Interactions between Cdx genes and retinoic acid modulate early cardiogenesis.
ABSTRACT: Cdx transcription factors regulate embryonic positional identities and have crucial roles in anteroposterior patterning (AP) processes of all three germ layers. Previously we have shown that the zebrafish homologues cdx1a and cdx4 redundantly regulate posterior mesodermal derivatives inducing embryonic blood cell fate specification and patterning of the embryonic kidney. Here we hypothesize that cdx factors restrict formation of anterior mesodermal derivatives such as cardiac cells by imposing posterior identity to developing mesodermal cells. We show that ectopic expression of Cdx1 or Cdx4 applied during the brief window of mesoderm patterning in differentiating murine embryonic stem cell (ESC) strongly suppresses cardiac development as assayed by expression of cardiac genes and formation of embryoid bodies (EB) containing "beating" cell clusters. Conversely, in loss-of-function studies performed in cdx-deficient zebrafish embryos, we observed a dose-dependent expansion of tbx5a(+) anterior-lateral plate mesoderm giving rise to cardiac progenitors. However, further cardiac development of these mesodermal cells required additional suppression of the retinoic acid (RA) pathway, possibly due to differential activity of inhibitory RA signals in cdx mutants. Together, our data suggest that cdx proteins affect cardiogenesis by regulating the formation of cardiogenic mesoderm and together with the RA pathway control the early development of cardiac precursor cells.
Project description:Hematopoietic development during embryogenesis involves the interaction of extrinsic signaling pathways coupled to an intrinsic cell fate that is regulated by cell-specific transcription factors. Retinoic acid (RA) has been linked to stem cell self-renewal in adults and also participates in yolk sac blood island formation. Here, we demonstrate that RA decreases gata1 expression and blocks primitive hematopoiesis in zebrafish (Danio rerio) embryos, while increasing expression of the vascular marker, fli1. Treatment with an inhibitor of RA biosynthesis or a retinoic acid receptor antagonist increases gata1(+) erythroid progenitors in the posterior mesoderm of wild-type embryos and anemic cdx4(-/-) mutants, indicating a link between the cdx-hox signaling pathway and RA. Overexpression of scl, a DNA binding protein necessary for hematopoietic development, rescues the block of hematopoiesis induced by RA. We show that these effects of RA and RA pathway inhibitors are conserved during primitive hematopoiesis in murine yolk sac explant cultures and embryonic stem cell assays. Taken together, these data indicate that RA inhibits the commitment of mesodermal cells to hematopoietic fates, functioning downstream of cdx4 and upstream of scl. Our studies establish a new connection between RA and scl during development that may participate in stem cell self-renewal and hematopoietic differentiation.
Project description:Cdx genes (Cdx1, Cdx2, and Cdx4) encode a family of caudal-related transcription factors that mediate anterior-posterior patterning during embryogenesis through Hox gene regulation. Homologues in the zebrafish have been shown to play key roles in blood formation. To define the role of Cdx genes during embryonic hematopoiesis in a mammalian system, we examined the hematopoietic potential of Cdx-deficient mouse embryonic stem cells (ESCs) in vitro and in vivo. Individual Cdx-deficient ESCs exhibited impaired embryonic hematopoietic progenitor formation and altered Hox gene expression, most notably for Cdx2 deficiency. A more severe hematopoietic defect was observed with compound Cdx deficiency than loss of function of any single Cdx gene. Reduced hematopoietic progenitor formation of ESCs deficient in multiple Cdx genes could be rescued by ectopic expression of Cdx4, concomitant with partially restored Hox gene expression. These results reveal an essential and partially redundant role for multiple Cdx genes during embryonic hematopoiesis in the mouse.
Project description:Cdx1, Cdx2, and Cdx4 comprise the caudal-like Cdx gene family in mammals, whose homologues regulate hematopoietic development in zebrafish. Previously, we reported that overexpression of Cdx4 enhances hematopoietic potential from murine embryonic stem cells (ESCs). Here we compare the effect of ectopic Cdx1, Cdx2, and Cdx4 on the differentiation of murine ESC-derived hematopoietic progenitors. The 3 Cdx genes differentially influence the formation and differentiation of hematopoietic progenitors within a CD41(+)c-kit(+) population of embryoid body (EB)-derived cells. Cdx1 and Cdx4 enhance, whereas Cdx2 strongly inhibits, the hematopoietic potential of CD41(+)ckit(+) EB-derived cells, changes that are reflected by effects on hematopoietic lineage-specific and Hox gene expression. When we subject stromal cell and colony assay cultures of EB-derived hematopoietic progenitors to ectopic expression of Cdx genes, Cdx4 dramatically enhances, whereas Cdx1 and Cdx2 both inhibit hematopoietic activity, probably by blocking progenitor differentiation. These data demonstrate distinct effects of Cdx genes on hematopoietic progenitor formation and differentiation, insights that we are using to facilitate efforts at in vitro culture of hematopoietic progenitors from ESC. The behavior of Cdx genes in vitro suggests how derangement of these developmental regulators might contribute to leukemogenesis.
Project description:Despite the fundamental importance of patterning along the dorsal-ventral (DV) and anterior-posterior (AP) axes during embryogenesis, uncertainty exists in the orientation of these axes for the mesoderm. Here we examine the origin and formation of the zebrafish kidney, a ventrolateral mesoderm derivative, and show that AP patterning of the non-axial mesoderm occurs across the classic gastrula stage DV axis while DV patterning aligns along the animal-vegetal pole. We find that BMP signalling acts early to establish broad anterior and posterior territories in the non-axial mesoderm while retinoic acid (RA) functions later, but also across the classic DV axis. Our data support a model in which RA on the dorsal side of the embryo induces anterior kidney fates while posterior kidney progenitors are protected ventrally by the RA-catabolizing enzyme Cyp26a1. This work clarifies our understanding of vertebrate axis orientation and establishes a new paradigm for how the kidney and other mesodermal derivatives arise during embryogenesis.
Project description:Deletion of caudal/cdx genes alters hox gene expression and causes defects in posterior tissues and hematopoiesis. Yet, the defects in hox gene expression only partially explain these phenotypes. To gain deeper insight into Cdx4 function, we performed chromatin immunoprecipitation sequencing (ChIP-seq) combined with gene-expression profiling in zebrafish, and identified the transcription factor spalt-like 4 (sall4) as a Cdx4 target. ChIP-seq revealed that Sall4 bound to its own gene locus and the cdx4 locus. Expression profiling showed that Cdx4 and Sall4 coregulate genes that initiate hematopoiesis, such as hox, scl, and lmo2. Combined cdx4/sall4 gene knockdown impaired erythropoiesis, and overexpression of the Cdx4 and Sall4 target genes scl and lmo2 together rescued the erythroid program. These findings suggest that auto- and cross-regulation of Cdx4 and Sall4 establish a stable molecular circuit in the mesoderm that facilitates the activation of the blood-specific program as development proceeds.
Project description:The spinal cord is a unique vertebrate feature that originates, together with the hindbrain, from the caudal neural plate. Whereas the hindbrain subdivides into rhombomeres, the spinal cord remains unsegmented. We have identified Cdx transcription factors as key determinants of the spinal cord region in zebrafish. Loss of Cdx1a and Cdx4 functions causes posterior expansion of the hindbrain at the expense of the unsegmented spinal cord. By contrast, cdx4 overexpression in the hindbrain impairs rhombomere segmentation and patterning and induces the expression of spinal cord-specific genes. Using cell transplantation, we demonstrate that Cdx factors function directly within the neural ectoderm to specify spinal cord. Overexpression of 5' Hox genes fails to rescue hindbrain and spinal cord defects associated with cdx1a/cdx4 loss-of-function, suggesting a Hox-independent mechanism of spinal cord specification. In the absence of Cdx function, the caudal neural plate retains hindbrain characteristics and remains responsive to surrounding signals, particularly retinoic acid, in a manner similar to the native hindbrain. We propose that by preventing the posterior-most region of the neural plate from following a hindbrain developmental program, Cdx factors help determine the size of the prospective hindbrain and spinal cord territories.
Project description:cdx4, a caudal-related homeodomain-containing transcription factor, functions as a regulator of hox genes, thereby playing a critical role in anterior-posterior (A-P) patterning during embryogenesis. In zebrafish, homozygous deletion of the cdx4 gene results in a mutant phenotype known as kugelig, with aberrant A-P patterning and severe anemia characterized by decreased gata1 expression in the posterior lateral mesoderm. To identify pathways that interact with cdx4 during primitive hematopoiesis, we conducted a chemical genetic screen in the cdx4 mutant background for compounds that increase gata1 expression in cdx4 mutants. Among 2640 compounds that were tested, we discovered two compounds that rescued gata1 expression in the cdx4-mutant embryos. The strongest rescue was observed with bergapten, a psoralen compound found in bergamont oil. Another member of the psoralen family, 8-methoxypsoralen, was also found to rescue gata1 expression in cdx4-mutant embryos. The psoralen compounds also disrupted normal A-P patterning of embryos. These compounds modify the cdx4-mutant phenotype and will help elucidate signaling pathways that act downstream or parallel to the cdx4-hox pathway.
Project description:TBX5 is essential for limb and heart development. Mutations in TBX5 are associated with Holt-Oram syndrome in humans. Due to the teleost specific genome duplication, zebrafish have two copies of TBX5: tbx5a and tbx5b. Both of these genes are expressed in regions of the lateral plate mesoderm and retina. In this study, we perform comparative RNA sequencing analysis on zebrafish embryos during the stages of lateral plate mesoderm migration. This work shows that knockdown of the Tbx5 paralogues results in altered gene expression in many tissues outside of the lateral plate mesoderm, especially in the somitic mesoderm and the intermediate mesoderm. Specifically, knockdown of tbx5b results in changes in somite size, in the differentiation of vasculature progenitors and in later patterning of trunk blood vessels.
Project description:Despite two decades of studies documenting the in vitro blood-forming potential of murine embryonic stem cells (ESCs), achieving stable long-term blood engraftment of ESC-derived hematopoietic stem cells in irradiated mice has proven difficult. We have exploited the Cdx-Hox pathway, a genetic program important for blood development, to enhance the differentiation of ESCs along the hematopoietic lineage. Using an embryonic stem cell line engineered with tetracycline-inducible Cdx4, we demonstrate that ectopic Cdx4 expression promotes hematopoietic mesoderm specification, increases hematopoietic progenitor formation, and, together with HoxB4, enhances multilineage hematopoietic engraftment of lethally irradiated adult mice. Clonal analysis of retroviral integration sites confirms a common stem cell origin of lymphoid and myeloid populations in engrafted primary and secondary mice. These data document the cardinal stem cell features of self-renewal and multilineage differentiation of ESC-derived hematopoietic stem cells.
Project description:Kidney function depends on the nephron, which comprises a blood filter, a tubule that is subdivided into functionally distinct segments, and a collecting duct. How these regions arise during development is poorly understood. The zebrafish pronephros consists of two linear nephrons that develop from the intermediate mesoderm along the length of the trunk. Here we show that, contrary to current dogma, these nephrons possess multiple proximal and distal tubule domains that resemble the organization of the mammalian nephron. We examined whether pronephric segmentation is mediated by retinoic acid (RA) and the caudal (cdx) transcription factors, which are known regulators of segmental identity during development. Inhibition of RA signaling resulted in a loss of the proximal segments and an expansion of the distal segments, while exogenous RA treatment induced proximal segment fates at the expense of distal fates. Loss of cdx function caused abrogation of distal segments, a posterior shift in the position of the pronephros, and alterations in the expression boundaries of raldh2 and cyp26a1, which encode enzymes that synthesize and degrade RA, respectively. These results suggest that the cdx genes act to localize the activity of RA along the axis, thereby determining where the pronephros forms. Consistent with this, the pronephric-positioning defect and the loss of distal tubule fate were rescued in embryos doubly-deficient for cdx and RA. These findings reveal a novel link between the RA and cdx pathways and provide a model for how pronephric nephrons are segmented and positioned along the embryonic axis.