The Dlx5 homeobox gene is essential for vestibular morphogenesis in the mouse embryo through a BMP4-mediated pathway

In the mouse embryo, Dlx5 is expressed in the otic placode and vesicle, and later in the semicircular canals of the inner ear. In mice homozygous for a null Dlx5/LacZ allele, a severe dysmorphogenesis of the vestibular region is observed, characterized by the absence of semicircular canals and the shortening of the endolymphatic duct. Minor defects are observed in the cochlea, although Dlx5 is not expressed in this region. Cristae formation is severely impaired; however, sensory epithelial cells, recognized by calretinin immunostaining, are present in the vestibular epithelium of Dlx5(-/-) mice. The maculae of utricle and saccule are present but cells appear sparse and misplaced. The abnormal morphogenesis of the semicircular canals is accompanied by an altered distribution of proliferating and apoptotic cells. In the Dlx5(-/-) embryos, no changes in expression of Nkx5.1(Hmx3), Pax2, and Lfng have been seen, while expression of bone morphogenetic protein-4 (Bmp4) was drastically reduced. Notably, BMP4 has been shown to play a fundamental role in vestibular morphogenesis of the chick embryo. We propose that development of the semicircular canals and the vestibular inner ear requires the independent control of several homeobox genes, which appear to exert their function via tight regulation of BPM4 expression and the regional organization of cell differentiation, proliferation, and apoptosis.     

Utility of HoxB2 enhancer‐mediated Cre activity for functional studies in the developing inner ear

The rhombomere 4(r4)‐restricted expression of the mouse Hoxb2 gene is regulated by a 1.4‐kb enhancer‐containing fragment. Here, we showthat transgenic mouse lines expressing cre driven by this fragment (B2‐r4‐Cre), activated the R26R Cre reporter in rhombomere 4 and the second branchial arch, the epithelium of the first branchial arch, apical ectodermal ridge of the limb buds and the tail region. Of particular interest is Cre activity in the developing inner ear. Cre activity was found in the preotic field and otic placode at E8.5 and otocyst at E9.5–E12.5, in the cochleovestibular and facio‐acoustic ganglia at E10.5 and the vestibular and spiral ganglia and all the otic epithelia derived from the otocyst at E15.5 and P0. Our data suggest that the B2‐r4‐Cre transgenic mice provide an important tool for conditional gene manipulation and lineage tracing in the inner ear. In combination with other transgenic lines expressing cre exclusively in the otic vesicle, the relative contributions of the hindbrain, periotic mesenchyme and otic epithelium in otic development can be dissected. genesis 47:361–365, 2009. © 2009 Wiley‐Liss, Inc.     

The expression of LIM‐homeobox genes,Lhx1 and Lhx5, in the forebrain is essential for neural retina differentiation

Elucidating the mechanisms underlying eye development is essential for advancing the medical treatment of eye‐related disorders. The primordium of the eye is an optic vesicle (OV), which has a dual potential for generation of the developing neural retina and retinal pigment epithelium. However, the factors that regulate the differentiation of the retinal primordium remain unclear. We have previously shown that overexpression of Lhx1 and Lhx5, members of the LIM‐homeobox genes, induced the formation of a second neural retina from the presumptive pigmented retina of the OV. However, the precise timing of Lhx1 expression required for neural retina differentiation has not been clarified. Moreover, RNA interference of Lhx5 has not been previously reported. Here, using a modified electroporation method, we show that, Lhx1 expression in the forebrain around stage 8 is required for neural retina formation. In addition, we have succeeded in the knockdown of Lhx5 expression, resulting in conversion of the neural retina region to a pigment vesicle‐like tissue, which indicates that Lhx5 is also required for neural retina differentiation, which correlates temporally with the activity of Lhx1. These results suggest that Lhx1 and Lhx5 in the forebrain regulate neural retina differentiation by suppressing the development of the retinal pigment epithelium, before the formation of the OV.     

Regulatory networks that function to specify flower meristems require the function of homeobox genes PENNYWISE and POUND-FOOLISH in Arabidopsis

Flowering is a major developmental phase change that transforms the fate of the shoot apical meristem (SAM) from a leaf-bearing vegetative meristem to that of a flower-producing inflorescence meristem. In Arabidopsis, floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)–FD complex and the flower meristem identity gene, LEAFY ( LFY ). Two redundant functioning homeobox genes, PENNYWISE ( PNY ) and POUND-FOOLISH ( PNF ), which are expressed in the vegetative and inflorescence SAM, regulate patterning events during reproductive development, including floral specification. To determine the role of PNY and PNF in the floral specification network, we characterized the genetic relationship of these homeobox genes with LFY and FT . Results from this study demonstrate that LFY functions downstream of PNY and PNF. Ectopic expression of LFY promotes flower formation in pny pnf plants, while the flower specification activity of ectopic FT is severely attenuated. Genetic analysis shows that when mutations in pny and pnf genes are combined with lfy , a synergistic phenotype is displayed that significantly reduces floral specification and alters inflorescence patterning events. In conclusion, results from this study support a model in which PNY and PNF promote LFY expression during reproductive development. At the same time, the flower formation activity of FT is dependent upon the function of PNY and PNF.     

Molecular strategies in the evolution of mammalian dental patterning

The acquisition of masticatory capability by mammals allowed a better processing of food and a consequent increase in the efficiency of nutrients intake by the digestive system. The development of tooth classes and variations in tooth number can be considered intrinsic characteristics of mammalian dentition. These features allowed species to develop specialized dentitions, creating new adaptive zones. Comparative developmental data from knockout mutant mice and human tooth agenesis present new insights on the molecular strategies that permitted rapid phenotypic differentiation, adaptation and speciation of mammalian dentition.     

Dlx5/6-enhancer directed expression of Cre recombinase in the pharyngeal arches and brain

Dlx5 and Dlx6, two members of the Distalless gene family, are required for development of numerous tissues during embryogenesis, including facial and limb development. This gene pair is expressed in tandem, transcribed toward each other and separated by a short intergenic region containing multiple putative enhancers. Targeted inactivation of Dlx5 and Dlx6 in mice results in multiple developmental defects in craniofacial and limb structures, suggesting that these genes are crucial for aspects of both neural crest and nonneural crest development. To further investigate potential developmental roles of Dlx5 and Dlx6, we used one of the Dlx5/6 intergenic enhancers to drive Cre recombinase expression in transgenic mice. Crossing Dlx5/6-Cre transgenic mice with mice from the R26R strain results in beta-galactosidase staining in the apical ectodermal ridge, brain, and neural crest-derived mesenchyme of the pharyngeal arches, with staining in term embryos observed in the facial skeleton and specific brain structures. However, in contrast to endogenous expression patterns of Dlx5 and Dlx6, Cre expression within the pharyngeal arches occurs during a very narrow window in early development. Our studies suggest that Dlx5/6-Cre mice may prove useful both in further understanding the function and regulation of Distalless genes during development and in studies of gene function in conditional knockout mice.     

Evolutionary change of the numbers of homeobox genes in bilateral animals

It has been known that the conservation or diversity of homeobox genes is responsible for the similarity and variability of some of the morphological or physiological characters among different organisms. To gain some insights into the evolutionary pattern of homeobox genes in bilateral animals, we studied the change of the numbers of these genes during the evolution of bilateral animals. We analyzed 2,031 homeodomain sequences compiled from 11 species of bilateral animals ranging from Caenorhabditis elegans to humans. Our phylogenetic analysis using a modified reconciled-tree method suggested that there were at least about 88 homeobox genes in the common ancestor of bilateral animals. About 50-60 genes of them have left at least one descendant gene in each of the 11 species studied, suggesting that about 30-40 genes were lost in a lineage-specific manner. Although similar numbers of ancestral genes have survived in each species, vertebrate lineages gained many more genes by duplication than invertebrate lineages, resulting in more than 200 homeobox genes in vertebrates and about 100 in invertebrates. After these gene duplications, a substantial number of old duplicate genes have also been lost in each lineage. Because many old duplicate genes were lost, it is likely that lost genes had already been differentiated from other groups of genes at the time of gene loss. We conclude that both gain and loss of homeobox genes were important for the evolutionary change of phenotypic characters in bilateral animals.     

Distinct roles of Fgf8, Foxi1, Dlx3b and Pax8/2 during otic vesicle induction and maintenance in medaka

The development of the vertebrate inner ear is a complex process that has been investigated in several model organisms. In this work, we examined genetic interactions regulating early development of otic structures in medaka. We demonstrate that misexpression of Fgf8, Dlx3b and Foxi1 during early gastrulation is sufficient to produce ectopic otic vesicles. Combined misexpression strongly increases the appearance of this phenotype. By using a heat-inducible promoter we were furthermore able to separate the regulatory interactions among Fgf8, Foxi1, Dlx3b, Pax8 and Pax2 genes, which are active during different stages of early otic development. In the preplacodal stage we suggest a central position of Foxi1 within a regulatory network of early patterning genes including Dlx3b and Pax8. Different pathways are active after the placodal stage with Dlx3b playing a central role. There Dlx3b regulates members of the Pax-Six-Eya-Dach network and also strongly affects the early dorsoventral marker genes Otx1 and Gbx2.     

The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells

In Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell-cell interactions within the developing Drosophila ventral nerve cord.     

Greater epithelial ridge cells are the principal organoid-forming progenitors of the mouse cochlea

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