Tmem2 restricts atrioventricular canal differentiation by regulating degradation of hyaluronic acid.

BACKGROUND: Atrioventricular valve development relies upon the precisely defined dimensions of the atrioventricular canal (AVC). Current models suggest that Wnt signaling plays an important role atop a pathway that promotes AVC development. The factors that confine AVC differentiation to the appropriate location, however, are less well understood. RESULTS: Transmembrane protein 2 (Tmem2) is a key player in restricting AVC differentiation: in zebrafish, tmem2 mutants display an expansion of AVC characteristics, but the molecular mechanism of Tmem2 function in this context remains unclear. Through structure-function analysis, we demonstrate that the extracellular portion of Tmem2 is crucial for its role in restricting AVC boundaries. Importantly, the Tmem2 ectodomain contains regions implicated in the depolymerization of hyaluronic acid (HA). We find that tmem2 mutant hearts exhibit excess HA deposition alongside broadened distribution of Wnt signaling. Moreover, addition of ectopic hyaluronidase can restore the restriction of AVC differentiation in tmem2 mutants. Finally, we show that alteration of a residue important for HA depolymerization impairs the efficacy of Tmem2 function during AVC development. CONCLUSIONS: Taken together, our data support a model in which HA degradation, regulated by Tmem2, limits the distribution of Wnt signaling and thereby confines the differentiation of the AVC.

Tmem2 regulates cell-matrix interactions that are essential for muscle fiber attachment.

Skeletal muscle morphogenesis depends upon interactions between developing muscle fibers and the extracellular matrix (ECM) that anchors fibers to the myotendinous junction (MTJ). The pathways that organize the ECM and regulate its engagement by cell-matrix adhesion complexes (CMACs) are therefore essential for muscle integrity. Here, we demonstrate the impact of transmembrane protein 2 (tmem2) on cell-matrix interactions during muscle morphogenesis in zebrafish. Maternal-zygotic tmem2 mutants (MZtmem2) exhibit muscle fiber detachment, in association with impaired laminin organization and ineffective fibronectin degradation at the MTJ. Similarly, disorganized laminin and fibronectin surround MZtmem2 cardiomyocytes, which could account for their hindered movement during cardiac morphogenesis. In addition to ECM defects, MZtmem2 mutants display hypoglycosylation of α-dystroglycan within the CMAC, which could contribute to the observed fiber detachment. Expression of the Tmem2 ectodomain can rescue aspects of the MZtmem2 phenotype, consistent with a possible extracellular function of Tmem2. Together, our results suggest that Tmem2 regulates cell-matrix interactions by affecting both ECM organization and CMAC activity. These findings evoke possible connections between the functions of Tmem2 and the etiologies of congenital muscular dystrophies, particularly dystroglycanopathies.

Cardiac outflow morphogenesis depends on effects of retinoic acid signaling on multiple cell lineages.

BACKGROUND: Retinoic acid (RA), the bioactive derivative of vitamin A, is essential for vertebrate heart development. Both excess and reduced RA signaling lead to cardiovascular malformations affecting the outflow tract (OFT). To address the cellular mechanisms underlying the effects of RA signaling during OFT morphogenesis, we used transient maternal RA supplementation to rescue the early lethality resulting from inactivation of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene. RESULTS: By embryonic day 13.5, all rescued Raldh2(-/-) hearts exhibit severe, reproducible OFT septation defects, although wild-type and Raldh2(+/-) littermates have normal hearts. Cardiac neural crest cells (cNCC) were present in OFT cushions of Raldh2(-/-) mutant embryos but ectopically located in the periphery of the endocardial cushions, rather than immediately underlying the endocardium. Excess mesenchyme was generated by Raldh2(-/-) mutant endocardium, which displaced cNCC derivatives from their subendocardial, medial position. CONCLUSIONS: RA signaling affects not only cNCC numbers but also their position relative to endocardial mesenchyme during the septation process. Our study shows that inappropriate coordination between the different cell types of the OFT perturbs its morphogenesis and leads to a severe congenital heart defect, persistent truncus arteriosus.

[Potential of the zebrafish model to study congenital muscular dystrophies]. / Le poisson zèbre - Un modèle d'étude des dystrophies musculaires congénitales.

In order to better understand the complexity of congenital muscular dystrophies (CMD) and develop new strategies to cure them, it is important to establish new disease models. Due to its numerous helpful attributes, the zebrafish has recently become a very powerful animal model for the study of CMD. For some CMD, this vertebrate model is phenotypically closer to human pathology than the murine model. Over the last few years, researchers have developed innovative techniques to screen rapidly and on a large scale for muscle defects in zebrafish. Furthermore, new genome editing techniques in zebrafish make possible the identification of new disease models. In this review, the major attributes of zebrafish for CMD studies are discussed and the principal models of CMD in zebrafish are highlighted.

The novel transmembrane protein Tmem2 is essential for coordination of myocardial and endocardial morphogenesis.

Coordination between adjacent tissues plays a crucial role during the morphogenesis of developing organs. In the embryonic heart, two tissues - the myocardium and the endocardium - are closely juxtaposed throughout their development. Myocardial and endocardial cells originate in neighboring regions of the lateral mesoderm, migrate medially in a synchronized fashion, collaborate to create concentric layers of the heart tube, and communicate during formation of the atrioventricular canal. Here, we identify a novel transmembrane protein, Tmem2, that has important functions during both myocardial and endocardial morphogenesis. We find that the zebrafish mutation frozen ventricle (frv) causes ectopic atrioventricular canal characteristics in the ventricular myocardium and endocardium, indicating a role of frv in the regional restriction of atrioventricular canal differentiation. Furthermore, in maternal-zygotic frv mutants, both myocardial and endocardial cells fail to move to the midline normally, indicating that frv facilitates cardiac fusion. Positional cloning reveals that the frv locus encodes Tmem2, a predicted type II single-pass transmembrane protein. Homologs of Tmem2 are present in all examined vertebrate genomes, but nothing is known about its molecular or cellular function in any context. By employing transgenes to drive tissue-specific expression of tmem2, we find that Tmem2 can function in the endocardium to repress atrioventricular differentiation within the ventricle. Additionally, Tmem2 can function in the myocardium to promote the medial movement of both myocardial and endocardial cells. Together, our data reveal that Tmem2 is an essential mediator of myocardium-endocardium coordination during cardiac morphogenesis.

Hox genes define distinct progenitor sub-domains within the second heart field.

Much of the heart, including the atria, right ventricle and outflow tract (OFT) is derived from a progenitor cell population termed the second heart field (SHF) that contributes progressively to the embryonic heart during cardiac looping. Several studies have revealed anterior-posterior patterning of the SHF, since the anterior region (anterior heart field) contributes to right ventricular and OFT myocardium whereas the posterior region gives rise to the atria. We have previously shown that Retinoic Acid (RA) signal participates to this patterning. We now show that Hoxb1, Hoxa1, and Hoxa3, as downstream RA targets, are expressed in distinct sub-domains within the SHF. Our genetic lineage tracing analysis revealed that Hoxb1, Hoxa1 and Hoxa3-expressing cardiac progenitor cells contribute to both atria and the inferior wall of the OFT, which subsequently gives rise to myocardium at the base of pulmonary trunk. By contrast to Hoxb1(Cre), the contribution of Hoxa1-enhIII-Cre and Hoxa3(Cre)-labeled cells is restricted to the distal regions of the OFT suggesting that proximo-distal patterning of the OFT is related to SHF sub-domains characterized by combinatorial Hox genes expression. Manipulation of RA signaling pathways showed that RA is required for the correct deployment of Hox-expressing SHF cells. This report provides new insights into the regulatory gene network in SHF cells contributing to the atria and sub-pulmonary myocardium.

Endogenous retinoic acid regulates cardiac progenitor differentiation.

Retinoic acid (RA) has several established functions during cardiac development, including actions in the fetal epicardium required for myocardial growth. An open question is if retinoid effects are limited to growth factor stimulation pathway(s) or if additional actions on uncommitted progenitor/stem populations might drive cardiac differentiation. Here we report the dual effects of RA deficiency on cardiac growth factor signaling and progenitor/stem biology using the mouse retinaldehyde dehydrogenase 2 (Raldh2) knockout model. Although early heart defects in Raldh2(-/-) embryos result from second-heart-field abnormalities, it is unclear whether this role is transient or whether RA has sustained effects on cardiac progenitors. To address this, we used transient maternal RA supplementation to overcome early Raldh2(-/-) lethality. By embryonic day 11.5-14.5, Raldh2(-/-) hearts exhibited reduced venticular compact layer outgrowth and altered coronary vessel development. Although reductions in Fgf2 and target pERK levels occurred, no alterations in Wnt/beta-catenin expression were observed. Cell proliferation is increased in compact zone myocardium, whereas cardiomyocyte differentiation is reduced, alterations that suggest progenitor defects. We report that the fetal heart contains a reservoir of stem/progenitor cells, which can be isolated by their ability to efflux a fluorescent dye and that retinoid signaling acts on this fetal cardiac side population (SP). Raldh2(-/-) hearts display increased SP cell numbers, with selective increases in expression of cardiac progenitor cell markers and reduced differentiation marker levels. Hence, although lack of RA signaling increases cardiac SP numbers, simultaneous reductions in Fgf signaling reduce cardiomyocyte differentiation, possibly accounting for long-term defects in myocardial growth.

Decreased levels of embryonic retinoic acid synthesis accelerate recovery from arterial growth delay in a mouse model of DiGeorge syndrome.

RATIONALE: Loss of Tbx1 and decrease of retinoic acid (RA) synthesis result in DiGeorge/velocardiofacial syndrome (DGS/VCFS)-like phenotypes in mouse models, including defects in septation of the outflow tract of the heart and anomalies of pharyngeal arch-derived structures including arteries of the head and neck, laryngeal-tracheal cartilage, and thymus/parathyroid. Wild-type levels of T-box transcription factor (Tbx)1 and RA signaling are required for normal pharyngeal arch artery development. Recent studies have shown that reduction of RA or loss of Tbx1 alters the contribution of second heart field (SHF) progenitor cells to the elongating heart tube. OBJECTIVE: Here we tested whether Tbx1 and the RA signaling pathway interact during the deployment of the SHF and formation of the mature aortic arch. METHODS AND RESULTS: Molecular markers of the SHF, neural crest and smooth muscle cells, were analyzed in Raldh2;Tbx1 compound heterozygous mutants. Our results revealed that the SHF and outflow tract develop normally in Raldh2(+/-);Tbx1(+/-) embryos. However, we found that decreased levels of RA accelerate the recovery from arterial growth delay observed in Tbx1(+/-) mutant embryos. This compensation coincides with the differentiation of smooth muscle cells in the 4th pharyngeal arch arteries, and is associated with severity of neural crest cell migration defects observed in these mutants. CONCLUSIONS: Our data suggest that differences in levels of embryonic RA may contribute to the variability in great artery anomalies observed in DGS/VCFS patients.

Retinoic acid deficiency alters second heart field formation.

Retinoic acid (RA), the active derivative of vitamin A, has been implicated in various steps of cardiovascular development. The retinaldehyde dehydrogenase 2 (RALDH2) enzyme catalyzes the second oxidative step in RA biosynthesis and its loss of function creates a severe embryonic RA deficiency. Raldh2(-/-) knockout embryos fail to undergo heart looping and have impaired atrial and sinus venosus development. To understand the mechanism(s) producing these changes, we examined the contribution of the second heart field (SHF) to pharyngeal mesoderm, atria, and outflow tract in Raldh2(-/-) embryos. RA deficiency alters SHF gene expression in two ways. First, Raldh2(-/-) embryos exhibited a posterior expansion of anterior markers of the SHF, including Tbx1, Fgf8, and the Mlc1v-nlacZ-24/Fgf10 reporter transgene as well as of Islet1. This occurred at early somite stages, when cardiac defects became irreversible in an avian vitamin A-deficiency model, indicating that endogenous RA is required to restrict the SHF posteriorly. Explant studies showed that this expanded progenitor population cannot differentiate properly. Second, RA up-regulated cardiac Bmp expression levels at the looping stage. The contribution of the SHF to both inflow and outflow poles was perturbed under RA deficiency, creating a disorganization of the heart tube. We also investigated genetic cross-talk between Nkx2.5 and RA signaling by generating double mutant mice. Strikingly, Nkx2.5 deficiency was able to rescue molecular defects in the posterior region of the Raldh2(-/-) mutant heart, in a gene dosage-dependent manner.