Differential Regulation of CASZ1 Protein Expression During Cardiac and Skeletal Muscle Development

BACKGROUND: The zinc-finger transcription factor CASZ1 is required for differentiation of a distinct population of cardiomyocytes during development. However, expression of Casz1 mRNA is detected throughout the developing heart, suggesting the spatial regulation of CASZ1 occurs at the protein level. Relatively little is known about posttranscriptional regulation of Casz1 in the heart. RESULTS: We generated antibodies that specifically recognize CASZ1 in developing Xenopus embryos, and performed immunofluorescence analysis of CASZ1 during cardiac development. CASZ1 was detected throughout the developing myocardium. CASZ1 was restricted to terminally differentiated cardiomyocytes, and was down-regulated in cells that re-enter the cell cycle. We determined that CASZ1 expression correlated with terminal differentiation in cardiac muscle cells, skeletal muscle cells, and lymph-heart musculature. CONCLUSIONS: This study indicates that spatially distinct expression of CASZ1 protein may be due to posttranscriptional control of Casz1 mRNA during cardiac development. The results of this study provide insights into the role of Casz1 in cardiac function and in the differentiation of other cell types, including skeletal muscle and lymph heart

Budgett's frog (Lepidobatrachus laevis): A new amphibian embryo for developmental biology

AbstractThe large size and rapid development of amphibian embryos has facilitated ground-breaking discoveries in developmental biology. Here, we describe the embryogenesis of the Budgett's frog (Lepidobatrachus laevis), an unusual species with eggs that are over twice the diameter of laboratory Xenopus, and embryos that can tolerate higher temperatures to develop into a tadpole four times more rapidly. In addition to detailing their early development, we demonstrate that, like Xenopus, these embryos are amenable to explant culture assays and can express exogenous transcripts in a tissue-specific manner. Moreover, the steep developmental trajectory and large scale of Lepidobatrachus make it exceptionally well-suited for morphogenesis research. For example, the developing organs of the Budgett's frog are massive compared to those of most model species, and are composed of larger individual cells, thereby affording increased subcellular resolution of early vertebrate organogenesis. Furthermore, we found that complete limb regeneration, which typically requires months to achieve in most vertebrate models, occurs in a matter of days in the Budgett's tadpole, which substantially accelerates the pace of experimentation. Thus, the unusual combination of the greater size and speed of the Budgett's frog model provides inimitable advantages for developmental studies—and a novel inroad to address the mechanisms of spatiotemporal scaling during evolution

RNA-seq in the tetraploid Xenopus laevis enables genome-wide insight in a classic developmental biology model organism

Advances in sequencing technology have significantly advanced the landscape of developmental biology research. The dissection of genetic networks in model and nonmodel organisms has been greatly enhanced with high-throughput sequencing technologies. RNA-seq has revolutionized the ability to perform developmental biology research in organisms without a published genome sequence. Here, we describe a protocol for developmental biologists to perform RNA-seq on dissected tissue or whole embryos. We start with the isolation of RNA and generation of sequencing libraries. We further show how to interpret and analyze the large amount of sequencing data that is generated in RNA-seq. We explore the abilities to examine differential expression, gene duplication, transcript assembly, alternative splicing and SNP discovery. For the purposes of this article, we use Xenopus laevis as the model organism to discuss uses of RNA-seq in an organism without a fully annotated genome sequence

Xenopus: An emerging model for studying congenital heart disease

Congenital heart defects affect nearly 1% of all newborns and are a significant cause of infant death. Clinical studies have identified a number of congenital heart syndromes associated with mutations in genes that are involved in the complex process of cardiogenesis. The African clawed frog, Xenopus, has been instrumental in studies of vertebrate heart development and provides a valuable tool to investigate the molecular mechanisms underlying human congenital heart diseases. In this review, we discuss the methodologies that make Xenopus an ideal model system to investigate heart development and disease. We also outline congenital heart conditions linked to cardiac genes that have been well-studied in Xenopus and describe some emerging technologies that will further aid in the study of these complex syndromes

Congenital heart disease protein 5 associates with CASZ1 to maintain myocardial tissue integrity

The identification and characterization of the cellular and molecular pathways involved in the differentiation and morphogenesis of specific cell types of the developing heart are crucial to understanding the process of cardiac development and the pathology associated with human congenital heart disease. Here, we show that the cardiac transcription factor CASTOR (CASZ1) directly interacts with congenital heart disease 5 protein (CHD5), which is also known as tryptophan-rich basic protein (WRB), a gene located on chromosome 21 in the proposed region responsible for congenital heart disease in individuals with Down's syndrome. We demonstrate that loss of CHD5 in Xenopus leads to compromised myocardial integrity, improper deposition of basement membrane, and a resultant failure of hearts to undergo cell movements associated with cardiac formation. We further report that CHD5 is essential for CASZ1 function and that the CHD5-CASZ1 interaction is necessary for cardiac morphogenesis. Collectively, these results establish a role for CHD5 and CASZ1 in the early stages of vertebrate cardiac development

Casz1 is required for cardiomyocyte G1-to-S phase progression during mammalian cardiac development

Organ growth occurs through the integration of external growth signals during the G1 phase of the cell cycle to initiate DNA replication. Although numerous growth factor signals have been shown to be required for the proliferation of cardiomyocytes, genetic studies have only identified a very limited number of transcription factors that act to regulate the entry of cardiomyocytes into S phase. Here, we report that the cardiac para-zinc-finger protein CASZ1 is expressed in murine cardiomyocytes. Genetic fate mapping with an inducible Casz1 allele demonstrates that CASZ1-expressing cells give rise to cardiomyocytes in the first and second heart fields. We show through the generation of a cardiac conditional null mutation that Casz1 is essential for the proliferation of cardiomyocytes in both heart fields and that loss of Casz1 leads to a decrease in cardiomyocyte cell number. We further report that the loss of Casz1 leads to a prolonged or arrested S phase, a decrease in DNA synthesis, an increase in phospho-RB and a concomitant decrease in the cardiac mitotic index. Taken together, these studies establish a role for CASZ1 in mammalian cardiomyocyte cell cycle progression in both the first and second heart fields

Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT)

The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development

CASZ1 Promotes Vascular Assembly and Morphogenesis through the Direct Regulation of an EGFL7/RhoA-Mediated Pathway

The formation of the vascular system is essential for embryonic development and homeostasis. However, transcriptional control of this process is not fully understood. Here we report an evolutionarily conserved role for the transcription factor CASZ1 in blood vessel assembly and morphogenesis. In the absence of CASZ1, Xenopus embryos fail to develop a branched and lumenized vascular system, and CASZ1-depleted human endothelial cells display dramatic alterations in adhesion, morphology, and sprouting. Mechanistically, we show CASZ1 directly regulates Epidermal Growth Factor-Like Domain 7 (Egfl7). We further demonstrate that defects of CASZ1 or EGFL7-depleted cells are in part due to diminished RhoA expression and impaired focal adhesion localization. Moreover, these abnormal endothelial cell behaviors in CASZ1-depleted cells can be rescued by restoration of Egfl7. Collectively, these studies show CASZ1 is required to directly regulate a unique EGFL7/RhoA-mediated pathway to promote vertebrate vascular development

Progranulin signaling in sepsis, community-acquired bacterial pneumonia and COVID-19: a comparative, observational study

BACKGROUND Progranulin is a widely expressed pleiotropic growth factor with a central regulatory effect during the early immune response in sepsis. Progranulin signaling has not been systematically studied and compared between sepsis, community-acquired pneumonia (CAP), COVID-19 pneumonia and a sterile systemic inflammatory response (SIRS). We delineated molecular networks of progranulin signaling by next-generation sequencing (NGS), determined progranulin plasma concentrations and quantified the diagnostic performance of progranulin to differentiate between the above-mentioned disorders using the established biomarkers procalcitonin (PCT), interleukin-6 (IL-6) and C-reactive protein (CRP) for comparison. METHODS The diagnostic performance of progranulin was operationalized by calculating AUC and ROC statistics for progranulin and established biomarkers in 241 patients with sepsis, 182 patients with SIRS, 53 patients with CAP, 22 patients with COVID-19 pneumonia and 53 healthy volunteers. miRNAs and mRNAs in blood cells from sepsis patients (n = 7) were characterized by NGS and validated by RT-qPCR in an independent cohort (n = 39) to identify canonical gene networks associated with upregulated progranulin at sepsis onset. RESULTS Plasma concentrations of progranulin (ELISA) in patients with sepsis were 57.5 (42.8-84.9, Q25-Q75) ng/ml and significantly higher than in CAP (38.0, 33.5-41.0~ng/ml, p < 0.001), SIRS (29.0, 25.0-35.0~ng/ml, p < 0.001) and the healthy state (28.7, 25.5-31.7~ng/ml, p < 0.001). Patients with COVID-19 had significantly higher progranulin concentrations than patients with CAP (67.6, 56.6-96.0 vs. 38.0, 33.5-41.0~ng/ml, p < 0.001). The diagnostic performance of progranulin for the differentiation between sepsis vs. SIRS (n = 423) was comparable to that of procalcitonin. AUC was 0.90 (95% CI = 0.87-0.93) for progranulin and 0.92 (CI = 0.88-0.96, p = 0.323) for procalcitonin. Progranulin showed high discriminative power to differentiate bacterial CAP from COVID-19 (sensitivity 0.91, specificity 0.94, AUC 0.91 (CI = 0.8-1.0) and performed significantly better than PCT, IL-6 and CRP. NGS and partial RT-qPCR confirmation revealed a transcriptomic network of immune cells with upregulated progranulin and sortilin transcripts as well as toll-like-receptor 4 and tumor-protein 53, regulated by miR-16 and others. CONCLUSIONS Progranulin signaling is elevated during the early antimicrobial response in sepsis and differs significantly between sepsis, CAP, COVID-19 and SIRS. This suggests that progranulin may serve as a novel indicator for the differentiation between these disorders. TRIAL REGISTRATION Clinicaltrials.gov registration number NCT03280576 Registered November 19, 2015

Chondrotoxicity of Local Anesthetics: Liposomal Bupivacaine Is Less Chondrotoxic than Standard Bupivacaine

Objective. The purpose of this study is to determine whether (1) liposomal bupivacaine is chondrotoxic; (2) the chondrotoxicity of liposomal bupivacaine differs from standard bupivacaine; and (3) chondrotoxic effects are time dependent. Materials and Methods. We obtained 72 10 mm articular cartilage plugs from 12 fresh bovine distal femoral knee joints and exposed them to either saline, 0.5% bupivacaine, or liposomal bupivacaine for either 30 or 90 minutes. Twenty-four hours after treatment, chondrocyte viability was measured with the use of a fluorescent live/dead assay. An ANOVA test of variance was performed followed by a Holm–Sidak test to make pairwise comparisons across conditions. Student’s t-test was used to compare means. Results. Percent viability of cells exposed to liposomal bupivacaine for 30 minutes was less versus saline control (53.9% ± 21.5% vs. 73.7 ± 18.4%, p=0.035), and this remained significant at 90 minutes (49.1% ± 20.3% vs. 67.2% ± 25.6%, p<0.001). Liposomal bupivacaine had less chondrotoxic effects when compared with bupivacaine after 90 minutes, with greater viability (49.1% ± 20.3% vs. 21.4% ± 14.0%, p=0.003). Chondrotoxicity was found to be time dependent within the bupivacaine group (percent viability at 30 min: 45.5 ± 18.2%, 90 min: 21.4 ± 14.0%, p=0.001); however, liposomal bupivacaine did not demonstrate a significant time-dependent chondrotoxic relationship (p=0.583). Conclusions. Bupivacaine and liposomal bupivacaine are both toxic to chondrocytes. Liposomal bupivacaine is less chondrotoxic than standard bupivacaine and does not demonstrate a time-dependent toxicity