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Purpose: The purpose of this study was to investigate if FDG-PET and DWI identify the same or different targets for dose escalation in the GTV of HN cancer patients. Additionally, the dose coverage of DWI-targets in an FDG-PET-based dose painting plan was analyzed., Materials and Methods: Eighteen HN cancer patients underwent FDG-PET and DWI exams, which were converted to standardized uptake value (SUV)- and apparent diffusion coefficient (ADC)-maps. The correspondence between the two imaging modalities was determined on a voxel-level using Spearman's correlation coefficient (ρ). Dose painting plans were optimized based on the 50% isocontour of the maximum SUV ( SUV(50%max)). Dose coverage was analyzed in three different SUV- and three different ADC-targets using the mean dose and the near-minimum and near-maximum doses., Results: The average maximum SUV was 13.9 and the mean ADC was 1.17 · 10(-3) mm(2)/s. The average ρ between SUV and ADC was -0.2 (range: -0.6 to 0.4). The ADC-targets were only partly overlapping the SUV(50%max)-target and the dose parameters were significantly smaller in the ADC-targets compared to the SUV(50%max)-target., Conclusions: FDG-PET and DWI contain different information, resulting in different targets. Further information about failure patterns and dose relations can be obtained by adding DWI to currently ongoing dose painting trials., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Modern radiation therapy techniques are exceptionally flexible in the deposition of radiation dose in a target volume. Complex distributions of dose can be delivered reliably, so that the tumor is exposed to a high dose, whereas nearby healthy structures can be avoided. As a result, an increase in curative dose is no longer invariably associated with an increased level of toxicity. This modern technology can be exploited further by modulating the required dose in space so as to match the variation in radiation sensitivity in the tumor. This approach is called dose painting. For dose painting to be effective, functional imaging techniques are essential to identify regions in a tumor that require a higher dose. Several techniques are available in nuclear medicine and radiology. In recent years, there has been a considerable research effort concerning the integration of magnetic resonance imaging (MRI) into the external radiotherapy workflow motivated by the superior soft tissue contrast as compared to computed tomography. In MRI, diffusion-weighted MRI reflects the cell density of tissue and thus may indicate regions with a higher tumor load. Dynamic contrast-enhanced MRI reflects permeability of the microvasculature and blood flow, correlated to the oxygenation of the tumor. These properties have impact on its radiation sensitivity. New questions must be addressed when these techniques are applied in radiation therapy: scanning in treatment position requires alternative solutions to the standard patient setup in the choice of receive coils compared to a diagnostic department. This standard positioning also facilitates repeated imaging. The geometrical accuracy of MR images is critical for high-precision radiotherapy. In particular, when multiparametric functional data are used for dose painting, quantification of functional parameters at a high spatial resolution becomes important. In this review, we will address these issues and describe clinical developments in MRI-guided dose painting., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aims: Crypts can be found with cardiovascular magnetic resonance imaging (CMR) in hypertrophic cardiomyopathy (HCM) mutation carriers without hypertrophy (carriers) using a modified two-chamber view through the inferoseptum, but also in other patients and healthy individuals with standard long-axis views. Since it is currently unknown if carriers display a specific crypt morphology, we compared crypts in carriers with other cardiac pathologies (controls). Besides, we aimed to determine the optimal imaging plane for the detection of crypts by comparing modified two-chamber views with standard long-axis views. Finally, we evaluated the accuracy of crypts to identify carriers in HCM family screening., Methods and Results: Standard CMR long-axis views with additional modified two-chamber views were prospectively performed in carriers (n= 43), consecutive CMR control patients (n= 252), and mutation-negative family members (n= 15). Crypts were found in 70% (30/43) of carriers and in 12% (31/252) of controls (P< 0.001). Crypts in carriers showed deeper penetrance into the myocardium compared with controls (74 ± 21% vs. 59 ± 22%, P< 0.01). Detection of two or more crypts had a sensitivity of 51% and specificity of 94% for carriership. Modified two-chamber views doubled the sensitivity to detect crypts compared with standard long-axis views. In family screening, ≥2 crypts had a 100% positive predictive value to identify carriers., Conclusions: Multiple crypts in the absence of left ventricular hypertrophy are highly specific for HCM mutation carriership and warrant clinical follow-up. A modified two-chamber view has a superior sensitivity compared with standard long-axis views for crypt detection. CMR may be of additional value to identify carriers in family screening.

Background: Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant condition caused by germline FLCN mutations, and characterised by fibrofolliculomas, pneumothorax and renal cancer. The renal cancer risk, cancer phenotype and pneumothorax risk of BHD have not yet been fully clarified. The main focus of this study was to assess the risk of renal cancer, the histological subtypes of renal tumours and the pneumothorax risk in BHD., Methods: In this study we present the clinical data of 115 FLCN mutation carriers from 35 BHD families., Results: Among 14 FLCN mutation carriers who developed renal cancer 7 were <50 years at onset and/or had multifocal/bilateral tumours. Five symptomatic patients developed metastatic disease. Two early-stage cases were diagnosed by surveillance. The majority of tumours showed characteristics of both eosinophilic variants of clear cell and chromophobe carcinoma. The estimated penetrance for renal cancer and pneumothorax was 16% (95% minimal confidence interval: 6-26%) and 29% (95% minimal confidence interval: 9-49%) at 70 years of age, respectively. The most frequent diagnosis in families without identified FLCN mutations was familial multiple discoid fibromas., Conclusion: We confirmed a high yield of FLCN mutations in clinically defined BHD families, we found a substantially increased lifetime risk of renal cancer of 16% for FLCN mutation carriers. The tumours were metastatic in 5 out of 14 patients and tumour histology was not specific for BHD. We found a pneumothorax risk of 29%. We discuss the implications of our findings for diagnosis and management of BHD.

Purpose: We investigated radiation-induced changes in the salivary glands, 6 weeks after RT, using MRI., Materials and Methods: Eighteen oropharyngeal cancer patients were treated with salivary gland sparing IMRT. All patients received a 3T MRI exam before and 6 weeks after the end of RT, including a T(1)-weighted (T(1)w), a T(2)-weighted (T(2)w), and a dynamic contrast-enhanced (DCE) MRI. For both time points separately, the parotid and submandibular glands were delineated on the MR images. Differences in median signal intensity and signal variation within the glands were tested for significance. Correlations were studied between the MR changes and the planned RT dose., Results: The volume of the glands reduced significantly by 25%. The T(1)w signal decreased by 10% and the T(2)w signal increased by 23%. The k(ep) value decreased, while the v(e) increased. A correlation of the changes in T(2)w signal with the mean dose was found in both glands., Conclusions: Overall radiation-induced changes and volume loss were observed in the parotid and submandibular gland using MR. The observed differences indicated an increased water content such as found in oedema. The overall changes could be related to the mean dose, with a slightly greater impact in the high dose area., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Aims: We investigated the presence of a clinical diagnosis of hypertrophic cardiomyopathy (HCM), risk factors for sudden cardiac death (SCD), and cardiac events during follow-up in predictively tested-not known to have a clinical diagnosis of HCM before the DNA test-carriers of a sarcomeric gene mutation and associations with age and gender to determine the best cardiological screening strategy., Methods and Results: One hundred and thirty-six (30%) of 446 mutation carriers were diagnosed with HCM at one or more cardiological evaluation(s). Male gender and higher age were associated with manifest disease. Incidence of newly diagnosed manifest HCM was <10% per person-year under the age of 40 years and >10% in older carriers, although numbers were small in carriers <15 years. Twenty-three percent of carriers, with and without manifest disease, had established risk factor(s) for SCD (no significant difference). During an average follow-up of 3.5 ± 1.7 years two carriers, both with manifest disease, died suddenly (0.13% per person-year). A high-risk status for SCD (≥2 risk factors and manifest HCM) was present in 17 carriers during follow-up (2.4% per person-year). Age but not gender was associated with a high-risk status for SCD., Conclusion: Thirty percent of carriers had or developed manifest HCM after predictive DNA testing and risk factors for SCD were frequently present. Our data suggest that the SCD risk is low and risk stratification for SCD can be omitted in carriers without manifest disease and that frequency of cardiological evaluations can possibly be decreased in carriers between 15 and 40 years as long as hypertrophy is absent.

Pathogenic mutations in CYLD can be identified in patients affected with Brooke-Spiegler syndrome, (Familial) Cylindromatosis or multiple familial trichoepithelioma. To date, only technologies which are able to identify small point mutations in CYLD, such as sequence and WAVE analysis, were used. Here we describe the identification of a larger rearrangement identified by Quantitative PCR analysis of CYLD, indicating that a combination of these technologies is necessary when searching for pathogenic mutations in CYLD.

Purpose: Besides sparing the parotid gland, sparing the submandibular gland is considered to be important in preventing xerostomia in head-and-neck cancer patients. Delineation of the submandibular gland at CT, and even on T(1)- and T(2)-weighted MR images, is difficult, due to low contrast with the surrounding tissues. MR sialography might be used for delineation., Methods and Materials: Sixteen oropharyngeal cancer patients received a CT and MRI exam as part of the standard treatment imaging protocol. Patients were scanned in their five-point RT immobilization mask. The MRI exam included T(1)- and T(2)-weighted MRI scans and an MR sialography scan. Thirty submandibular glands were delineated on only CT, on the combined CT and T(1)- and T(2)-weighted MRI scans and on all MR images. A Wilcoxon signed-rank test was performed to test if the delineated volumes were significantly different., Results: The delineated volume of the submandibular gland was 7.3mL in the CT-delineation, 7.1mL in the CT/MRI-delineation and 8.1mL in the MRI-delineation. The MRI-delineation was significantly larger than the other delineations (p<0.001). The differences were mainly located in the cranial direction., Conclusion: The delineation of the submandibular gland was improved in the cranial direction by using T(1)- and T(2)-weighted MRI and MR sialography, compared to the other delineations., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Purpose: To analyze the combined and updated results from the University of Michigan and University Medical Center Utrecht on normal tissue complication probability (NTCP) of the parotid gland 1 year after radiotherapy (RT) for head-and-neck (HN) cancer., Patients and Methods: A total of 222 prospectively analyzed patients with various HN malignancies were treated with conventional and intensity-modulated RT. Stimulated individual parotid gland flow rates were measured before RT and 1 year after RT using Lashley cups at both centers. A flow ratio <25% of pretreatment was defined as a complication. The data were fitted to the Lyman-Kutcher-Burman model., Results: A total of 384 parotid glands (Michigan: 157; Utrecht: 227 glands) was available for analysis 1 year after RT. Combined NTCP analysis based on mean dose resulted in a TD(50) (uniform dose leading to 50% complication probability) of 39.9 Gy and m (steepness of the curve) of 0.40. The resulting NTCP curve had good qualitative agreement with the combined clinical data. Mean doses of 25-30 Gy were associated with 17-26% NTCP., Conclusions: A definite NTCP curve for parotid gland function 1 year after RT is presented, based on mean dose. No threshold dose was observed, and TD(50) was equal to 40 Gy., (2010 Elsevier Inc. All rights reserved.)

The benefits of a patient-specific head support, developed to improve immobilization during radiotherapy, were determined in head and neck cancer patients. Cone-beam CTs were registered to the planning CT in five regions. Compared to the standard head support, the individual head support decreased the systematic and random errors of the inter- and intrafraction displacements and reduced deformations., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy (ARVD/C) Diagnostic Task Force Criteria (TFC) proposed in 1994 are highly specific but lack sensitivity. A new international task force modified criteria to improve diagnostic yield. A comparison of diagnosis by 1994 TFC versus newly proposed criteria in 3 patient groups was conducted., Methods and Results: In new TFC, scoring by major and minor criteria is maintained. Structural abnormalities are quantified and TFC highly specific for ARVD/C upgraded to major. Furthermore, new criteria are added: terminal activation duration of QRS > or = 55 ms, ventricular tachycardia with left bundle-branch block morphology and superior axis, and genetic criteria. Three groups were studied: (1) 105 patients with proven ARVD/C according to 1994 TFC, (2) 89 of their family members, and (3) 39 patients with probable ARVD/C (ie, 3 points by 1994 TFC). All were screened for pathogenic mutations in desmosomal genes. Three ARVD/C patients did not meet the new sharpened criteria on structural abnormalities and thereby did not fulfill new TFC. In 62 of 105 patients with proven ARVD/C, mutations were found: 58 in the gene encoding Plakophilin2 (PKP2), 3 in Desmoglein2, 3 in Desmocollin2, and 1 in Desmoplakin. Three patients had bigenic involvement. Ten additional relatives (11%) fulfilled new TFC: 9 (90%) were female, and all carried PKP2 mutations. No relatives lost diagnosis by application of new TFC. Of patients with probable ARVD/C, 25 (64%) fulfilled new TFC: 8 (40%) women and 14 (56%) carrying pathogenic mutations., Conclusions: In this first study applying new TFC to patients suspected of ARVD/C, 64% of probable ARVD/C patients and 11% of family members were additionally diagnosed. ECG criteria and pathogenic mutations especially contributed to new diagnosis. Newly proposed TFC have a major impact in increasing diagnostic yield of ARVD/C.

Background and Purpose: The submandibular glands are proposed to be important in preventing xerostomia in head-and-neck cancer patients. We investigated the feasibility of sparing the contralateral submandibular gland (cSMG) by reducing the dose to the contralateral planning target volume (PTV) and by reducing the clinical target volume (CTV)-to-PTV margin., Materials and Methods: Ten oropharyngeal cancer patients with a contralateral elective PTV were included in this planning study, using intensity modulated radiotherapy (IMRT). The effect on the mean dose to the cSMG of reducing the dose coverage to the contralateral elective PTV from 95 to 90% of the prescribed dose (54Gy in 1.8Gy daily fractions) was determined. The influence of reducing the margin for position uncertainty from 5 to 2mm was investigated., Results: The mean dose to the cSMG was reduced from 54Gy to approximately 40Gy if the dose coverage to the contralateral PTV was reduced to 90% of the prescribed dose. The estimated normal tissue complication probability (NTCP) was reduced below 50%. Reducing the margin from 5 to 2mm resulted in a decrease in the mean dose to the cSMG of approximately 6Gy., Conclusions: Reducing the mean dose to the cSMG below 40Gy is possible with a reasonable dose coverage of the contralateral elective PTV.

Nppa, encoding atrial natriuretic factor, is expressed in fetal atrial and ventricular myocardium and is downregulated in the ventricles after birth. During hypertrophy and heart failure, Nppa expression is reactivated in the ventricles and serves as a highly conserved marker of heart disease. The Nppa promoter has become a frequently used model to study mechanisms of cardiac gene regulation. Nevertheless, the regulatory sequences that provide the correct developmental pattern and ventricular reactivation during cardiac disease remain to be defined. We found that proximal Nppa fragments ranging from 250 bp to 16 kbp provide robust reporter gene activity in the atria and correct repression in the atrioventricular canal and the nodes of the conduction system in vivo. However, depending on fragment size and site of integration into the genome of mice, the fetal ventricular activity was either absent or present in an incorrect pattern. Furthermore, these fragments did not provide ventricular reactivation in heart disease models. These results indicate that the proximal promoter does not provide a physiologically relevant model for ventricular gene activity. In contrast, 2 modified bacterial artificial chromosome clones with partially overlapping genomic Nppa sequences provided appropriate reactivation of the green fluorescent protein reporter during pressure overload-induced hypertrophy and heart failure in vivo. However, only 1 of these bacterial artificial chromosomes provided correct fetal ventricular green fluorescent protein activity. These results show that distinct distal regulatory sequences and divergent regulatory pathways control fetal ventricular activity and reactivation of Nppa during cardiac disease, respectively.

Myosin heavy-chain (MHC) isoforms are major structural components of the contractile apparatus of the heart muscle. Their spatio-temporal patterns of expression have been used as a tool to dissect cardiac development and differentiation. Although extensively investigated, controversy still exists concerning the expression patterns of atrial (AMHC), ventricular (VMHC), and cardiac myosin heavy-chain (CMHC) during development in the heart. In this study, we describe that probe length, probe concentration, and staining time in the non-radioactive in situ hybridization procedure seriously influence the observed pattern of MHC expression and the subsequent interpretation, explaining the divergent opinions in the field. Using a variety of external and internal controls for the in situ hybridization procedure, we demonstrate that both AMHC and VMHC are expressed throughout the entire heart tube during early development. During subsequent development, VMHC becomes restricted to the ventricles, whereas AMHC remains expressed in the atria, and, at substantially lower levels, is detected in the ventricles. These results are discussed in the context of methodological constraints of demonstrating patterns of gene expression. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

The endocrine function of the heart was shown by the identification of a potent inhibitor of renal tubular NaCl reabsorbtion, atrial natriuretic peptide/factor (ANP), in atrial extracts. Since then, Nppa, the gene encoding ANP, has become an important tool for molecular and developmental biologists. Expression of Nppa is an early and specific marker for the differentiating working myocardium of the atria and ventricles of the developing heart. Around the time of birth, ventricular Nppa expression is down-regulated, to be reactivated in response to a variety of cardiovascular disorders. Many aspects of the regulatory pathways that control Nppa gene expression during cardiac development or disease have been revealed. However, several fundamental issues remain to be resolved. Particularly, the regulatory mechanisms underlying the ventricular Nppa expression in the embryonic heart and the reactivation in the failing ventricle remain unclear. Here we review current knowledge on the transcriptional regulation of the Nppa gene.

We identified and characterized the chicken natriuretic peptide precursor gene cluster and found its organization to be highly conserved compared with the mammalian Nppb-Nppa cluster. However, phylogenetic analysis indicated that the putative chicken natriuretic peptide precursor genes are the homologues of CNP-3 and Nppb, respectively. Comparative expression analysis revealed that, in human, mouse, and rat hearts, Nppb is a novel marker for the differentiating working myocardium. Its expression pattern is strikingly similar to that of Nppa before birth, and diverges only after birth. In contrast, whereas the chicken Nppb gene expression profile resembled that of mammalian Nppb, the CNP-3 gene showed very limited expression in the heart, not resembling the pattern of either Nppa or Nppb. These results show that, in chicken, the Nppa gene has been lost from the natriuretic peptide precursor gene cluster, whereas the CNP-3 gene has been retained.

Using degenerated PCR-primers to identify known and novel BMPs that are expressed in the developing chicken heart, we identified not only BMP2, -4, and -7 mRNA, but also the TGFbeta superfamily member cVg1. The expression pattern of cVg1 mRNA was determined during chicken development from HH4 to HH44. In early developmental stages, cVg1 mRNA is expressed in the primitive streak, paraxial mesoderm, developing somites, and developing neural tube. Subsequently, cVg1 mRNA is expressed in the developing central and peripheral nervous system, retina, auditory vesicle, notochord, lung alveoli, and olfactory mucosa. In the heart, cVg1 is initially expressed through the linear heart tube, but becomes restricted to the forming chamber myocardium, in an expression domain similar to that of atrial natriuretic factor (ANF) mRNA., (Copyright 2003 Wiley-Liss, Inc.)

In mouse, atrial natriuretic factor (ANF) gene expression was shown to be a marker for chamber formation within the embryonic heart. To gain insight into the process of chamber formation in the chicken embryonic heart, we analyzed the expression pattern of cANF during development. We found cANF to be specifically expressed in the myocardium of the morphologically distinguishable atrial and ventricular chambers, similar to ANF in mouse. cANF expression was never detected in the myocardium of the atrioventricular canal (AVC), inner curvature, and outflow tract (OFT), which is lined by endocardial cushions. Expression was strictly excluded from the interventricular myocardium and most proximal part of the bundle branches, as identified by the expression of Msx-2, whereas the rest of the bundle branches, trabeculae, and surrounding working myocardium did express cANF. The myocardium that forms de novo within the cushions after looping did not express cANF. At HH9 cANF expression was first observed in a subset of cardiomyocytes, which was localized ventrally in the fused heart tube and laterally in the unfused cardiac sheets. Together, these results show that cANF expression can be used to distinguish differentiated chamber (working) myocardium, including the peripheral ventricular conduction system, from embryonic myocardium. We conclude that differentiation of chamber myocardium takes place already at HH9 at the ventral side of the linear heart tube, possibly preceded by latero-medial signals in the unfused cardiac sheets., (Copyright 2002 Wiley-Liss, Inc.)

Mammalian homologues of the Drosophila Iroquois homeobox gene complex, involved in patterning and regionalization of differentiation, have recently been identified (Mech. Dev., 69 (1997) 169; Dev. Biol., 217 (2000) 266; Dev. Dyn., 218 (2000) 160; Mech. Dev., 91 (2000) 317; Dev. Biol., 224 (2000) 263; Genome Res., 10 (2000) 1453; Mech. Dev., 103 (2001) 193). The six members of the murine family were found to be organized in two cognate clusters of three genes each, Irx1, -2, -4 and Irx3, -5, -6, respectively (Peters et al., 2000). As a basis for further study of their regulation and function we performed a comparative analysis of the genomic organization and of the expression patterns of all six Irx genes. The genes are expressed in highly specific and regionalized patterns of ectoderm, mesoderm and endoderm derived tissues. In most tissues the pattern of expression of the clustered genes, especially of Irx1 and -2 and of Irx3 and -5, respectively, closely resembled each other while those of Irx4 and -6 were very divergent. Interestingly, the expression of cognate genes was found to be mutually exclusive in adjacent and interacting tissues of limb, heart and the laryncho-pharyncheal region. The results indicate that the Irx genes are coordinately regulated at the level of the cluster.

Iroquois (Irx) proteins comprise a family of homeodomain-containing transcription factors involved in patterning and regionalization of embryonic tissues in both vertebrates and invertebrates. The six murine Irx genes are organized in two clusters, each consisting of three genes. Irx1, Irx2 and Irx4 belong to the IrxA cluster on chromosome 13, whereas Irx3, Irx5 and Irx6, comprising the IrxB cluster, are located on chromosome 8 (Peters et al., Genome Res. 10 (2000) 1453). Developmental expression patterns have so far only been reported for five Irx genes. Here, we investigated the expression pattern of Irx6 during mouse morphogenesis and early organogenesis. The pattern was found to be much more restricted compared to the other five Irx genes, and its level of expression was much lower.

The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method.

We isolated cDNAs of mouse Iroquois-related homeobox genes Irx1, -2, -3, -4, and -5 and characterized their patterns of expression in the developing heart. Irx1 and Irx2 were found to be expressed specifically in the ventricular septum from the onset of its formation onward. In fetal stages, the expression of both genes appeared to gradually become confined to the myocardium of the atrioventricular bundle and bundle branches of the forming ventricular conduction system. Irx3 was found to be expressed specifically in the trabeculated myocardium of the ventricles. Irx4 expression was observed in a segment of the linear heart tube and the atrioventricular canal and ventricular myocardium including the inner curvature after looping, resembling the pattern of MLC2V. Transcripts for Irx5 were detected specifically in the endocardium lining the ventricular and atrial working myocardium that also expressed von Willebrand factor, but were absent from the endocardium of the endocardial cushions, i.e., the atrioventricular canal, inner curvature, and outflow tract. The spatiodevelopmental pattern of Irx5 matched that of ANF, a marker for the forming working myocardium of the chambers. Taken together, all members of the Irx gene family were found to be expressed in highly specific patterns in the developing mouse heart, suggesting a critical role in the specification of the distinct components of the four-chambered heart.