GATA4/5/6 family transcription factors are conserved determinants of cardiac versus pharyngeal mesoderm fate.

GATA4/5/6 transcription factors play essential, conserved roles in heart development. To understand how GATA4/5/6 modulates the mesoderm-to-cardiac fate transition, we labeled, isolated, and performed single-cell gene expression analysis on cells that express gata5 at precardiac time points spanning zebrafish gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamic gata5/6 expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was substantially altered. In addition to the expected absence of cardiac mesoderm, we confirmed a concomitant expansion of cranial-pharyngeal mesoderm. Moreover, Gata5/6 loss led to extensive changes in chromatin accessibility near cardiac and pharyngeal genes. Functional analyses in zebrafish and the tunicate Ciona, which has a single GATA4/5/6 homolog, revealed that GATA4/5/6 acts upstream of tbx1 to exert essential and cell-autonomous roles in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, cardiac and pharyngeal mesoderm fate choices are achieved through an evolutionarily conserved GATA4/5/6 regulatory network.

Altered VEGF Signaling Leads to Defects in Heart Tube Elongation and Omphalomesenteric Vein Fusion in Quail Embryos.

Formation of the endocardial and myocardial heart tubes involves precise cardiac progenitor sorting and tissue displacements from the primary heart field to the embryonic midline-a process that is dependent on proper formation of conjoining great vessels, including the omphalomesenteric veins (OVs) and dorsal aortae. Using a combination of vascular endothelial growth factor (VEGF) over- and under-activation, fluorescence labeling of cardiac progenitors (endocardial and myocardial), and time-lapse imaging, we show that altering VEGF signaling results in previously unreported myocardial, in addition to vascular and endocardial phenotypes. Resultant data show: (1) exogenous VEGF leads to truncated endocardial and myocardial heart tubes and grossly dilated OVs; (2) decreased levels of VEGF receptor 2 tyrosine kinase signaling result in a severe abrogation of the endocardial tube, dorsal aortae, and OVs. Surprisingly, only slightly altered myocardial tube fusion and morphogenesis is observed. We conclude that VEGF has direct effects on the VEGF receptor 2-bearing endocardial and endothelial precursors, and that altered vascular morphology of the OVs also indirectly results in altered myocardial tube formation. Anat Rec, 302:175-185, 2019. © 2018 Wiley Periodicals, Inc.

Mespaa can potently induce cardiac fates in zebrafish.

The Mesp family of transcription factors have been implicated in the early formation and migration of the cardiac lineage, although the precise molecular mechanisms underlying this process remain unknown. In this study we examine the function of Mesp family members in zebrafish cardiac development and find that Mespaa is remarkably efficient at promoting cardiac fates in normally non-cardiogenic cells. However, Mespaa is dispensable for normal cardiac formation. Despite no overt defects in cardiovascular specification, we find a consistent defect in cardiac laterality in mespaa null embryos. This is further exacerbated by the depletion of other mesp paralogues, highlighting a conserved role for the mesp family in left-right asymmetry, distinct from a function in cardiac specification. Despite an early requirement for mespaa to promote cardiogenesis, cells over-expressing mespaa are found to both exhibit unique cellular behaviors and activate the transcription of gata5 only after the completion of gastrulation. We propose that while mespaa remains capable of driving cardiac progenitor formation in zebrafish, it may not play an essential role in the cardiac regulatory network. Furthermore, the late activation of migration and cardiac gene transcription in mespaa over-expressing cells challenges previous studies on the timing of these events and provides intriguing questions for future study.

The endoderm and myocardium join forces to drive early heart tube assembly.

Formation of the muscular layer of the heart, the myocardium, involves the medial movement of bilateral progenitor fields; driven primarily by shortening of the endoderm during foregut formation. Using a combination of time-lapse imaging, microsurgical perturbations and computational modeling, we show that the speed of the medial-ward movement of the myocardial progenitors is similar, but not identical to that of the adjacent endoderm. Further, the extracellular matrix microenvironment separating the two germ layers also moves with the myocardium, indicating that collective tissue motion and not cell migration drives tubular heart assembly. Importantly, as myocardial cells approach the midline, they perform distinct anterior-directed movements relative to the endoderm. Based on the analysis of microincision experiments and computational models, we propose two characteristic, autonomous morphogenetic activities within the early myocardium: 1) an active contraction of the medial portion of the heart field and 2) curling- the tendency of the unconstrained myocardial tissue to form a spherical surface with a concave ventral side. In the intact embryo, these deformations are constrained by the endoderm and the adjacent mesoderm, nevertheless the corresponding mechanical stresses contribute to the proper positioning of myocardial primordia.

Active cell and ECM movements during development.

Dynamic imaging of the extracellular matrix (ECM) and cells can reveal how tissues are formed. Displacement differences between cells and the adjacent ECM scaffold can be used to establish active movements of mesenchymal cells. Cells can also generate large-scale tissue movements in which cell and ECM displacements are shared. We describe computational methods for analyzing multi-spectral time-lapse image sequences. The resulting data can distinguish between local "active" cellular motion versus large-scale, tissue movements, both of which occur during organogenesis. The movement data also provide the basis for construction of realistic biomechanical models and computer simulations of in vivo tissue formation.

Convective tissue movements play a major role in avian endocardial morphogenesis.

Endocardial cells play a critical role in cardiac development and function, forming the innermost layer of the early (tubular) heart, separated from the myocardium by extracellular matrix (ECM). However, knowledge is limited regarding the interactions of cardiac progenitors and surrounding ECM during dramatic tissue rearrangements and concomitant cellular repositioning events that underlie endocardial morphogenesis. By analyzing the movements of immunolabeled ECM components (fibronectin, fibrillin-2) and TIE1 positive endocardial progenitors in time-lapse recordings of quail embryonic development, we demonstrate that the transformation of the primary heart field within the anterior lateral plate mesoderm (LPM) into a tubular heart involves the precise co-movement of primordial endocardial cells with the surrounding ECM. Thus, the ECM of the tubular heart contains filaments that were associated with the anterior LPM at earlier developmental stages. Moreover, endocardial cells exhibit surprisingly little directed active motility, that is, sustained directed movements relative to the surrounding ECM microenvironment. These findings point to the importance of large-scale tissue movements that convect cells to the appropriate positions during cardiac organogenesis.

An N- and C-terminal truncated isoform of zinc finger X-linked duplicated C protein represses MHC class II transcription.

The zinc finger X-linked duplicated A (ZXDA) and ZXDC proteins are both required for robust transcription of major histocompatibility complex class II (MHC II) genes. Aside from the full length ZXDC mRNA transcript, at least one additional mRNA is produced by the ZXDC gene, in which transcription initiates within the first exon and terminates within the seventh intron. The protein product produced from this transcript, which we have named ZXDC2, is truncated on both the N- and C-terminus. We demonstrate here that ZXDC2 functions to repress MHC II transcription induced in HeLa cells treated with IFN-gamma. We further demonstrate that ZXDC2 interacts with both ZXDA and ZXDC, suggesting a mechanism by which ZXDC2 may inhibit MHC II transcription. These studies not only provide additional support for the role of ZXD proteins in regulating MHC II transcription, but also demonstrate a unique mechanism for the synthesis of a mRNA isoform.

The zinc finger proteins ZXDA and ZXDC form a complex that binds CIITA and regulates MHC II gene transcription.

The transcription of major histocompatibility complex class II (MHC II) genes depends critically upon the activity of the class II trans-activator (CIITA) protein. We previously described a novel CIITA-binding protein named zinc finger X-linked duplicated family member C (ZXDC) that contributes to the activity of CIITA and the transcription of MHC II genes. Here, we examined the contribution of a closely related family member of ZXDC, the ZXDA protein, to MHC II gene transcription. ZXDA has a domain organization similar to ZXDC, containing ten zinc fingers and a transcriptional activation domain. Knockdown and overexpression of ZXDA demonstrated its importance in the transcriptional activation of MHC II genes. We found that ZXDA and ZXDC can self-associate, and also form a complex with each other. The regions of the two proteins that contain zinc fingers mediate these interactions. Importantly, we found that the ZXDA-ZXDC complex interacts with CIITA, rather than either protein alone. Given our additional finding that ZXDC is present at MHC II promoters in HeLa cells, prior to and after treatment with IFN-gamma, it appears that ZXDA and ZXDC form an important regulatory complex for MHC II gene transcription.