Combinatorial gene regulation by Bmp and Wnt in zebrafish posterior mesoderm formation

Combinatorial signaling is an important mechanism that allows the embryo to utilize overlapping signaling pathways to specify different territories. In zebrafish, the Wnt and Bmp pathways interact to regulate the formation of the posterior body. In order to understand how this works mechanistically, we have identified tbx6 as a posterior mesodermal gene activated by both of these signaling pathways. We isolated a genomic fragment from the tbx6 gene that recapitulates the endogenous tbx6 expression, and used this to ask how the Bmp and Wnt signaling pathways combine to regulate gene expression. We find that the tbx6 promoter utilizes distinct domains to integrate the signaling inputs from each pathway, including multiple Tcf/LEF sites and a novel Bmp-response element. Surprisingly, we found that overexpression of either signaling pathway can activate the tbx6 promoter and the endogenous gene, whereas inputs from both pathways are required for the normal pattern of expression. These results demonstrate that both Bmp and Wnt are present at submaximal levels, which allows the pathways to function combinatorially. We present a model in which overlapping Wnt and Bmp signals in the ventrolateral region activate the expression of tbx6 and other posterior mesodermal genes, leading to the formation of posterior structures.     

Transgenic zebrafish reveal stage-specific roles for Bmp signaling in ventral and posterior mesoderm development

Bone morphogenetic protein (Bmp) signaling is crucial for the formation and patterning of zebrafish ventral and posterior mesoderm. Mutants defective in the Bmp pathway have expanded trunk muscle, abnormal tails and severely impaired development of ventral mesodermal derivatives such as vasculature, blood and pronephros. As Bmps continue to be expressed in the ventral and posterior mesoderm after gastrulation, it is likely that Bmp signaling continues to play an important developmental role during outgrowth of the posterior body. However, because Bmp signaling plays an essential role during the gastrula stages, it has not been possible with mutants or standard disruption techniques to determine the later functions of the Bmp pathway. To study the role of Bmp signaling in the ventral and posterior mesoderm during trunk and tail outgrowth, we generated a transgenic zebrafish line containing a heatshock-inducible dominant-negative Bmp receptor-GFP fusion. Our data show that Bmps are important for tail organizer formation and for patterning the ventral mesoderm during early gastrulation. However, from mid-gastrulation to the early somitogenesis stages, Bmp signaling is important for ventral tail fin development and for preventing secondary tail formation. We conclude that the role of Bmp signaling in the ventral and posterior mesoderm changes as gastrulation proceeds.     

Interaction of Wnt and caudal-related genes in zebrafish posterior body formation

Although Wnt signaling plays an important role in body patterning during early vertebrate embryogenesis, the mechanisms by which Wnts control the individual processes of body patterning are largely unknown. In zebrafish, wnt3a and wnt8 are expressed in overlapping domains in the blastoderm margin and later in the tailbud. The combined inhibition of Wnt3a and Wnt8 by antisense morpholino oligonucleotides led to anteriorization of the neuroectoderm, expansion of the dorsal organizer, and loss of the posterior body structure-a more severe phenotype than with inhibition of each Wnt alone-indicating a redundant role for Wnt3a and Wnt8. The ventrally expressed homeobox genes vox, vent, and ved mediated Wnt3a/Wnt8 signaling to restrict the organizer domain. Of posterior body-formation genes, expression of the caudal-related cdx1a and cdx4/kugelig, but not bmps or cyclops, was strongly reduced in the wnt3a/wnt8 morphant embryos. Like the wnt3a/wnt8 morphant embryos, cdx1a/cdx4 morphant embryos displayed complete loss of the tail structure, suggesting that Cdx1a and Cdx4 mediate Wnt-dependent posterior body formation. We also found that cdx1a and cdx4 expression is dependent on Fgf signaling. hoxa9a and hoxb7a expression was down-regulated in the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos, and in embryos with defects in Fgf signaling. Fgf signaling was required for Cdx-mediated hoxa9a expression. Both the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos failed to promote somitogenesis during mid-segmentation. These data indicate that the cdx genes mediate Wnt signaling and play essential roles in the morphogenesis of the posterior body in zebrafish.     

Characterization of the retinoic acid receptor genes raraa, rarab and rarg during zebrafish development

Retinoic acid signaling is important for patterning the central nervous system, paired appendages, digestive tract, and other organs. To begin to investigate retinoic acid signaling in zebrafish, we determined orthologies between zebrafish and tetrapod retinoic acid receptors (Rars) and examined the expression patterns of rar genes during embryonic development. Analysis of phylogenies and conserved syntenies showed that the three cloned zebrafish rar genes include raraa and rarab, which are co-orthologs of tetrapod Rara, and rarg, which is the zebrafish ortholog of tetrapod Rarg. We did not, however, find an ortholog of Rarb. RNA in situ hybridization experiments showed that rarab and rarg, are maternally expressed. Zygotic expression of raraa occurs predominantly in the hindbrain, lateral mesoderm, and tailbud. Zygotic expression of rarab largely overlaps that of raraa, except that in later stages rarab is expressed more broadly in the brain and in the pectoral fin bud and pharyngeal arches. Zygotic expression of zebrafish rarg also overlaps the other two genes, but it is expressed more strongly in the posterior hindbrain beginning in late somitogenesis as well as in neural crest cells in the pharyngeal arches. Thus, these three genes have largely overlapping expression patterns and a few gene-specific expression domains. Knowledge of these expression patterns will guide the interpretation of the roles these genes play in development.     

The Wnt-activated Xiro1 gene encodes a repressor that is essential for neural development and downregulates Bmp4

In the early Xenopus embryo, the Xiro homeodomain proteins of the Iroquois (Iro) family control the expression of proneural genes and the size of the neural plate. We report that Xiro1 functions as a repressor that is strictly required for neural differentiation, even when the BMP4 pathway is impaired. We also show that Xiro1 and Bmp4 repress each other. Consistently, Xiro1 and Bmp4 have complementary patterns of expression during gastrulation. The expression of Xiro1 requires Wnt signaling. Thus, Xiro1 is probably a mediator of the known downregulation of Bmp4 by Wnt signaling.     

Temtamy preaxial brachydactyly syndrome is caused by loss-of-function mutations in chondroitin synthase 1, a potential target of BMP signaling

Altered Bone Morphogenetic Protein (BMP) signaling leads to multiple developmental defects, including brachydactyly and deafness. Here we identify chondroitin synthase 1 (CHSY1) as a potential mediator of BMP effects. We show that loss of human CHSY1 function causes autosomal-recessive Temtamy preaxial brachydactyly syndrome (TPBS), mainly characterized by limb malformations, short stature, and hearing loss. After mapping the TPBS locus to chromosome 15q26-qterm, we identified causative mutations in five consanguineous TPBS families. In zebrafish, antisense-mediated chsy1 knockdown causes defects in multiple developmental processes, some of which are likely to also be causative in the etiology of TPBS. In the inner ears of zebrafish larvae, chsy1 is expressed similarly to the BMP inhibitor dan and in a complementary fashion to bmp2b. Furthermore, unrestricted Bmp2b signaling or loss of Dan activity leads to reduced chsy1 expression and, during epithelial morphogenesis, defects similar to those that occur upon Chsy1 inactivation, indicating that Bmp signaling affects inner-ear development by repressing chsy1. In addition, we obtained strikingly similar zebrafish phenotypes after chsy1 overexpression, which might explain why, in humans, brachydactyly can be caused by mutations leading either to loss or to gain of BMP signaling.     

Tfap2a and Foxd3 regulate early steps in the development of the neural crest progenitor population

The neural crest is a stem cell-like population exclusive to vertebrates that gives rise to many different cell types including chondrocytes, neurons and melanocytes. Arising from the neural plate border at the intersection of Wnt and Bmp signaling pathways, the complexity of neural crest gene regulatory networks has made the earliest steps of induction difficult to elucidate. Here, we report that tfap2a and foxd3 participate in neural crest induction and are necessary and sufficient for this process to proceed. Double mutant tfap2a (mont blanc, mob) and foxd3 (mother superior, mos) mob;mos zebrafish embryos completely lack all neural crest-derived tissues. Moreover, tfap2a and foxd3 are expressed during gastrulation prior to neural crest induction in distinct, complementary, domains; tfap2a is expressed in the ventral non-neural ectoderm and foxd3 in the dorsal mesendoderm and ectoderm. We further show that Bmp signaling is expanded in mob;mos embryos while expression of dkk1, a Wnt signaling inhibitor, is increased and canonical Wnt targets are suppressed. These changes in Bmp and Wnt signaling result in specific perturbations of neural crest induction rather than general defects in neural plate border or dorso-ventral patterning. foxd3 overexpression, on the other hand, enhances the ability of tfap2a to ectopically induce neural crest around the neural plate, overriding the normal neural plate border limit of the early neural crest territory. Although loss of either Tfap2a or Foxd3 alters Bmp and Wnt signaling patterns, only their combined inactivation sufficiently alters these signaling gradients to abort neural crest induction. Collectively, our results indicate that tfap2a and foxd3, in addition to their respective roles in the differentiation of neural crest derivatives, also jointly maintain the balance of Bmp and Wnt signaling in order to delineate the neural crest induction domain.