Bi-potential neuromesodermal progenitors (NMPs) produce both neural and paraxial mesodermal progenitors in the trunk and tail during vertebrate body elongation. We show that Sall4, a pluripotency-related transcription factor gene, has multiple roles in regulating NMPs and their descendants in post-gastrulation mouse embryos. Sall4 deletion using TCre caused body/tail truncation, reminiscent of early depletion of NMPs, suggesting a role of Sall4 in NMP maintenance. This phenotype became significant at the time of the trunk-to-tail transition, suggesting that Sall4 maintenance of NMPs enables tail formation. Sall4 mutants exhibit expanded neural and reduced mesodermal tissues, indicating a role of Sall4 in NMP differentiation balance. Mechanistically, we show that Sall4 promotion of WNT/β-catenin signaling contributes to NMP maintenance and differentiation balance. RNA-Seq and SALL4 ChIP-Seq analyses support the notion that Sall4 regulates both mesodermal and neural development. Furthermore, in the mesodermal compartment, genes regulating presomitic mesoderm differentiation are downregulated in Sall4 mutants. In the neural compartment, we show that differentiation of NMPs towards post-mitotic neuron is accelerated in Sall4 mutants. Our results collectively provide evidence supporting the role of Sall4 in regulating NMPs and their descendants.

Zebrafish possess the remarkable ability to regenerate injured hearts as adults, which contrasts the very limited ability in mammals. Although very limited, mammalian hearts do in fact have measurable levels of cardiomyocyte regeneration. Therefore, elucidating mechanisms of zebrafish heart regeneration would provide information of naturally occurring regeneration to potentially apply to mammalian studies, in addition to addressing this biologically interesting phenomenon in itself. Studies over the past 13 years have identified processes and mechanisms of heart regeneration in zebrafish. After heart injury, preexisting cardiomyocytes dedifferentiate, enter the cell cycle, and repair the injured myocardium. This process requires interaction with epicardial cells, endocardial cells, and vascular endothelial cells. Epicardial cells envelope the heart, while endocardial cells make up the inner lining of the heart. They provide paracrine signals to cardiomyocytes to regenerate the injured myocardium, which is vascularized during heart regeneration. In addition, accumulating results suggest that local migration of these major cardiac cell types have roles in heart regeneration. In this review, we summarize the characteristics of various heart injury methods used in the research community and regeneration of the major cardiac cell types. Then, we discuss local migration of these cardiac cell types and immune cells during heart regeneration.

Mechanisms of haematopoietic and cardiac patterning remain poorly understood. Here we show that the BMP and Wnt signalling pathways are integrated in an endoglin (Eng)-dependent manner in cardiac and haematopoietic lineage specification. Eng is expressed in early mesoderm and marks both haematopoietic and cardiac progenitors. In the absence of Eng, yolk sacs inappropriately express the cardiac marker, Nkx2.5. Conversely, high levels of Eng in vitro and in vivo increase haematopoiesis and inhibit cardiogenesis. Levels of Eng determine the activation of both BMP and Wnt pathways, which are integrated downstream of Eng by phosphorylation of Smad1 by Gsk3. By interrogating Eng-dependent Wnt-mediated transcriptional changes, we identify Jdp2 as a key Eng-dependent Wnt target, sufficient to establish haematopoietic fate in early mesoderm when BMP and Wnt crosstalk is disturbed. These studies provide mechanistic insight into the integration of BMP and Wnt signalling in the establishment of haematopoietic and cardiac progenitors during embryogenesis.

Mutations in the SALL4 gene cause human syndromes with defects in multiple organs. Sall4 expression declines rapidly in post-gastrulation mouse embryos, and our understanding of the requirement of Sall4 in animal development is still limited. To assess the contributions of Sall4 expressing cells to developing mouse embryos, we monitored temporal changes of the contribution of Sall4 lineages using a Sall4 GFP-CreERT2 knock-in mouse line and recombination-dependent reporter lines. By administering tamoxifen at various time points we observed that the contributions of Sall4 lineages to the axial level were rapidly restricted from the entire body to the posterior part of the body. The contribution to forelimbs, hindlimbs, craniofacial structures and external genitalia also declined after gastrulation with different temporal dynamics. We also detected Sall4 lineage contributions to the extra-embryonic tissues, such as the yolk sac and umbilical cord, in a temporal manner. These Sall4 lineage contributions provide insights into potential roles of Sall4 during mammalian embryonic development. In postnatal males, long-term lineage tracing detected Sall4 lineage contributions to the spermatogonial stem cell pool during spermatogenesis. The Sall4 GFP-CreERT2 line can serve as a tool to monitor spatial-temporal contributions of Sall4 lineages as well as to perform gene manipulations in Sall4-expressing lineages.

Gli3 is a major regulator of Hedgehog signaling during limb development. In the anterior mesenchyme, GLI3 is proteolytically processed into GLI3R, a truncated repressor form that inhibits Hedgehog signaling. Although numerous studies have identified mechanisms that regulate Gli3 function in vitro, it is not completely understood how Gli3 function is regulated in vivo. In this study, we show a novel mechanism of regulation of GLI3R activities in limb buds by Gata6, a member of the GATA transcription factor family. We show that conditional inactivation of Gata6 prior to limb outgrowth by the Tcre deleter causes preaxial polydactyly, the formation of an anterior extra digit, in hindlimbs. A recent study suggested that Gata6 represses Shh transcription in hindlimb buds. However, we found that ectopic Hedgehog signaling precedes ectopic Shh expression. In conjunction, we observed Gata6 and Gli3 genetically interact, and compound heterozygous mutants develop preaxial polydactyly without ectopic Shh expression, indicating an additional prior mechanism to prevent polydactyly. These results support the idea that Gata6 possesses dual roles during limb development: enhancement of Gli3 repressor function to repress Hedgehog signaling in the anterior limb bud, and negative regulation of Shh expression. Our in vitro and in vivo studies identified that GATA6 physically interacts with GLI3R to facilitate nuclear localization of GLI3R and repressor activities of GLI3R. Both the genetic and biochemical data elucidates a novel mechanism by Gata6 to regulate GLI3R activities in the anterior limb progenitor cells to prevent polydactyly and attain proper development of the mammalian autopod.