Ccdc103 promotes myeloid cell proliferation and migration independent of motile cilia.

The pathology of primary ciliary dyskinesia (PCD) is predominantly attributed to impairment of motile cilia. However, PCD patients also have perplexing functional defects in myeloid cells, which lack motile cilia. Here, we show that coiled-coil domain-containing protein 103 (CCDC103), one of the genes that, when mutated, is known to cause PCD, is required for the proliferation and directed migration of myeloid cells. CCDC103 is expressed in human myeloid cells, where it colocalizes with cytoplasmic microtubules. Zebrafish ccdc103/schmalhans (smh) mutants have macrophages and neutrophils with reduced proliferation, abnormally rounded cell morphology and an inability to migrate efficiently to the site of sterile wounds, all of which are consistent with a loss of cytoplasmic microtubule stability. Furthermore, we demonstrate that direct interactions between CCDC103 and sperm associated antigen 6 (SPAG6), which also promotes microtubule stability, are abrogated by CCDC103 mutations from PCD patients, and that spag6 zebrafish mutants recapitulate the myeloid defects observed in smh mutants. In summary, we have illuminated a mechanism, independent of motile cilia, to explain functional defects in myeloid cells from PCD patients. This article has an associated First Person interview with the first author of the paper.

HDAC1-mediated repression of the retinoic acid-responsive gene ripply3 promotes second heart field development.

Coordinated transcriptional and epigenetic mechanisms that direct development of the later differentiating second heart field (SHF) progenitors remain largely unknown. Here, we show that a novel zebrafish histone deacetylase 1 (hdac1) mutant allele cardiac really gone (crg) has a deficit of ventricular cardiomyocytes (VCs) and smooth muscle within the outflow tract (OFT) due to both cell and non-cell autonomous loss in SHF progenitor proliferation. Cyp26-deficient embryos, which have increased retinoic acid (RA) levels, have similar defects in SHF-derived OFT development. We found that nkx2.5+ progenitors from Hdac1 and Cyp26-deficient embryos have ectopic expression of ripply3, a transcriptional co-repressor of T-box transcription factors that is normally restricted to the posterior pharyngeal endoderm. Furthermore, the ripply3 expression domain is expanded anteriorly into the posterior nkx2.5+ progenitor domain in crg mutants. Importantly, excess ripply3 is sufficient to repress VC development, while genetic depletion of Ripply3 and Tbx1 in crg mutants can partially restore VC number. We find that the epigenetic signature at RA response elements (RAREs) that can associate with Hdac1 and RA receptors (RARs) becomes indicative of transcriptional activation in crg mutants. Our study highlights that transcriptional repression via the epigenetic regulator Hdac1 facilitates OFT development through directly preventing expression of the RA-responsive gene ripply3 within SHF progenitors.

Nr2f-dependent allocation of ventricular cardiomyocyte and pharyngeal muscle progenitors.

Multiple syndromes share congenital heart and craniofacial muscle defects, indicating there is an intimate relationship between the adjacent cardiac and pharyngeal muscle (PM) progenitor fields. However, mechanisms that direct antagonistic lineage decisions of the cardiac and PM progenitors within the anterior mesoderm of vertebrates are not understood. Here, we identify that retinoic acid (RA) signaling directly promotes the expression of the transcription factor Nr2f1a within the anterior lateral plate mesoderm. Using zebrafish nr2f1a and nr2f2 mutants, we find that Nr2f1a and Nr2f2 have redundant requirements restricting ventricular cardiomyocyte (CM) number and promoting development of the posterior PMs. Cre-mediated genetic lineage tracing in nr2f1a; nr2f2 double mutants reveals that tcf21+ progenitor cells, which can give rise to ventricular CMs and PM, more frequently become ventricular CMs potentially at the expense of posterior PMs in nr2f1a; nr2f2 mutants. Our studies reveal insights into the molecular etiology that may underlie developmental syndromes that share heart, neck and facial defects as well as the phenotypic variability of congenital heart defects associated with NR2F mutations in humans.

Myogenesis in Drosophila melanogaster: Dissection of Distinct Muscle Types for Molecular Analysis.

Drosophila is a useful model organism for studying the molecular signatures that define specific muscle types during myogenesis. It possesses significant genetic conservation with humans for muscle disease causing genes and a lack of redundancy that simplifies functional analysis. Traditional molecular methods can be utilized to understand muscle developmental processes such as Western blots, in situ hybridizations, RT-PCR and RNAseq, to name a few. However, one challenge for these molecular methods is the ability to dissect different muscle types. In this protocol we describe some useful techniques for extracting muscles from the pupal and adult stages of development using flight and jump muscles as an example.

Absence of the Drosophila jump muscle actin Act79B is compensated by up-regulation of Act88F.

BACKGROUND: Actins are structural components of the cytoskeleton and muscle, and numerous actin isoforms are found in most organisms. However, many actin isoforms are expressed in distinct patterns allowing each actin to have a specialized function. Numerous studies have demonstrated that actin isoforms both can and cannot compensate for each other under specific circumstances. This allows for an ambiguity of whether isoforms are functionally distinct. RESULTS: In this study, we analyzed mutants of Drosophila Act79B, the predominant actin expressed in the adult jump muscle. Functional and structural analysis of the Act79B mutants found the flies to have normal jumping ability and sarcomere structure. Analysis of actin gene expression determined that expression of Act88F, an actin gene normally expressed in the flight muscles, was significantly up-regulated in the jump muscles of mutants. This indicated that loss of Act79B caused expansion of Act88F expression. When we created double mutants of Act79B and Act88F, this abolished the jump ability of the flies and resulted in severe defects in myofibril formation. CONCLUSIONS: These results indicate that Act88F can functionally substitute for Act79B in the jump muscle, and that the functional compensation in actin expression in the jump muscles only occurs through Act88F. Developmental Dynamics 247:642-649, 2018. © 2018 Wiley Periodicals, Inc.

Nr2f1a balances atrial chamber and atrioventricular canal size via BMP signaling-independent and -dependent mechanisms.

Determination of appropriate chamber size is critical for normal vertebrate heart development. Although Nr2f transcription factors promote atrial maintenance and differentiation, how they determine atrial size remains unclear. Here, we demonstrate that zebrafish Nr2f1a is expressed in differentiating atrial cardiomyocytes. Zebrafish nr2f1a mutants have smaller atria due to a specific reduction in atrial cardiomyocyte (AC) number, suggesting it has similar requirements to Nr2f2 in mammals. Furthermore, the smaller atria in nr2f1a mutants are derived from distinct mechanisms that perturb AC differentiation at the chamber poles. At the venous pole, Nr2f1a enhances the rate of AC differentiation. Nr2f1a also establishes the atrial-atrioventricular canal (AVC) border through promoting the differentiation of mature ACs. Without Nr2f1a, AVC markers are expanded into the atrium, resulting in enlarged endocardial cushions (ECs). Inhibition of Bmp signaling can restore EC development, but not AC number, suggesting that Nr2f1a concomitantly coordinates atrial and AVC size through both Bmp-dependent and independent mechanisms. These findings provide insight into conserved functions of Nr2f proteins and the etiology of atrioventricular septal defects (AVSDs) associated with NR2F2 mutations in humans.

Tcf7l1 proteins cell autonomously restrict cardiomyocyte and promote endothelial specification in zebrafish.

Tcf7l1 (formerly Tcf3) proteins are conserved transcription factors whose function as transcriptional repressors is relieved through interactions with ß-catenin. Although the functions of Tcf7l1 proteins have been studied in many developmental contexts, whether this conserved mediator of Wnt signaling is required for appropriate cardiomyocyte (CM) development has not been investigated. We find that Tcf7l1 proteins are necessary during two developmental periods to limit CM number in zebrafish embryos: prior to gastrulation and after the initial wave of CM differentiation. In contrast to partially redundant roles in anterior neural patterning, we find that Tcf7l1a and Tcf7l1b have non-redundant functions with respect to restricting CM specification during anterior mesodermal patterning, suggesting that between the two zebrafish Tcf7l1 paralogs there is a limit to the transcriptional repression provided during early CM specification. Using cell transplantation experiments, we determine that the Tcf7l1 paralogs are required cell autonomously to restrict CM specification and promote endothelial cell (EC) specification, which is overtly similar to the ability of Wnt signaling to direct a transformation between these progenitors in embryonic stem cells. Therefore, these results argue that during anterior-posterior patterning of the mesoderm Tcf7l1 proteins are cell autonomously required to limit Wnt signaling, which balances CM and EC progenitor specification within the anterior lateral plate mesoderm. This study expands our understanding of the in vivo developmental requirements of Tcf7l1 proteins and the mechanisms directing CM development in vertebrates.

Distinct phases of Wnt/ß-catenin signaling direct cardiomyocyte formation in zebrafish.

Normal heart formation requires reiterative phases of canonical Wnt/ß-catenin (Wnt) signaling. Understanding the mechanisms by which Wnt signaling directs cardiomyocyte (CM) formation in vivo is critical to being able to precisely direct differentiated CMs from stem cells in vitro. Here, we investigate the roles of Wnt signaling in zebrafish CM formation using heat-shock inducible transgenes that increase and decrease Wnt signaling. We find that there are three phases during which CM formation is sensitive to modulation of Wnt signaling through the first 24 h of development. In addition to the previously recognized roles for Wnt signaling during mesoderm specification and in the pre-cardiac mesoderm, we find a previously unrecognized role during CM differentiation where Wnt signaling is necessary and sufficient to promote the differentiation of additional atrial cells. We also extend the previous studies of the roles of Wnt signaling during mesoderm specification and in pre-cardiac mesoderm. Importantly, in pre-cardiac mesoderm we define a new mechanism where Wnt signaling is sufficient to prevent CM differentiation, in contrast to a proposed role in inhibiting cardiac progenitor (CP) specification. The inability of the CPs to differentiate appears to lead to cell death through a p53/Caspase-3 independent mechanism. Together with a report for an even later role for Wnt signaling in restricting proliferation of differentiated ventricular CMs, our results indicate that during the first 3days of development in zebrafish there are four distinct phases during which CMs are sensitive to Wnt signaling.