Bapx1 homeobox gene gain-of-function mice show preaxial polydactyly and activated Shh signaling in the developing limb.

To explore Bapx1 homeobox gene function in embryonic control of development, we employed a gain-of-function approach to complement our previous loss-of-function mutant analysis. We show that transgenic mice overexpressing Bapx1 are affected by skeletal defects including hindlimb preaxial polydactyly and tibial hypoplasia. Bapx1 overexpression generates limb anteroposterior patterning defects including induction of Shh signaling and ectopic activation of functions downstream of Shh signaling into the anterior region of the autopod. Moreover, Bapx1 overexpression stimulates formation of limb prechondrogenic condensations. We also show that Shh is reciprocally able to activate Bapx1 expression in mouse embryos as the orthologous hedgehog (hh) does with the bagpipe/Bapx1 gene in Drosophila. Our results indicate that Bapx1 can modulate appendicular skeletal formation, that the genetic hierarchy between Shh/hh and Bapx1/bagpipe has been conserved during evolution, and that in mouse embryos these two genes can influence one another in a genetically reciprocal manner. We conclude that it is reasonable to expect overexpression of Bapx1 in certain forms of polydactyly.     

The mouse homeobox gene Not is required for caudal notochord development and affected by the truncate mutation

The floating head (flh) gene in zebrafish encodes a homeodomain protein, which is essential for notochord formation along the entire body axis. flh orthologs, termed Not genes, have been isolated from chick and Xenopus, but no mammalian ortholog has yet been identified. Truncate (tc) is an autosomal recessive mutation in mouse that specifically disrupts the development of the caudal notochord. Here, we demonstrate that truncate arose by a mutation in the mouse Not gene. The truncate allele (Nottc) contains a point mutation in the homeobox of Not that changes a conserved Phenylalanine residue in helix 1 to a Cysteine (F20C), and significantly destabilizes the homeodomain. Reversion of F20C in one allele of homozygous tc embryonic stem (ES) cells is sufficient to restore normal notochord formation in completely ES cell-derived embryos. We have generated a targeted mutation of Not by replacing most of the Not coding sequence, including the homeobox with the eGFP gene. The phenotype of NoteGFP/eGFP, NoteGFP/tc, and Nottc/tc embryos is very similar but slightly more severe in NoteGFP/eGFP than in Nottc/tc embryos. This confirms allelism of truncate and Not, and indicates that tc is not a complete null allele. Not expression is abolished in Foxa2 and T mutant embryos, suggesting that Not acts downstream of both genes during notochord development. This is in contrast to zebrafish embryos, in which flh interacts with ntl (zebrafish T) in a regulatory loop and is essential for development of the entire notochord, and suggests that different genetic control circuits act in different vertebrate species during notochord formation.     

Spectrum of mutations and genotype-phenotype analysis in Currarino syndrome

The triad of a presacral tumour, sacral agenesis and anorectal malformation constitutes the Currarino syndrome which is caused by dorsal-ventral patterning defects during embryonic development. The syndrome occurs in the majority of patients as an autosomal dominant trait associated with mutations in the homeobox gene HLXB9 which encodes the nuclear protein HB9. However, genotype-phenotype analyses have been performed only in a few families and there are no reports about the specific impact of HLXB9 mutations on HB9 function. We performed a mutational analysis in 72 individuals from nine families with Currarino syndrome. We identified a total of five HLXB9 mutations, four novel and one known mutation, in four out of four families and one out of five sporadic cases. Highly variable phenotypes and a low penetrance with half of all carriers being clinically asymptomatic were found in three families, whereas affected members of one family showed almost identical phenotypes. However, an obvious genotype-phenotype correlation was not found. While HLXB9 mutations were diagnosed in 23 patients, no mutation or microdeletion was detected in four sporadic patients with Currarino syndrome. The distribution pattern of here and previously reported HLXB9 mutations indicates mutational predilection sites within exon 1 and the homeobox. Furthermore, sequence homology to Drosophila homeobox genes suggest that some of these mutations located within the homeobox may alter the DNA-binding specificity of HB9 while those in sequences homologous to a recently identified NLS motif of the human homeobox gene PDX-1 may impair nuclear translocation of the mutated protein.     

Beyond the Hox: how widespread is homeobox gene clustering?

The arrangement of Hox genes into physical clusters is fundamental to the patterning of animal body plans, through the phenomenon of colinearity. Other homeobox genes are often described as dispersed, implying they are not arranged into clusters. Contrary to this view, however, two clusters of non-Hox homeobox genes have been reported: the amphioxus ParaHox gene cluster and the Drosophila 93D/E cluster (referred to here as the NKL cluster). Here I examine the antiquity of these gene clusters, their conservation and their pattern of evolution in vertebrate genomes. I argue that the ParaHox gene cluster arose early in animal evolution, and duplicated in vertebrates to give the four clusters in human and mouse genomes. The NKL cluster is also ancient, and also duplicated to yield four descendent clusters in mammalian genomes. The NKL and Hox gene clusters were originally chromosomal neighbours, within an ancient and extensive array of at least 30 related homeobox genes. There is no necessary relationship between clustering and colinearity, although it is argued that the ParaHox gene cluster does show modified spatial colinearity. A novel hypothesis for the evolution of ParaHox gene expression in deuterostomes is presented.     

Beyond the Hox: how widespread is homeobox gene clustering?

The arrangement of Hox genes into physical clusters is fundamental to the patterning of animal body plans, through the phenomenon of colinearity. Other homeobox genes are often described as dispersed, implying they are not arranged into clusters. Contrary to this view, however, two clusters of non-Hox homeobox genes have been reported: the amphioxus ParaHox gene cluster and the Drosophila 93D/E cluster (referred to here as the NKL cluster). Here I examine the antiquity of these gene clusters, their conservation and their pattern of evolution in vertebrate genomes. I argue that the ParaHox gene cluster arose early in animal evolution, and duplicated in vertebrates to give the four clusters in human and mouse genomes. The NKL cluster is also ancient, and also duplicated to yield four descendent clusters in mammalian genomes. The NKL and Hox gene clusters were originally chromosomal neighbours, within an ancient and extensive array of at least 30 related homeobox genes. There is no necessary relationship between clustering and colinearity, although it is argued that the ParaHox gene cluster does show modified spatial colinearity. A novel hypothesis for the evolution of ParaHox gene expression in deuterostomes is presented.     

Role of programmed cell death in normal neuronal development and function

The consequences of eliminating the process of programmed cell death during the development of the nervous system is examined by reviewing studies in the genetic model organisms Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus, where mutations of cell death genes have eliminated or reduced programmed cell death in the nervous system. In many cases, genetic elimination of cell death leads to embryonic mortality or gross anatomical malformations; however, there are cases where animals develop normally but with excess neurons and glia in the nervous system. Undead cells either differentiate and function as working neurons, in some instances being of smaller size, or fail to differentiate and lack normal connections with their targets. Changes in motor control and sensory processing are generally not observed, except for during the most complex of behaviors. Examination of organisms where death genes have been genetically eliminated reveals that programmed cell death may play an important role in sculpting gross brain structure during early development of the neural tube. In contrast, the consequences of preventing neuronal cell death at later developmental stages (e.g. during vertebrate synapse formation) are just beginning to be understood.     

The Dlx5 and Dlx6 homeobox genes are essential for craniofacial,axial, and appendicular skeletal development

Dlx homeobox genes are mammalian homologs of the Drosophila Distal-less (Dll) gene. The Dlx/Dll gene family is of ancient origin and appears to play a role in appendage development in essentially all species in which it has been identified. In Drosophila, Dll is expressed in the distal portion of the developing appendages and is critical for the development of distal structures. In addition, human Dlx5 and Dlx6 homeobox genes have been identified as possible candidate genes for the autosomal dominant form of the split-hand/split-foot malformation (SHFM), a heterogeneous limb disorder characterized by missing central digits and claw-like distal extremities. Targeted inactivation of Dlx5 and Dlx6 genes in mice results in severe craniofacial, axial, and appendicular skeletal abnormalities, leading to perinatal lethality. For the first time, Dlx/Dll gene products are shown to be critical regulators of mammalian limb development, as combined loss-of-function mutations phenocopy SHFM. Furthermore, spatiotemporal-specific transgenic overexpression of Dlx5, in the apical ectodermal ridge of Dlx5/6 null mice can fully rescue Dlx/Dll function in limb outgrowth.     

Developmental patterning genes and their conserved functions: from model organisms to humans

Molecular and genetic evidence accumulated during the past 20 years in the field of developmental biology indicates that different animals possess many common genetic systems for embryonic patterning. In this review we describe the conserved functions of such developmental patterning genes and their relevance for human pathological conditions. Special attention is given to the Hox genetic system, involved in establishing cell identities along the anterior-posterior axis of all higher metazoans. We also describe other conserved genetic systems, such as the involvement of Pax6 genes in eye development and the role of Nkx2.5-type proteins in heart development. Finally, we outline some fascinating problems at the forefront of the studies of developmental patterning genes and show how knowledge obtained from model genetic organisms such as Drosophila helps to explain normal human morphogenesis and the genetic basis of some birth defects.