Signals derived from the underlying mesoderm are dispensable for zebrafish neural crest induction

Signals from the non-neural ectoderm, the neural ectoderm, and the underlying mesoderm have all been implicated in the induction of neural crest. Bone morphogenetic protein (BMP) signaling in particular has an important role in this process; however, it is unclear whether this activity of BMP is due to its effects on patterning the underlying mesoderm, to its ability to establish a competent neural plate boundary zone, or to the direct specification of neural crest at intermediate levels of activity within a BMP gradient. We show neural crest induction occurs in zebrafish in the absence of involuted mesoderm, indicating that this tissue and signals derived from it are dispensable for the formation of neural crest. Dorsal-involuted mesoderm is a major source of secreted BMP antagonists, and the activity of BMP signaling is thought to depend on the presence of the opposing activity of these antagonists. We find that the three BMP antagonists known to be expressed during gastrulation in zebrafish, noggin1, follistatin, and chordin, are dispensable for neural crest induction. These results suggest that mechanisms for restricting the spatio-temporal pattern of BMP expression may compensate for the loss of secreted BMP antagonist activity in establishing dorso-ventral patterning, neural induction, and the neural crest.     

Role of BMP signaling and the homeoprotein Iroquois in the specification of the cranial placodal field

Different types of placodes originate at the anterior border of the neural plate but it is still an unresolved question whether individual placodes arise as distinct ectodermal specializations in situ or whether all or a subset of the placodes originate from a common preplacodal field. We have analyzed the expression and function of the homeoprotein Iro1 in Xenopus and zebrafish embryos, and we have compared its expression with several preplacodal and placodal markers. Our results indicate that the iro1 genes are expressed in the preplacodal region, being one of the earliest markers for this area. We show that an interaction between the neural plate and the epidermis is able to induce the expression of several preplacodal markers, including Xiro1, by a similar mechanism to that previously shown for neural crest induction. In addition, we analyzed the role of BMP in the specification of the preplacodal field by studying the expression of the preplacodal markers Six1, Xiro1, and several specific placodal markers. We experimentally modified the level of BMP activity by three different methods. First, we implanted beads soaked with noggin in early neurula stage Xenopus embryos; second, we injected the mRNA that encodes a dominant negative of the BMP receptor into Xenopus and zebrafish embryos; and third, we grafted cells expressing chordin into zebrafish embryos. The results obtained using all three methods show that a reduction in the level of BMP activity leads to an expansion of the preplacodal and placodal region similar to what has been described for neural crest regions. By using conditional constructs of Xiro1, we performed gain and loss of function experiments. We show that Xiro1 play an important role in the specification of both the preplacodal field as well as individual placodes. We have also used inducible dominant negative and activator constructs of Notch signaling components to analyze the role of these factors on placodal development. Our results indicate that the a precise level of BMP activity is required to induce the neural plate border, including placodes and neural crest cells, that in this border the iro1 gene is activated, and that this activation is required for the specification of the placodes.     

Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals

At the border of the neural plate, the induction of the neural crest can be achieved by interactions with the epidermis, or with the underlying mesoderm. Wnt signals are required for the inducing activity of the epidermis in chick and amphibian embryos. Here, we analyze the molecular mechanisms of neural crest induction by the mesoderm in Xenopus embryos. Using a recombination assay, we show that prospective paraxial mesoderm induces a panel of neural crest markers (Slug, FoxD3, Zic5 and Sox9), whereas the future axial mesoderm only induces a subset of these genes. This induction is blocked by a dominant negative (dn) form of FGFR1. However, neither dnFGFR4a nor inhibition of Wnt signaling prevents neural crest induction in this system. Among the FGFs, FGF8 is strongly expressed by the paraxial mesoderm. FGF8 is sufficient to induce the neural crest markers FoxD3, Sox9 and Zic5 transiently in the animal cap assay. In vivo, FGF8 injections also expand the Slug expression domain. This suggests that FGF8 can initiate neural crest formation and cooperates with other DLMZ-derived factors to maintain and complete neural crest induction. In contrast to Wnts, eFGF or bFGF, FGF8 elicits neural crest induction in the absence of mesoderm induction and without a requirement for BMP antagonists. In vivo, it is difficult to dissociate the roles of FGF and WNT factors in mesoderm induction and neural patterning. We show that, in most cases, effects on neural crest formation were parallel to altered mesoderm or neural development. However, neural and neural crest patterning can be dissociated experimentally using different dominant-negative manipulations: while Nfz8 blocks both posterior neural plate formation and neural crest formation, dnFGFR4a blocks neural patterning without blocking neural crest formation. These results suggest that different signal transduction mechanisms may be used in neural crest induction, and anteroposterior neural patterning.     

Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction.

The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. Here, the roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, was transformed into neural crest by posteriorizing signals. This transformation was mimicked by bFGF, Wnt-8, or retinoic acid treatment and was also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells was also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor was shown to directly affect the ectoderm. We thereby propose a two-step model for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. We discuss a unifying model where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction.     

Zic2 is required for neural crest formation and hindbrain patterning during mouse development

The Zic genes are the vertebrate homologues of the Drosophila pair rule gene odd-paired. It has been proposed that Zic genes play several roles during neural development including mediolateral segmentation of the neural plate, neural crest induction, and inhibition of neurogenesis. Initially during mouse neural development Zic2 is expressed throughout the neural plate while later on expression in the neurectoderm becomes restricted to the lateral region of the neural plate. A hypomorphic allele of Zic2 has demonstrated that in the mouse Zic2 is required for the timing of neurulation. We have isolated a new allele of Zic2 that behaves as a loss of function allele. Analysis of this mutant reveals two further functions for Zic2 during early neural development. Mutation of Zic2 results in a delay of neural crest production and a decrease in the number of neural crest cells that are produced. These defects are independent of mediolateral segmentation of the neurectoderm and of dorsal neurectoderm proliferation, both of which occur normally in the mutant embryos. Additionally Zic2 is required during hindbrain patterning for the normal development of rhombomeres 3 and 5. This work provides the first genetic evidence that the Zic genes are involved in neural crest production and the first demonstration that Zic2 functions during hindbrain patterning.