RESUMEN
Wnt signaling is a crucial developmental pathway involved in early development as well as stem-cell maintenance in adults and its misregulation leads to numerous diseases. Thus, understanding the regulation of this pathway becomes vitally important. Axin2 and Nkd1 are widely utilized negative feedback regulators in Wnt signaling where Axin2 functions to destabilize cytoplasmic ß-catenin, and Nkd1 functions to inhibit the nuclear localization of ß-catenin. Here, we set out to further understand how Axin2 and Nkd1 regulate Wnt signaling by creating axin2gh1/gh1, nkd1gh2/gh2 single mutants and axin2gh1/gh1;nkd1gh2/gh2 double mutant zebrafish using sgRNA/Cas9. All three Wnt regulator mutants were viable and had impaired heart looping, neuromast migration defects, and behavior abnormalities in common, but there were no signs of synergy in the axin2gh1/gh1;nkd1gh2/gh2 double mutants. Further, Wnt target gene expression by qRT-PCR and RNA-seq, and protein expression by mass spectrometry demonstrated that the double axin2gh1/gh1;nkd1gh2/gh2 mutant resembled the nkd1gh2/gh2 phenotype demonstrating that Nkd1 functions downstream of Axin2. In support of this, the data further demonstrates that Axin2 uniquely alters the properties of ß-catenin-dependent transcription having novel readouts of Wnt activity compared with nkd1gh2/gh2 or the axin2gh1/gh1;nkd1gh2/gh2 double mutant. We also investigated the sensitivity of the Wnt regulator mutants to exacerbated Wnt signaling, where the single mutants displayed characteristic heightened Wnt sensitivity, resulting in an eyeless phenotype. Surprisingly, this phenotype was rescued in the double mutant, where we speculate that cross-talk between Wnt/ß-catenin and Wnt/Planar Cell Polarity pathways could lead to altered Wnt signaling in some scenarios. Collectively, the data emphasizes both the commonality and the complexity in the feedback regulation of Wnt signaling.
Asunto(s)
Proteína Axina , Vía de Señalización Wnt , Proteínas de Pez Cebra , Pez Cebra , beta Catenina , Animales , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteína Axina/metabolismo , Proteína Axina/genética , beta Catenina/metabolismo , Proteínas Portadoras , Mutación/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
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.
Asunto(s)
Factor de Transcripción GATA4 , Pez Cebra , Animales , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Factor de Transcripción GATA5/genética , Factor de Transcripción GATA5/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
The question of how the heart develops, and the genetic networks governing this process have become intense areas of research over the past several decades. This research is propelled by classical developmental studies and potential clinical applications to understand and treat congenital conditions in which cardiac development is disrupted. Discovery of the tinman gene in Drosophila, and examination of its vertebrate homolog Nkx2.5, along with other core cardiac transcription factors has revealed how cardiac progenitor differentiation and maturation drives heart development. Careful observation of cardiac morphogenesis along with lineage tracing approaches indicated that cardiac progenitors can be divided into two broad classes of cells, namely the first and second heart fields, that contribute to the heart in two distinct waves of differentiation. Ample evidence suggests that the fate of individual cardiac progenitors is restricted to distinct cardiac structures quite early in development, well before the expression of canonical cardiac progenitor markers like Nkx2.5. Here we review the initial specification of cardiac progenitors, discuss evidence for the early patterning of cardiac progenitors during gastrulation, and consider how early gene expression programs and epigenetic patterns can direct their development. A complete understanding of when and how the developmental potential of cardiac progenitors is determined, and their potential plasticity, is of great interest developmentally and also has important implications for both the study of congenital heart disease and therapeutic approaches based on cardiac stem cell programming.