Zebrafish msxB, msxC and msxE function together to refine the neural-nonneural border and regulate cranial placodes and neural crest development

The zebrafish muscle segment homeobox genes msxB, msxC and msxE are expressed in partially overlapping domains in the neural crest and preplacodal ectoderm. We examined the roles of these msx genes in early development. Disrupting individual msx genes causes modest variable defects, whereas disrupting all three produces a reproducible severe phenotype, suggesting functional redundancy. Neural crest differentiation is blocked at an early stage. Preplacodal development begins normally, but placodes arising from the msx expression domain later show elevated apoptosis and are reduced in size. Cell proliferation is normal in these tissues. Unexpectedly, Msx-deficient embryos become ventralized by late gastrulation whereas misexpression of msxB dorsalizes the embryo. These effects appear to involve Distal-less (Dlx) protein activity, as loss of dlx3b and dlx4b suppresses ventralization in Msx-depleted embryos. At the same time, Msx-depletion restores normal preplacodal gene expression to dlx3b-dlx4b mutants. These data suggest that mutual antagonism between Msx and Dlx proteins achieves a balance of function required for normal preplacodal differentiation and placement of the neural-nonneural border.     

Regulation of Msx genes by a Bmp gradient is essential for neural crest specification

There is evidence in Xenopus and zebrafish embryos that the neural crest/neural folds are specified at the border of the neural plate by a precise threshold concentration of a Bmp gradient. In order to understand the molecular mechanism by which a gradient of Bmp is able to specify the neural crest, we analyzed how the expression of Bmp targets, the Msx genes, is regulated and the role that Msx genes has in neural crest specification. As Msx genes are directly downstream of Bmp, we analyzed Msx gene expression after experimental modification in the level of Bmp activity by grafting a bead soaked with noggin into Xenopus embryos, by expressing in the ectoderm a dominant-negative Bmp4 or Bmp receptor in Xenopus and zebrafish embryos, and also through Bmp pathway component mutants in the zebrafish. All the results show that a reduction in the level of Bmp activity leads to an increase in the expression of Msx genes in the neural plate border. Interestingly, by reaching different levels of Bmp activity in animal cap ectoderm, we show that a specific concentration of Bmp induces msx1 expression to a level similar to that required to induce neural crest. Our results indicate that an intermediate level of Bmp activity specifies the expression of Msx genes in the neural fold region. In addition, we have analyzed the role that msx1 plays on neural crest specification. As msx1 has a role in dorsoventral pattering, we have carried out conditional gain- and loss-of-function experiments using different msx1 constructs fused to a glucocorticoid receptor element to avoid an early effect of this factor. We show that msx1 expression is able to induce all other early neural crest markers tested (snail, slug, foxd3) at the time of neural crest specification. Furthermore, the expression of a dominant negative of Msx genes leads to the inhibition of all the neural crest markers analyzed. It has been previously shown that snail is one of the earliest genes acting in the neural crest genetic cascade. In order to study the hierarchical relationship between msx1 and snail/slug we performed several rescue experiments using dominant negatives for these genes. The rescuing activity by snail and slug on neural crest development of the msx1 dominant negative, together with the inability of msx1 to rescue the dominant negatives of slug and snail strongly argue that msx1 is upstream of snail and slug in the genetic cascade that specifies the neural crest in the ectoderm. We propose a model where a gradient of Bmp activity specifies the expression of Msx genes in the neural folds, and that this expression is essential for the early specification of the neural crest.     

Progression from mitotic catastrophe to germ cell death in Caenorhabditis elegans lis-1 mutants requires the spindle checkpoint

Deletion of the lissencephaly disease gene LIS-1 in humans causes an extreme disorganization of the brain associated with significant reduction in cortical neurons. Here we show that deletion or RNA interference (RNAi) of Caenorhabditis elegans lis-1 results in a reduction in germline nuclei and causes a variety of cellular, developmental, and neurological defects throughout development. Our analysis of the germline defects suggests that the reduction in nuclei number stems from dysfunctional mitotic spindles resulting in cell cycle arrest and eventually programmed cell death (apoptosis). Deletion of the spindle checkpoint gene mdf-1 blocks lis-1(lf)-induced cell cycle arrest and germline apoptosis, placing the spindle checkpoint pathway upstream of the programmed cell death pathway. These results suggest that apoptosis may contribute to the cell-sparse pathology of lissencephaly.     

Neural crests are actively precluded from the anterior neural fold by a novel inhibitory mechanism dependent on Dickkopf1 secreted by the prechordal mesoderm

It is known the interactions between the neural plate and epidermis generate neural crest (NC), but it is unknown why the NC develops only at the lateral border of the neural plate and not in the anterior fold. Using grafting experiments we show that there is a previously unidentified mechanism that precludes NC from the anterior region. We identify prechordal mesoderm as the tissue that inhibits NC in the anterior territory and show that the Wnt/beta-catenin antagonist Dkk1, secreted by this tissue, is sufficient to mimic this NC inhibition. We show that Dkk1 is required for preventing the formation of NC in the anterior neural folds as loss-of-function experiments using a Dkk1 blocking antibody in Xenopus as well as the analysis of Dkk1-null mouse embryos transform the anterior neural fold into NC. This can be mimicked by Wnt/beta-catenin signaling activation without affecting the anterior posterior patterning of the neural plate, or placodal specification. Finally, we show that the NC cells induced at the anterior neural fold are able to migrate and differentiate as normal NC. These results demonstrate that anterior regions of the embryo lack NC because of a mechanism, conserved from fish to mammals, that suppresses Wnt/beta-catenin signaling via Dkk1.     

Role of programmed cell death in patterning the Drosophila antennal arista

Programmed cell death is a critical process for the patterning and sculpting of organs during development. The Drosophila arista, a feather-like structure at the tip of the antenna, is composed of a central core and several lateral branches. A homozygous viable mutation in the thread gene, which encodes an inhibitor of apoptosis protein, produces a branchless arista. We have found that mutations in the proapoptotic gene hid lead to numerous extra branches, suggesting that the level of cell death determines the number of branches in the arista. Consistent with this idea, we have found that thread mutants show excessive cell death restricted to the antennal imaginal disc during the middle third instar larval stage. These findings point to a narrow window of development in which regulation of programmed cell death is essential to the proper formation of the arista.     

A Xenopus tribbles orthologue is required for the progression of mitosis and for development of the nervous system

The product of the Drosophila gene tribbles inhibits cell division in the ventral furrow of the embryo and thereby allows the normal prosecution of gastrulation. Cell division is also absent in involuting dorsal mesoderm during gastrulation in Xenopus, and to ask whether the two species employ similar mechanisms to coordinate morphogenesis and the cell cycle, we isolated a putative Xenopus homologue of tribbles which we call Xtrb2. Extensive cDNA cloning identified long and short forms of Xtrb2, termed Xtrb2-L and Xtrb2-S, respectively. Xtrb2 is expressed maternally and in mesoderm and ectoderm at blastula and gastrula stages. Later, it is expressed in dorsal neural tube, eyes, and cephalic neural crest. Time-lapse imaging of GFP-tagged Xtrb2-L suggests that during cell division, it is associated with mitotic spindles. Knockdown of Xtrb2 by antisense morpholino oligonucleotides (MOs) disrupted synchronous cell divisions during blastula stages, apparently as a result of delayed progression through mitosis and cytokinesis. At later stages, tissues expressing the highest levels of Xtrb2 were most markedly affected by morpholino knockdown, with perturbation of neural crest and eye development.     

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.