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.     

Xiro homeoproteins coordinate cell cycle exit and primary neuron formation by upregulating neuronal-fate repressors and downregulating the cell-cycle inhibitor XGadd45-gamma

The iroquois (iro) homeobox genes participate in many developmental processes both in vertebrates and invertebrates, among them are neural plate formation and neural patterning. In this work, we study in detail Xenopus Iro (Xiro) function in primary neurogenesis. We show that misexpression of Xiro genes promotes the activation of the proneural gene Xngnr1 but suppresses neuronal differentiation. This is probably due to upregulation of at least two neuronal-fate repressors: XHairy2A and XZic2. Accordingly, primary neurons arise at the border of the Xiro expression domains. In addition, we identify XGadd45-gamma as a new gene repressed by Xiro. XGadd45-gamma encodes a cell-cycle inhibitor and is expressed in territories where cells will exit mitosis, such as those where primary neurons arise. Indeed, XGadd45-gamma misexpression causes cell cycle arrest. We conclude that, during Xenopus primary neuron formation, in Xiro expressing territories neuronal differentiation is impaired, while in adjacent cells, XGadd45-gamma may help cells stop dividing and differentiate as neurons.     

Bar homeobox genes are latitudinal prepattern genes in the developing Drosophila notum whose expression is regulated by the concerted functions of decapentaplegic and wingless

In Drosophila notum, the expression of achaete-scute proneural genes and bristle formation have been shown to be regulated by putative prepattern genes expressed longitudinally. Here, we show that two homeobox genes at the Bar locus (BarH1 and BarH2) may belong to a different class of prepattern genes expressed latitudinally, and suggest that the developing notum consists of checker-square-like subdomains, each governed by a different combination of prepattern genes. BarH1 and BarH2 are coexpressed in the anterior-most notal region and regulate the formation of microchaetae within the region of BarH1/BarH2 expression through activating achaete-scute. Presutural macrochaetae formation also requires Bar homeobox gene activity. Bar homeobox gene expression is restricted dorsally and posteriorly by Decapentaplegic signaling, while the ventral limit of the expression domain of Bar homeobox genes is determined by wingless whose expression is under the control of Decapentaplegic signaling.     

Identification and expression of zebrafish Iroquois homeobox gene irx1

Iroquois homeoproteins are prepatterning factors that positively regulate proneural genes and control neurogenesis. We have identified a zebrafish Iroquois gene, irx1, which is highly homologous to Xenopus Xiro1, Gallus c-Irx1 and mouse Irx1. Expression of irx1 was initially detected at the bud stage. By 16 h post-fertilization (hpf), irx1 expression was exclusively limited to the prospective midbrain and hindbrain. By 24 hpf, irx1 expression was clearly detected in the acousticovestibual ganglia, tectum, tegmentum, cerebellum and rhombomere 1 but not in rhombomere 2 or mid-hindbrain boundary.     

Her5 acts as a prepattern factor that blocks neurogenin1 and coe2 expression upstream of Notch to inhibit neurogenesis at the midbrain-hindbrain boundary

Neurogenesis in both vertebrates and invertebrates is tightly controlled in time and space involving both positive and negative regulators. We report here that the bHLH factor Her5 acts as a prepattern gene to prevent neurogenesis in the anlage of the midbrain/hindbrain boundary in the zebrafish neural plate. This involves selective suppression of both neurogenin1 (ngn1) and coe2 mRNA expression in a process that is independent of Notch signalling, and where inhibition of either ngn1 or coe2 expression is sufficient to prevent neuronal differentiation across the midbrain-hindbrain boundary. A ngn1 transgene faithfully responds to Her5 and deletion analysis of the transgene identifies an E-box in a ngn1 upstream enhancer to be required for repression by Her5. Together our data demonstrate a role of Her5 as a prepattern factor in the spatial definition of proneural domains in the zebrafish neural plate, in a manner similar to its Drosophila homologue Hairy.     

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.     

Control of neural precursor specification by proneural proteins in the CNS of Drosophila.

Formation of neural precursors in Drosophila is determined by proneural genes. The distinctive pattern of expression of some genes of the achaete-scute complex in the embryonic neuroectoderm has prompted the speculation that they could also function in the specification of neural precursor identity in the CNS. To test this hypothesis, we have analysed the capacity of different proneural proteins to promote the development of a particular CNS precursor, the MP2 precursor. Our results indicate that: (i) all known proneural proteins are similarly able to support the formation of a neural precursor at the position of MP2; (ii) different proneural proteins promote the expression of different characteristics of MP2; and (iii) a totally normal specification of the MP2 fate can only be attained by the proneural genes achaete or scute.     

Patterning of proneuronal and inter-proneuronal domains by hairy- and enhancer of split-related genes in zebrafish neuroectoderm

In teleosts and amphibians, the proneuronal domains, which give rise to primary-motor, primary-inter and Rohon-Beard (RB) neurons, are established at the beginning of neurogenesis as three longitudinal stripes along the anteroposterior axis in the dorsal ectoderm. The proneuronal domains are prefigured by the expression of basic helix-loop-helix (bHLH) proneural genes, and separated by domains (inter-proneuronal domains) that do not express the proneural genes. Little is known about how the formation of these domains is spatially regulated. We have found that the zebrafish hairy- and enhancer of split-related (Her) genes her3 and her9 are expressed in the inter-proneuronal domains, and are required for their formation. her3 and her9 expression was not regulated by Notch signaling, but rather controlled by positional cues, in which Bmp signaling is involved. Inhibition of Her3 or Her9 by antisense morpholino oligonucleotides led to ectopic expression of the proneural genes in part of the inter-proneuronal domains. Combined inhibition of Her3 and Her9 induced ubiquitous expression of proneural and neuronal genes in the neural plate, and abolished the formation of the inter-proneuronal domains. Furthermore, inhibition of Her3/Her9 and Notch signaling led to ubiquitous and homogeneous expression of proneural and neuronal genes in the neural plate, revealing that Her3/Her9 and Notch signaling have distinct roles in neurogenesis. These data indicate that her3 and her9 function as prepattern genes that link the positional dorsoventral polarity information in the posterior neuroectoderm to the spatial regulation of neurogenesis.     

Control of neurogenesis--lessons from frogs, fish and flies

Two types of genes activated by neural inducers have been identified, those that lead to the activation of proneural genes and those that limit the activity of these genes to specific domains in the neural plate. The analysis of these genes has begun to fill gaps in our understanding of events that lead from neural induction to the generation of neurons within three longitudinal columns in the Xenopus and zebrafish neural plate.     

A region of the vertebrate neural plate in which neighbouring cells can adopt neural or epidermal fates

Cells in the neurogenic region of the fly, Drosophila melanogaster, become either neural stem cells or epidermis and the selection of the former requires the activity of the proneural genes [1]. In contrast, it is commonly thought that all cells in the vertebrate neural plate contribute to the neural tube and that consequently there is no need for the selection of individual neural precursors (e.g., [2]). Here we present a detailed fate map of the chick caudal neural plate (CNP), a cell population that generates the posterior hindbrain and spinal cord. We show that this is a unique region of the neural plate where neighbouring cells can contribute to neural tube or epidermis. Further, neural tube precursors leave the CNP in an approximate rostro-caudal order and give rise to discrete portions of the neural tube where they or their progeny behave as neural stem cells [3]. Our data suggest that neural and epidermal cell fates are acquired on a cell-by-cell basis within the CNP and thus in a manner strikingly similar to that in the fly. Indeed, the assignment of neural cell fate in this region may prove to be mediated by the functional homologue of the fly proneural genes (chick achaete-scute homologue 4, cash4), which is expressed heterogeneously within this cell population [4].     

The expression and function of the achaete-scute genes in Tribolium castaneum reveals conservation and variation in neural pattern formation and cell fate specification

The study of achaete-scute (ac/sc) genes has recently become a paradigm to understand the evolution and development of the arthropod nervous system. We describe the identification and characterization of the ac/sc genes in the coleopteran insect species Tribolium castaneum. We have identified two Tribolium ac/sc genes - achaete-scute homolog (Tc-ASH) a proneural gene and asense (Tc-ase) a neural precursor gene that reside in a gene complex. Focusing on the embryonic central nervous system we find that Tc-ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies we show that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. Furthermore, we find that Tc-ase is expressed in all neural precursors suggesting an important and conserved role for asense genes in insect nervous system development. Our analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development.