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.

Single-minded 2 is required for left-right asymmetric stomach morphogenesis.

The morphogenesis of left-right (LR) asymmetry is a crucial phase of organogenesis. In the digestive tract, the development of anatomical asymmetry is first evident in the leftward curvature of the stomach. To elucidate the molecular events that shape this archetypal laterality, we performed transcriptome analyses of the left versus right sides of the developing stomach in frog embryos. Besides the known LR gene pitx2, the only gene found to be expressed asymmetrically throughout all stages of curvature was single-minded 2 (sim2), a Down Syndrome-related transcription factor and homolog of a Drosophila gene (sim) required for LR asymmetric looping of the fly gut. We demonstrate that sim2 functions downstream of LR patterning cues to regulate key cellular properties and behaviors in the left stomach epithelium that drive asymmetric curvature. Our results reveal unexpected convergent cooption of single-minded genes during the evolution of LR asymmetric morphogenesis, and have implications for dose-dependent roles of laterality factors in non-laterality-related birth defects.

INTACT Proteomics in Xenopus.

Analysis of the molecular mechanisms driving cell specification, differentiation, and other cellular processes can be difficult due to the heterogeneity of tissues and organs. Therefore, it is critical to isolate pure cell populations in order to properly assess the function of certain cell types in the context of a tissue. This protocol describes use of the INTACT (isolation of nuclei tagged in specific cell types) method in Xenopus, followed by proteomics analysis of nuclear protein complexes. The INTACT protocol utilizes two transgenes: (1) a three-part nuclear targeting fusion (NTF) consisting of a nuclear envelope protein (Nup35) that targets the NTF to the nuclear membrane, an enhanced green fluorescent protein (EGFP) cassette for NTF visualization in live animals, and a biotin ligase receptor protein (BLRP) that provides a substrate for the biotinylation of the NTF, and (2) the E. coli ligase BirA (which biotinylates the NTF) tagged to mCherry (for visualization). Either or both transgenes are driven by a tissue-specific promoter, making this protocol easily adaptable to proteomics analyses of immunoprecipitated complexes from INTACT-isolated nuclei of multiple tissue types to determine the composition of protein complexes in pure cell populations.

The left-right asymmetry of liver lobation is generated by Pitx2c-mediated asymmetries in the hepatic diverticulum.

Internal organs exhibit left-right asymmetric sizes, shapes and anatomical positions, but how these different lateralities develop is poorly understood. Here we use the experimentally tractable Xenopus model to uncover the morphogenetic events that drive the left-right asymmetrical lobation of the liver. On the right side of the early hepatic diverticulum, endoderm cells become columnar and apically constricted, forming an expanded epithelial surface and, ultimately, an enlarged right liver lobe. In contrast, the cells on the left side become rounder, and rearrange into a compact, stratified architecture that produces a smaller left lobe. Side-specific gain- and loss-of-function studies reveal that asymmetric expression of the left-right determinant Pitx2c elicits distinct epithelial morphogenesis events in the left side of the diverticulum. Surprisingly, the cellular events induced by Pitx2c during liver development are opposite those induced in other digestive organs, suggesting divergent cellular mechanisms underlie the formation of different lateralities.

Stomach curvature is generated by left-right asymmetric gut morphogenesis.

Left-right (LR) asymmetry is a fundamental feature of internal anatomy, yet the emergence of morphological asymmetry remains one of the least understood phases of organogenesis. Asymmetric rotation of the intestine is directed by forces outside the gut, but the morphogenetic events that generate anatomical asymmetry in other regions of the digestive tract remain unknown. Here, we show in mouse and Xenopus that the mechanisms that drive the curvature of the stomach are intrinsic to the gut tube itself. The left wall of the primitive stomach expands more than the right wall, as the left epithelium becomes more polarized and undergoes radial rearrangement. These asymmetries exist across several species, and are dependent on LR patterning genes, including Foxj1, Nodal and Pitx2 Our findings have implications for how LR patterning manifests distinct types of morphological asymmetries in different contexts.

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

The 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.

Promotion of bone morphogenetic protein signaling by tetraspanins and glycosphingolipids.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor ß (TGFß) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like "Sma/Mab" signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer 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.

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.

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.

A role of the LIN-12/Notch signaling pathway in diversifying the non-striated egg-laying muscles in C. elegans.

The proper formation and function of an organ is dependent on the specification and integration of multiple cell types and tissues. An example of this is the Caenorhabditis elegans hermaphrodite egg-laying system, which requires coordination between the vulva, uterus, neurons, and musculature. While the genetic constituents of the first three components have been well studied, little is known about the molecular mechanisms underlying the specification of the egg-laying musculature. The egg-laying muscles are non-striated in nature and consist of sixteen cells, four each of type I and type II vulval muscles and uterine muscles. These 16 non-striated muscles exhibit distinct morphology, location, synaptic connectivity and function. Using an RNAi screen targeting the putative transcription factors in the C. elegans genome, we identified a number of novel factors important for the diversification of these different types of egg-laying muscles. In particular, we found that RNAi knockdown of lag-1, which encodes the sole C. elegans ortholog of the transcription factor CSL (CBF1, Suppressor of Hairless, LAG-1), an effector of the LIN-12/Notch pathway, led to the production of extra type I vulval muscles. Similar phenotypes were also observed in animals with down-regulation of the Notch receptor LIN-12 and its DSL (Delta, Serrate, LAG-2) ligand LAG-2. The extra type I vulval muscles in animals with reduced LIN-12/Notch signaling resulted from a cell fate transformation of type II vulval muscles to type I vulval muscles. We showed that LIN-12/Notch was activated in the undifferentiated type II vulval muscle cells by LAG-2/DSL that is likely produced by the anchor cell (AC). Our findings provide additional evidence highlighting the roles of LIN-12/Notch signaling in coordinating the formation of various components of the functional C. elegans egg-laying system. We also identify multiple new factors that play critical roles in the proper specification of the different types of egg-laying muscles.

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.

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 non-model 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.

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 (CASTOR) 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 that 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 that CASZ1 is required to directly regulate an EGFL7/RhoA-mediated pathway to promote vertebrate vascular development.

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.

The FoxF/FoxC factor LET-381 directly regulates both cell fate specification and cell differentiation in C. elegans mesoderm development.

Forkhead transcription factors play crucial and diverse roles in mesoderm development. In particular, FoxF and FoxC genes are, respectively, involved in the development of visceral/splanchnic mesoderm and non-visceral mesoderm in coelomate animals. Here, we show at single-cell resolution that, in the pseudocoelomate nematode C. elegans, the single FoxF/FoxC transcription factor LET-381 functions in a feed-forward mechanism in the specification and differentiation of the non-muscle mesodermal cells, the coelomocytes (CCs). LET-381/FoxF directly activates the CC specification factor, the Six2 homeodomain protein CEH-34, and functions cooperatively with CEH-34/Six2 to directly activate genes required for CC differentiation. Our results unify a diverse set of studies on the functions of FoxF/FoxC factors and provide a model for how FoxF/FoxC factors function during mesoderm development.

A conserved Six-Eya cassette acts downstream of Wnt signaling to direct non-myogenic versus myogenic fates in the C. elegans postembryonic mesoderm.

The subdivision of mesodermal cells into muscle and non-muscle cells is crucial to animal development. In the C. elegans postembryonic mesoderm, this subdivision is a result of an asymmetric cell division that leads to the formation of striated body wall muscles and non-muscle coelomocytes. Here we report that the Six homeodomain protein CEH-34 and its cofactor Eyes Absent, EYA-1, function synergistically to promote the non-muscle fate in cells also competent to form muscles. We further show that the asymmetric expression of ceh-34 and eya-1 is regulated by a combination of 1) mesodermal intrinsic factors MAB-5, HLH-1 and FOZI-1, 2) differential POP-1 (TCF/LEF) transcriptional activity along the anterior-posterior axis, and 3) coelomocyte competence factor(s). These factors are conserved in both vertebrates and invertebrates, suggesting a conserved paradigm for mesoderm development in metazoans.

Mesodermal expression of the C. elegans HMX homolog mls-2 requires the PBC homolog CEH-20.

Metazoan development proceeds primarily through the regulated expression of genes encoding transcription factors and components of cell signaling pathways. One way to decipher the complex developmental programs is to assemble the underlying gene regulatory networks by dissecting the cis-regulatory modules that direct temporal-spatial expression of developmental genes and identify corresponding trans-regulatory factors. Here, we focus on the regulation of a HMX homoebox gene called mls-2, which functions at the intersection of a network that regulates cleavage orientation, cell proliferation and fate specification in the Caenorhabditis elegans postembryonic mesoderm. In addition to its transient expression in the postembryonic mesodermal lineage, the M lineage, mls-2 expression is detected in a subset of embryonic cells, in three pairs of head neurons and transiently in the somatic gonad. Through mutational analysis of the mls-2 promoter, we identified two elements (E1 and E2) involved in regulating the temporal-spatial expression of mls-2. In particular, we showed that one of the elements (E1) required for mls-2 expression in the M lineage contains two critical putative PBC-Hox binding sites that are evolutionarily conserved in C. briggsae and C. remanei. Furthermore, the C. elegans PBC homolog CEH-20 is required for mls-2 expression in the M lineage. Our data suggest that mls-2 might be a direct target of CEH-20 in the M lineage and that the regulation of CEH-20 on mls-2 is likely Hox-independent.

A Zn-finger/FH2-domain containing protein, FOZI-1, acts redundantly with CeMyoD to specify striated body wall muscle fates in the Caenorhabditis elegans postembryonic mesoderm.

Striated muscle development in vertebrates requires the redundant functions of multiple members of the MyoD family. Invertebrates such as Drosophila and Caenorhabditis elegans contain only one MyoD homolog in each organism. Earlier observations suggest that factors outside of the MyoD family might function redundantly with MyoD in striated muscle fate specification in these organisms. However, the identity of these factors has remained elusive. Here, we describe the identification and characterization of FOZI-1, a putative transcription factor that functions redundantly with CeMyoD (HLH-1) in striated body wall muscle (BWM) fate specification in the C. elegans postembryonic mesoderm. fozi-1 encodes a novel nuclear-localized protein with motifs characteristic of both transcription factors and actin-binding proteins. We show that FOZI-1 shares the same expression pattern as CeMyoD in the postembryonic mesodermal lineage, the M lineage, and that fozi-1-null mutants exhibit similar M lineage-null defects to those found in animals lacking CeMyoD in the M lineage (e.g. loss of a fraction of M lineage-derived BWMs). Interestingly, fozi-1-null mutants with a reduced level of CeMyoD lack most, if not all, M lineage-derived BWMs. Our results indicate that FOZI-1 and the Hox factor MAB-5 function redundantly with CeMyoD in the specification of the striated BWM fate in the C. elegans postembryonic mesoderm, implicating a remarkable level of complexity for the production of a simple striated musculature in C. elegans.

An antagonistic role for the C. elegans Schnurri homolog SMA-9 in modulating TGFbeta signaling during mesodermal patterning.

In C. elegans, the Sma/Mab TGFbeta signaling pathway regulates body size and male tail patterning. SMA-9, the C. elegans homolog of Schnurri, has been shown to function as a downstream component to mediate the Sma/Mab TGFbeta signaling pathway in these processes. We have discovered a new role for SMA-9 in dorsoventral patterning of the C. elegans post-embryonic mesoderm, the M lineage. In addition to a small body size, sma-9 mutant animals exhibit a dorsal-to-ventral fate transformation within the M lineage. This M lineage defect of sma-9 mutants is unique in that animals carrying mutations in all other known components of the TGFbeta pathway exhibit no M lineage defects. Surprisingly, mutations in the core components of the Sma/Mab TGFbeta signaling pathway suppressed the M lineage defects of sma-9 mutants without suppressing their body size defects. We show that this suppression specifically happens within the M lineage. Our studies have uncovered an unexpected role of SMA-9 in antagonizing the TGFbeta signaling pathway during mesodermal patterning, suggesting a novel mode of function for the SMA-9/Schnurri family of proteins.