low luciferase activity in 3×FLAG-BHLHA9-expressing HeLa stable cells as compared with that in control HeLa stable cells (Fig. 5c). Furthermore, endogenous TP63 expression, including TA-p63 and ΔN-p63 expression, was downregulated in 3×FLAG-BHLHA9-expressing HeLa stable cells (Fig. 5d). Both results suggest that Bhlha9 directly sup- presses the expression of TP63 (Trp63), including TA-p63 and ΔN-p63. Our findings suggest that Bhlha9 maintains the AER formation by regulating TP63 (Trp63) expression and its downstream genes including Fgf8, which is known as an inhibitor of interdigital cell apoptosis, in the progress zone. The results suggest that this occurs at an earlier embryonic stage than the interdigital apoptosis stage, and that this function might contribute to finger morphogenesis.
Fig. 5. BHLHA9 directly regulates TP63 expression.
a Transactivating TP63 (TA-p63) and nontransactivating TP63 (ΔN-p63) promoter driven luciferase reporter gene constructs. Each 4-kb upstream fragment from the first protein coding exon of TA-p63 and ΔN-p63 was selected and cloned into a nanoluciferase (NLuc) vector. b, c Luciferase assay using 3×FLAG-BHLHA9-expressing HeLa stable cells. HeLa stable cells were transfected with TA-p63 and ΔN-p63 promoter driven luciferase reporter constructs for luciferase assays. 3×FLAG-BHLHA9 overexpression against control HeLa stable cells (set to 1.0) was sufficiently detected on the basis of in vitro analysis (b) (n = 4). The relative ratio of each TP63-promoter-driven luciferase activity against control HeLa stable cells (set to 1.0) is presented as the mean ± standard deviation (n = 4). The luciferase activity significantly reduced with the overexpression of 3×FLAG-BHLHA9 (c). An asterisk represents statistical significance (p < 0.05) as assessed by Student’s t test. d Endogenous TP63 expression in 3×FLAG-BHLHA9-expressing HeLa cells. Expression of TP63, including TA-p63 and ΔN-p63, relative to that of β-Actin in control HeLa cells or 3×FLAG-BHLHA9-expressing HeLa cells was assessed by quantitative real-time PCR. The relative ratios against control cells (set to 1.0) are presented as the mean ± standard deviation (n = 4). An asterisk represents statistical significance (p < 0.05), as assessed by Student’s t test. TSS transcription start site
Discussion
We have reported for the first time that endogenous Bhlha9 was prominently expressed at embryonic stage E10.5 on both the dorsal surface and the ventral surface, covering the progress zone near the AER (Fig. 1). Moreover, abnormalities in limb development, specifically, syndactyly in the forelimb bud and poliosis in the hindlimb finger, were found in Bhlha9-knockout mice (Fig. 2). On the basis of these observations, we hypothesized that BHLHA9 acts as a transcription factor during an early embryonic stage rather than during the interdigital cell apoptosis stage. As such, we determined that two AER-formation-related genes, Trp63 and Fgf8, exhibit transient aberrant expression patterns in Bhlha9-knockout mouse limb buds at E10.5, but not at E13.5 (Figs. 3, S2). It has been reported that the AER formation regresses after E12.5 [21]. Previous reports and our findings indicate that Bhlha9 transiently regulates the expression of Trp63 and its downstream genes in the AER and the progress zone during embryonic stages E10.5–E11.5, but not at later developmental stages.
Limb development results from gradients of signaling molecules in three spatial dimensions: proximodigital, anteroposterior, and dorsoventral [22]. Specifically, for the development of the anteroposterior dimension, the AER contains a specialized cell cluster that expresses TP63 (Trp63) and Fgf8. Failure to form the AER leads to a truncation of limb skeletal structures [23, 24]. The forelimbs of Trp63-knockout mice exhibit the abnormal expression of several genes, including Fgf8, and severe malformations such as a missing radius and autopod [5, 6]. It was also noted that mutations underlying SHFM4 exist in the TP63 gene [3]. Therefore, TP63 (Trp63) is thought to act as an AER maintenance gene. Similarly, Fgf8 is also considered as an essential genes induced by TP63 (Trp63) for AER structure [6, 25]. Cre-mediated inactivation of Fgf8 in the early limb ectoderm resulted in a substantial reduction in limb bud size, a delay in Shh expression, dysregulation of Fgf4 expression, and abnormalities in skeletal elements [26]. Furthermore, fibroblast growth factor (FGF) molecules, including FGF8, which is produced in the AER, suppress bone morphogenetic protein production in the interdigital tissue and act as inhibitors for the interdigital cell apoptosis [27]. Once the AER regresses, FGF production in the AER declines and bone morphogenetic protein (BMP) expression in the interdigital tissue is upregulated, leading to interdigital cell apoptosis [28–30]. Consequently, studies on the expression and maintenance of TP63 (Trp63) and Fgf8 are necessary to enhance our understanding of AER function, finger morphogenesis, and pathogenesis of SHFM and SHFLD.
Our investigation has revealed for the first time that disruption of Bhlha9 expression in the progress zone leads to the upregulation and dysregulation of Trp63 and Fgf8 in the AER and the progress zone at E10.5 rather than at the interdigital cell apoptosis stage (Figs. 3, 4). Furthermore, the AER of the wild-type mouse had a characteristic convex structure, which was not clearly detected in Bhlha9-null limb buds. These observations indicate that Bhlha9 expressed in the progress zone mainly contributes to the maintenance of AER formation by regulating Trp63 expression in the AER and the progress zone at E10.5. This notion was corroborated by in vitro analysis, which included luciferase assays and qPCR (Fig. 5). A previous publication indicated that interdigital cell apoptosis was inhibited in Bhlha9-null limb [15]. Interdigital cell apoptosis normally occurs after AER regression [28–30]. Therefore, we speculated that cell death in the AER of Bhlha9-null limb buds is abnormal compared with that in wild-type mouse limb buds. However, no significant difference in AER cell death was found in Bhlha9-knockout mouse limb buds on the basis of Nile blue sulfate staining, which is used to detect apoptotic cells (Fig. S3). On the basis of previous reports and our finding of Trp63 and Fgf8 overexpression in Bhlha9-null limb buds, we suggest that abnormal AER formation and overexpression of Fgf8, which is an interdigital cell apoptosis inhibitor, induced by Trp63 in the Bhlha9-null AER and progress zone inhibits interdigital cell apoptosis, but not AER regression, leading to the syndactyly (Fig. 6). Consequently, our observations suggest that abnormal expression of Bhlha9 leads to SHFM and SHFLD via dysregulation of AER formation and associated gene expression.
Fig. 6. Bhlha9-knockout mouse limb buds.
In the wild-type mouse limb bud, Trp63 and Fgf8 are expressed in the apical ectodermal ridge (AER), and this structure exhibits a characteristic convex shape. However, Trp63 and Fgf8 are overexpressed and dysregulated in Bhlha9-null AERs and progress zones via aberrant regulation of Trp63 expression by Bhlha9. Consequently, overexpression of fibroblast growth factor 8 (FGF8) in Bhlha9-knockout mouse limbs inhibits interdigital cell apoptosis, leading to the syndactyly. BMP bone morphogenetic protein, E embryonic day, KO knockout, PZ progress zone, WT wild type
Supplementary Material
Acknowledgments
This work was supported by Core Research for the Evolutionary Science and Technology (CREST) funding from the Japan Science and Technology Agency (JST), AMED-CREST from the Japan Agency for Medical Research and Development (AMED), KAKENHI (Grant Numbers 26113008, 15H02560, and 15K15544) from the Japan Society for the Promotion of Science, Grants from the National Institutes of Health (Grant Numbers AR050631 and AR065379), the Naito Foundation, and a Bristol-Myers K.K. RA Clinical Investigation Grant to Hiroshi Asaha. The funders had no role in study design, data collection and analysis, the decision to publish the findings, or preparation of the manuscript. We thank Natsuko Izumi and Daiki Fukuchi for technical assistance.
Footnotes
Conflict of interest
The authors declare that they have no conflict of interest.
References
- 1.Gurrieri F, Everman DB (2013) Clinical, genetic, and molecular aspects of split-hand/foot malformation: an update. Am J Med Genet A 11:2860–2872 [DOI] [PubMed] [Google Scholar]
- 2.Ianakiev P, Kilpatrick MW, Toudjarska I, Basel D, Beighton P, Tsipouras P (2000) Split-hand/split-foot malformation is caused by mutations in the p63 gene on 3q27. Am J Hum Genet 1:59–66 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.van Bokhoven H, Hamel BC, Bamshad M, Sangiorgi E, Gurrieri F et al. (2001) p63 gene mutations in EEC syndrome, limb-mammary syndrome, and isolated split hand-split foot malformation suggest a genotype-phenotype correlation. Am J Hum Genet 3:481–492 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dotsch V, Andrews NC, Caput D, McKeon F (1998) p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 3:305–316 [DOI] [PubMed] [Google Scholar]
- 5.Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, McKeon F (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 6729:714–718 [DOI] [PubMed] [Google Scholar]
- 6.Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 6729:708–713 [DOI] [PubMed] [Google Scholar]
- 7.Baquero-Montoya C, Gil-Rodriguez MC, Hernandez-Marcos M, Teresa-Rodrigo ME, Vicente-Gabas A, Bernal ML, Casale CH, Bueno-Lozano G, Bueno-Martinez I, Queralt E, Villa O, Hernando-Davalillo C, Armengol L, Gomez-Puertas P, Puisac B, Selicorni A, Ramos FJ, Pie J (2014) Severe ipsilateral musculoskeletal involvement in a Cornelia de Lange patient with a novel NIPBL mutation. Eur J Med Genet 9:503–509 [DOI] [PubMed] [Google Scholar]
- 8.Capra V, Mirabelli-Badenier M, Stagnaro M, Rossi A, Tassano E, Gimelli S, Gimelli G (2012) Identification of a rare 17p13.3 duplication including the BHLHA9 and YWHAE genes in a family with developmental delay and behavioural problems. BMC Med Genet 13:93. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Klopocki E, Lohan S, Doelken SC, Stricker S, Ockeloen CW et al. (2012) Duplications of BHLHA9 are associated with ectrodactyly and tibia hemimelia inherited in non-Mendelian fashion. J Med Genet 2:119–125 [DOI] [PubMed] [Google Scholar]
- 10.Luk HM, Wong VC, Lo IF, Chan KY, Lau ET, Kan AS, Tang MH, Tang WF, She WM, Chu YW, Sin WK, Chung BH (2014) A prenatal case of split-hand malformation associated with 17p13.3 triplication—a dilemma in genetic counseling. Eur J Med Genet 2–3:81–84 [DOI] [PubMed] [Google Scholar]
- 11.Nagata E, Kano H, Kato F, Yamaguchi R, Nakashima S et al. (2014) Japanese founder duplications/triplications involving BHLHA9 are associated with split-hand/foot malformation with or without long bone deficiency and Gollop-Wolfgang complex. Orphanet J Rare Dis doi: 10.1186/s13023-014-0125-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Nagata E, Haga N, Fujisawa Y, Fukami M, Nishimura G, Ogata T (2015) Femoral-tibial-digital malformations in a boy with the Japanese founder triplication of BHLHA9. Am J Med Genet A 12:3226–3228 [DOI] [PubMed] [Google Scholar]
- 13.Petit F, Andrieux J, Demeer B, Collet LM, Copin H, Boudry-Labis E, Escande F, Manouvrier-Hanu S, Mathieu-Dramard M (2013) Split-hand/foot malformation with long-bone deficiency and BHLHA9 duplication: two cases and expansion of the phenotype to radial agenesis. Eur J Med Genet 2:88–92 [DOI] [PubMed] [Google Scholar]
- 14.Petit F, Jourdain AS, Andrieux J, Baujat G, Baumann C, Beneteau C, David A, Faivre L, Gaillard D, Gilbert-Dussardier B, Jouk PS, Le Caignec C, Loget P, Pasquier L, Porchet N, Holder-Espinasse M, Manouvrier-Hanu S, Escande F (2014) Split hand/foot malformation with long-bone deficiency and BHLHA9 duplication: report of 13 new families. Clin Genet 5:464–469 [DOI] [PubMed] [Google Scholar]
- 15.Schatz O, Langer E, Ben-Arie N (2014) Gene dosage of the transcription factor fingerin (bHLHA9) affects digit development and links syndactyly to ectrodactyly. Hum Mol Genet 20:5394–5401 [DOI] [PubMed] [Google Scholar]
- 16.Malik S, Percin FE, Bornholdt D, Albrecht B, Percesepe A, Koch MC, Landi A, Fritz B, Khan R, Mumtaz S, Akarsu NA, Grzeschik KH (2014) Mutations affecting the BHLHA9 DNA binding domain cause MSSD, mesoaxial synostotic syndactyly with phalangeal reduction, Malik–Percin type. Am J Hum Genet 6:649–659 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Yokoyama S, Hashimoto M, Shimizu H, Ueno-Kudoh H, Uchibe K, Kimura I, Asahara H (2008) Dynamic gene expression of Lin28 during embryonic development in mouse and chicken. Gene Expr Patterns 3:155–160 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Miyaki S, Sato T, Inoue A, Otsuki S, Ito Y, Yokoyama S, Kato Y, Takemoto F, Nakasa T, Yamashita S, Takada S, Lotz MK, Ueno-Kudo H, Asahara H (2010) MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes Dev 11:1173–1185 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Moon AM, Capecchi MR (2000) Fgf8 is required for outgrowth and patterning of the limbs. Nat Genet 4:455–459 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Mahmood R, Bresnick J, Hornbruch A, Mahony C, Morton N, Colquhoun K, Martin P, Lumsden A, Dickson C, Mason I (1995) A role for FGF-8 in the initiation and maintenance of vertebrate limb bud outgrowth. Curr Biol 7:797–806 [DOI] [PubMed] [Google Scholar]
- 21.Casanova JC, Uribe V, Badia-Careaga C, Giovinazzo G, Torres M, Sanz-Ezquerro JJ (2011) Apical ectodermal ridge morphogenesis in limb development is controlled by Arid3b-mediated regulation of cell movements. Development 6:1195–1205 [DOI] [PubMed] [Google Scholar]
- 22.Zeller R, Lopez-Rios J, Zuniga A (2009) Vertebrate limb bud development: moving towards integrative analysis of organogenesis. Nat Rev Genet 12:845–858 [DOI] [PubMed] [Google Scholar]
- 23.Saunders JW Jr (1948) The proximo-distal sequence of origin of the parts of the chick wing and the role of the ectoderm. J Exp Zool 3:363–403 [DOI] [PubMed] [Google Scholar]
- 24.Summerbell D (1974) A quantitative analysis of the effect of excision of the AER from the chick limb-bud. J Embryol Exp Morphol 3:651–660 [PubMed] [Google Scholar]
- 25.Guerrini L, Costanzo A, Merlo GR (2011) A symphony of regulations centered on p63 to control development of ectoderm-derived structures. J Biomed Biotechnol 2011:864904. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Lewandoski M, Sun X, Martin GR (2000) Fgf8 signalling from the AER is essential for normal limb development. Nat Genet 4:460–463 [DOI] [PubMed] [Google Scholar]
- 27.Pajni-Underwood S, Wilson CP, Elder C, Mishina Y, Lewandoski M (2007) BMP signals control limb bud interdigital programmed cell death by regulating FGF signaling. Development 12:2359–2368 [DOI] [PubMed] [Google Scholar]
- 28.Guha U, Gomes WA, Kobayashi T, Pestell RG, Kessler JA (2002) In vivo evidence that BMP signaling is necessary for apoptosis in the mouse limb. Dev Biol 1:108–120 [DOI] [PubMed] [Google Scholar]
- 29.Ganan Y, Macias D, Duterque-Coquillaud M, Ros MA, Hurle JM (1996) Role of TGF beta s and BMPs as signals controlling the position of the digits and the areas of interdigital cell death in the developing chick limb autopod. Development 8:2349–2357 [DOI] [PubMed] [Google Scholar]
- 30.Merino R, Rodriguez-Leon J, Macias D, Ganan Y, Economides AN, Hurle JM (1999) The BMP antagonist gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb. Development 23:5515–5522 [DOI] [PubMed] [Google Scholar]
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