Mutual repression between msh and Iro-C is an essential component of the boundary between body wall and wing in Drosophila

During development, the imaginal wing disc of Drosophila is subdivided into territories separated by developmental boundaries. The best characterized boundaries delimit compartments defined by cell-lineage restrictions. Here, we analyze the formation of a boundary that does not rely on such restrictions, namely, that which separates the notum (body wall) and the wing hinge (appendage). It is known that the homeobox genes of the Iroquois complex (Iro-C) define the notum territory and that the distal limit of the Iro-C expression domain demarks the boundary between the notum and the wing hinge. However, it is unclear how this boundary is established and maintained. We now find that msh, a homeobox gene of the Msx family, is strongly expressed in the territory of the hinge contiguous to the Iro-C domain. Loss- and gain-of-function analyses show that msh maintains Iro-C repressed in the hinge, while Iro-C prevents high level expression of msh in the notum. Thus, a mutual repression between msh and Iro-C is essential to set the limit between the contiguous domains of expression of these genes and therefore to establish and/or maintain the boundary between body wall and wing. In addition, we find that msh is necessary for proper growth of the hinge territory and the differentiation of hinge structures. msh also participates in the patterning of the notum, where it is expressed at low levels.     

The Tribolium columnar genes reveal conservation and plasticity in neural precursor patterning along the embryonic dorsal-ventral axis

The Drosophila columnar genes are key regulators of neural precursor formation and patterning along the dorsal-ventral axis of the developing CNS and include ventral nerve cord defective (vnd), intermediate nerve cord defective (ind), muscle segment homeodomain (msh), and Epidermal growth factor receptor (Egfr). To investigate the evolution of neural pattern formation, we identified and determined the expression patterns of Tribolium vnd, ind, and msh, and found that they are expressed in the medial, intermediate, and lateral columns of the developing CNS, respectively, in patterns similar, but not identical, to their Drosophila orthologs. The pattern of Egfr activity suggests that the genetic regulatory mechanisms that initiate Tc-vnd expression are similar in Drosophila and Tribolium, whereas those that initiate Tc-ind have diverged. RNAi analyses of gene function show that Tc-vnd and Tc-ind promote the formation of medial and intermediate column neural precursors and that vnd-mediated repression of ind establishes the boundary between the medial and intermediate columns. These data suggest that columnar gene expression and function underlie neural pattern formation in Drosophila, Tribolium, and potentially all insects, but that subtle spatiotemporal differences in expression of these genes may produce species-specific morphological differences.     

Proximal-distal leg development in Drosophila requires the apterous gene and the Lim1 homologue dlim1

Proximal-distal leg development in Drosophila involves a battery of genes expressed and required in specific proximal-distal (PD) domains of the appendage. Here we report the characterisation of a new gene of this type, dlim1, a member of the Lhx family of genes whose proteins contain two Lim domains and a homeodomain. We show that the Lhx gene apterous (ap) is also required for PD leg development, and we study the functional interactions between ap, dlim1 and other PD genes during leg development. Our results show that a regulatory network formed by ap and dlim1 plus the homeobox genes aristaless and Bar specifies distal leg cell fates in Drosophila.     

Msx1 and Msx2 have shared essential functions in neural crest but may be dispensable in epidermis and axis formation in Xenopus

The homeodomain factors Msx1 and Msx2 are expressed in essentially identical patterns in the epidermis and neural crest of Xenopus embryos during neurula stages. Disruption of Msx1 and Msx2 RNA splicing with antisense morpholino oligonucleotides shows that both factors are also required for expression of the neural crest gene Slug. Loss of Msx1 can be compensated by overexpression of Msx2 and vice versa. Loss of Msx factors also leads to alterations in the expression boundaries for neural and epidermal genes, but does not prevent or reduce expression of epidermal keratin in ventrolateral ectoderm, nor is there a detectable effect on dorsal mesodermal marker gene expression. These results indicate that Msx1 and Msx2 are both essential for neural crest development, but that the two genes have the same function in this tissue. If Msx genes have important functions in epidermis or axial mesoderm induction, these functions must be shared with other regulatory proteins.     

Plasticity within the lateral somatic mesoderm of Drosophila embryos

Each of 30 Drosophila larval somatic muscles has its individual shape, insertion sites and innervation. From the very beginning, the formation of individual muscles is controlled by a set of muscle identity genes. The four lateral transverse muscles (LT1-LT4) are thought to be specified by the combinatorial activity of Krüppel (Kr), apterous (ap) and muscle specific homeobox (msh) genes whilst the activity of the ladybird (lb) genes is required for proper formation of the neighbouring segmental border muscle (SBM). We have recently shown that ectopic expression of lb changes the identity of Kr-expressing lateral muscle precursors and recruits them to form enlarged or duplicated SBMs. Here we report that loss of msh function leads to a similar transformation resulting in the overproduction of SBMs. Inversely, in msh gain of function embryos, the prospective SBM myoblasts change their identity resulting in the formation of enlarged lateral transverse muscles. These data indicate a key role for the msh and lb genes in the specification and diversification of myoblast lineages from the lateral domain, and reveal a plasticity of cell fate within the somatic mesoderm of Drosophila.     

The Lim homeobox gene Lhx2 is required for olfactory sensory neuron identity

Progenitor cells in the mouse olfactory epithelium generate over a thousand subpopulations of neurons, each expressing a unique odorant receptor (OR) gene. This event is under the control of spatial cues, since neurons in different epithelial regions are restricted to express region-specific subsets of OR genes. We show that progenitors and neurons express the LIM-homeobox gene Lhx2 and that neurons in Lhx2-null mutant embryos do not diversify into subpopulations expressing different OR genes and other region-restricted genes such as Nqo1 and Ncam2. Lhx2-/- embryos have, however, a normal distribution of Mash1-positive and neurogenin 1-positive neuronal progenitors that leave the cell cycle, acquire pan-neuronal traits and form axon bundles. Increased cell death in combination with increased expression of the early differentiation marker Neurod1, as well as reduced expression of late differentiation markers (Galphaolf and Omp), suggests that neuronal differentiation in the absence of Lhx2 is primarily inhibited at, or immediate prior to, onset of OR expression. Aberrant regional expression of early and late differentiation markers, taken together with unaltered region-restricted expression of the Msx1 homeobox gene in the progenitor cell layer of Lhx2-/- embryos, shows that Lhx2 function is not required for all aspects of regional specification of progenitors and neurons. Thus, these results indicate that a cell-autonomous function of Lhx2 is required for differentiation of progenitors into a heterogeneous population of individually and regionally specified mature olfactory sensory neurons.     

Msx1 is required for dorsal diencephalon patterning

The dorsal midline of the neural tube has recently emerged as a major signaling center for dorsoventral patterning. Msx genes are expressed at the dorsal midline, although their function at this site remains unknown. Using Msx1(nlacZ) mutant mice, we show that the normal expression domain of Msx1 is interrupted in the pretectum of mutant embryos. Morphological and gene expression data further indicate that a functional midline is not maintained along the whole prosomere 1 in Msx1 mutant mice. This results in the downregulation of genes expressed laterally to the midline in prosomere 1, confirming the importance of the midline as a signaling center. Wnt1 is essential for dorsoventral patterning of the neural tube. In the Msx1 mutant, Wnt1 is downregulated before the midline disappears, suggesting that its expression depends on Msx1. Furthermore, electroporation in the chick embryo demonstrates that Msx1 can induce Wnt1 expression in the diencephalon neuroepithelium and in the lateral ectoderm. In double Msx1/Msx2 mutants, Wnt1 expression is completely abolished at the dorsal midline of the diencephalon and rostral mesencephalon. This indicates that Msx genes may regulate Wnt1 expression at the dorsal midline of the neural tube. Based on these results, we propose a model in which Msx genes are intermediary between Bmp and Wnt at this site.     

The mouse SLIT family: secreted ligands for ROBO expressed in patterns that suggest a role in morphogenesis and axon guidance.

The Slit gene encodes a secreted molecule essential for neural development in Drosophila embryos. Here we report the identification of three Slit homologues in the mouse. We demonstrate that the mouse SLIT1 protein can bind ROBO1, a transmembrane receptor implicated in axon guidance. Both whole-mount and section in situ hybridization studies reveal unique and complementary patterns of expression of the three mouse Slit genes and of Robo1, both within the central nervous system and in other developing tissues. The complementary expression patterns of Slit and Robo1 and their in vitro interaction suggest a ligand-receptor relationship. The expression of all three Slit genes in the floor plate suggests that they are likely to share the same functional properties with their Drosophila homologue in midline neural development and axon guidance. The complementary expression of Slit and Robo1 in different subdivisions of the somites suggests their possible function in axon pathfinding and neural crest cell migration. The unique expression pattern in limb and other organs indicates additional potential functions of the Slit gene family.     

Genetic control of dorsoventral patterning and neuroblast specification in the Drosophila Central Nervous System

The Drosophila embryonic Central Nervous System (CNS) develops from the ventrolateral region of the embryo, the neuroectoderm. Neuroblasts arise from the neuroectoderm and acquire unique fates based on the positions in which they are formed. Previous work has identified six genes that pattern the dorsoventral axis of the neuroectoderm: Drosophila epidermal growth factor receptor (Egfr), ventral nerve cord defective (vnd), intermediate neuroblast defective (ind), muscle segment homeobox (msh), Dichaete and Sox-Neuro (SoxN). The activities of these genes partition the early neuroectoderm into three parallel longitudinal columns (medial, intermediate, lateral) from which three distinct columns of neural stem cells arise. Most of our knowledge of the regulatory relationships among these genes derives from classical loss of function analyses. To gain a more in depth understanding of Egfr-mediated regulation of vnd, ind and msh and investigate potential cross-regulatory interactions among these genes, we combined loss of function with ectopic activation of Egfr activity. We observe that ubiquitous activation of Egfr expands the expression of vnd and ind into the lateral column and reduces that of msh in the lateral column. Through this work, we identified the genetic criteria required for the development of the medial and intermediate column cell fates. We also show that ind appears to repress vnd, adding an additional layer of complexity to the genetic regulatory hierarchy that patterns the dorsoventral axis of the CNS. Finally, we demonstrate that Egfr and the genes of the achaete-scute complex act in parallel to regulate the individual fate of neural stem cells.     

Characterization of a Smad motif similar to Drosophila mad in the mouse Msx 1 promoter

Mouse Msx 1 gene, orthologous of the Drosophila msh, is involved in several developmental processes. BMP family members are major proteins in the regulation of Msx 1 expression. BMP signaling activates Smad 1/5/8 proteins, which associate to Smad 4 before translocating to the nucleus. Analysis of Msx 1 promoter revealed the presence of three elements similar to the consensus established for Mad, the Smad 1 Drosophila counterpart. Notably, such an element was identified in an enhancer important for Msx 1 regulation. Gel shift analysis demonstrated that proteins from 13.5 dpc embryo associate to this enhancer. Remarkably, supershift assays showed that Smad proteins are present in the complex. Purified Smad 1 and 4 also bind to this fragment. We demonstrate that functional binding sites in this enhancer are confined to the Mad motif and flanking region. Our data suggest that this Mad motif may be functional in response to BMP signaling.     

Characterization of Lhx9, a novel LIM/homeobox gene expressed by the pioneer neurons in the mouse cerebral cortex.

In order to explain the phenotype observed in Lhx2 mutant embryos, we previously proposed that an Lhx2 related gene might exist. We now have cloned a new LIM/homeobox gene called Lhx9. Lhx9 is closely related to Lhx2 and is expressed in the developing central nervous system (CNS). Lhx9 and Lhx2 have expression patterns that overlap in some areas but are distinct in others. Thus, in some developmental domains these two highly related proteins may be functionally redundant. Lhx9 is expressed in the pioneer neurons of the cerebral cortex, while Lhx2 is expressed throughout the cortical layers. Postnatally, Lhx9 is expressed in the inner nuclei of the cerebellum, while Lhx2 is in the granular layer. In the developing limbs, both genes are highly expressed in a similar pattern. Based on the expression pattern and the developmental regulation of Lhx9, we propose that Lhx9 may be involved in the specification or function of the pioneer neurons of the cerebral cortex. We show that both Lhx9 and Lhx2 bind the LIM domain binding protein Ldb1/Nli1/Clim2.     

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.