The Hemimelic extra toes mouse mutant: Historical perspective on unraveling mechanisms of dysmorphogenesis

Hemimelic extra toes (Hx) arose spontaneously as a dominant mutation in B10.D2/nSnJ mice in 1967. It specifically affects the appendicular skeleton, causing variable foreshortening of the tibia (radius) and preaxial polydactylism. Early anatomical studies revealed anterior overgrowth of the autopod, with decreased apoptosis and increased mitosis in the anterior apical ectodermal ridge and underlying mesenchyme; overextension of apoptosis in the central zeugopod accounted for hemimelia. The Hx mutant phenotype was coarsely mapped to mouse chromosome (Chr) 5 and closely linked to engrailed‐2 (En2) and Sonic hedgehog (Shh). This region is syntenic to human Chr 7q36 that harbors several dominant mutations affecting the hand. High‐resolution genome mapping identified the Hx mutation as a G → A base pair transition within Intron 5 of the murine Lmbr1 locus. The critical effect is on a multifunctional conserved regulatory element that acts as a limb‐specific, long‐distance cis‐acting enhancer of Shh expression. As such, the Hx mutant phenotype results from ectopic Shh signals at the anterior margin of the limb bud that directly or indirectly alter FGF4 signaling from the apical ectodermal ridge. Given significant advances in understanding of embryonic development in general and limb development in particular, this review article reveals how research that once attracted interest of teratologists has advanced across the decades to pinpoint a critical molecular lesion and reveal a potential mechanism of a specific malformation that is found commonly in experimental teratology. Birth Defects Research (Part C) 90:155–162, 2010. © 2010 Wiley‐Liss, Inc.     

Phylogenetic conservation of a limb-specific, <Emphasis Type="Italic">cis</Emphasis>-acting regulator of Sonic hedgehog (<Emphasis Type="Italic">Shh</Emphasis>)

Polarized expression of the Sonic hedgehog (Shh) gene in the posterior mesenchyme is essential for pattern formation in the appendages of higher vertebrates, from teleost fins to tetrapod limb buds. We report on a sequence in intron 5 of the Lmbr1 gene, which resides approximately 1 Mb from the Shh coding region in the mouse genome and is highly conserved among teleost fishes and throughout the tetrapod lineage. Positional cloning revealed that two mouse mutations, Hx and M100081, characterized by mirror-image digit duplication and ectopic anterior Shh expression, have base substitutions in this sequence. Absence of the conserved sequence in limbless reptiles and amphibians and a cis-trans test using the Hx and Shh KO alleles suggest that the sequence is a cis-acting regulator that controls the polarized expression of Shh. The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number: AB092986 to AB093004, AB093207, and AB114903.     

Essential mesenchymal role of small GTPase Rac1 in interdigital programmed cell death during limb development

Developing vertebrate limbs are often utilized as a model for studying pattern formation and morphogenetic cell death. Herein, we report that conditional deletion of Rac1, a member of the Rho family of proteins, in mouse limb bud mesenchyme led to skeletal deformities in the autopod and soft tissue syndactyly, with the latter caused by a complete absence of interdigital programmed cell death. Furthermore, the lack of interdigital programmed cell death and associated syndactyly was related to down-regulated gene expression of Bmp2, Bmp7, Msx1, and Msx2, which are known to promote apoptosis in the interdigital mesenchyme. Our findings from Rac1 conditional mutants indicate crucial roles for Rac1 in limb bud morphogenesis, especially interdigital programmed cell death.     

Mouse rump-white mutation associated with an inversion of Chromosome 5

The rump-white (Rw) mutation in the mouse was previously mapped as part of a cluster of spotting genes on Chromosome (Chr) 5 that includes the dominant spotting (W) and patch (Ph) loci. Recent studies have shown that the W locus encodes the KIT tyrosine kinase cell surface receptor and that Ph is a deletional mutation encompassing the platelet-derived growth factor receptor alpha subunit (Pdgfra) gene. However, the molecular basis of the Rw mutation remains to be established. We have analyzed an interspecific Mus spretus backcross segregating Rw and several loci proximal and distal to the W/Ph/Rw region to study the basis of this mutation. These studies indicated that loci within the En2 to Kit region of the chromosome do not recombine with one another even though they have been separated in other mapping studies presented here and elsewhere. We conducted a series of fluorescent in situ hybridization (FISH) studies with genomic probes to En2, Msx1, D5Buc1, and Kit to compare the physical order of these loci on the Rw and wild-type chromosomes. The Kit locus mapped to approximately the same region on both chromosomes of the Rw heterozygotes, while the positions of En2, Msx1, and D5Buc1 were reversed on the two chromosomes. Taken together, both the genetic and physical mapping data establish that the Rw mutation is associated with an inversion involving loci in the proximal region of Chromosome 5.     

A highly conserved Wnt-dependent TCF4 binding site within the proximal enhancer of the anti-myogenic Msx1 gene supports expression within Pax3-expressing limb bud muscle precursor cells

The product of the Msx1 gene is a potent inhibitor of muscle differentiation. Msx1 is expressed in muscle precursor cells of the limb bud that also express Pax3. It is thought that Msx1 may facilitate distal migration by delaying myogenesis in these cells. Despite the role played by Msx1 in inhibiting muscle differentiation, nothing is known of the mechanisms that support the expression of the Msx1 gene within limb bud muscle precursor cells. In the present study we have used a combination of comparative genomics, mouse transgenic analysis, in situ hybridisation and immunohistochemistry to identify a highly conserved and tissue-specific regulatory sub-domain within the previously characterised Msx1 gene proximal enhancer element that supports the expression of the Msx1 gene in Pax3-expressing mouse limb pre-muscle masses. Furthermore, using a combination of in situ hybridisation, in vivo ChIP assay and transgenic explant culture analysis we provide evidence that Msx1 expression in limb bud muscle precursor cells is dependent on the canonical Wnt/TCF signalling pathway that is important in muscle shape formation. The results of these studies provide evidence of a mechanistic link between the Wnt/TCF and the Msx1/Pax3/MyoD pathways within limb bud muscle precursor cells.     

<Emphasis Type="Italic">Msx1</Emphasis> and <Emphasis Type="Italic">Msx2</Emphasis>are required for endothelial-mesenchymal transformation of the atrioventricular cushions and patterning of the atrioventricular myocardium

Background  

Msx1 and Msx2, which belong to the highly conserved Nk family of homeobox genes, display overlapping expression patterns and redundant functions in multiple tissues and organs during vertebrate development. Msx1 and Msx2 have well-documented roles in mediating epithelial-mesenchymal interactions during organogenesis. Given that both Msx1 and Msx2 are crucial downstream effectors of Bmp signaling, we investigated whether Msx1 and Msx2 are required for the Bmp-induced endothelial-mesenchymal transformation (EMT) during atrioventricular (AV) valve formation.     

Genetic analysis of instability in Petunia hybrida

Summary In crossing experiments with Petunia hybrida, new mutations, some unstable, have been found in descendants of plants having an unstable allele of the anthocyanin gene An1. One of the unstable mutations affecting the new anthocyanin gene An11 was genetically analyzed, and it was subsequently established in which step of anthocyanin synthesis that An11 is involved. The discovery of new, unstable mutations at other loci indicates that in Petunia also a relation exists between unstable mutations and the presence of transposable elements in the genome. It was demonstrated that reverted alleles (an1 ^+/+) originating from unstable An1 alleles are less stable than the original wild-type allele An1, and that reversions do not increase the chances of occurrence of new, stable or unstable mutations at other loci. These results provide additional arguments in favour of the hypothesis posed in an earlier paper that reversions of unstable An1 alleles are not the result of excision of the inserted transposable element, but are due to the repair of secondary mutations induced by the insert in the regulatory region of the locus. Consequently, a reverted allele still contains the inserted element that may again induce mutations leading to inactivation of An1.     

Polydactyly in the Strong's luxoid mouse is suppressed by limb deformity alleles

The study of limb development has provided insight into pattern formation during vertebrate embryogenesis. Genetic approaches offer powerful ways to identify the critical molecules and their pathways of action required to execute a complex morphogenetic program. We have applied genetic analysis to the process of limb development by studying two mouse mutants, limb deformity (Id) and Strong's luxoid (Ist). These mutations confer contrasting phenotypic alterations to the anteroposterior limb pattern. The six mutant Id alleles are fully recessive and result in oligosyndactyly of all four limbs. By contrast, the two mutant Ist alleles result in a mirror-image polydactylous limb phenotype inherited in a semidominant fashion. Morphological and molecular analysis of embryonic limbs has shown that the Id and Ist alleles affect the extent and distribution of two key signaling centers differentially: the apical ectodermal ridge and the zone of polarizing activity. Molecular characterization of the Id gene has defined a new family of evolutionarily conserved proteins termed the formins. The underlying molecular defect in the Ist mutation has not been identified; however, both loci are tightly linked on mouse chromosome 2, suggesting the possibility that they may be allelic. In this study, we have used genetic analysis to examine the epistatic and allelic relationships of Id and Ist. We observed that in + Id/Ist + double heterozygotes, a single mutant Id allele is able to suppress the semi-dominant polydactylous Ist limb phenotype. By segregating the Ist and Id loci in a backcross, we observed that these loci recombine and are separated by a genetic distance of approximately 6 cM. Therefore, while our observations demonstrate a genetic interaction between Id and Ist, it is probable that Id and Ist are not allelic. Instead, Ist and Id may be operating either in a linear or in a parallel (bypass) genetic pathway to affect the limb signaling centers. © 1996 Wiley-Liss, Inc.     

Insight into a single halobacterium using a dual‐bacteriorhodopsin system with different functionally optimized pH ranges to cope with periplasmic pH changes associated with continuous light illumination

The light‐driven outward proton transporter assists energy production via an ATP synthase system best exemplified by the bacteriorhodopsin (BR) from Halobacterium salinarum, HsBR. As the only archaea able to survive in the resource‐limited ecosystem of the Dead Sea, Haloarcula marismortui has been reported to have a unique dual‐BR system, consisting of HmBRI and HmBRII, instead of only a single BR in a cell (solo‐BR). The contribution of this dual‐BR system to survival was investigated. First, native H. marismortui and H. salinarum cells were tested in water that had been adjusted to mimic the conditions of Dead Sea water. These archaea were shown to accumulate protons and reduce pH in their periplasmic regions, which disabled further proton transportation functionality in H. salinarum but not in H. marismortui. Then, pH‐dependent photocurrent measurements using purified BR proteins demonstrated that HsBR and HmBRI were functional at pH > 5.0 and that HmBRII was functional at pH > 4.0. Our results indicate that the dual‐HmBR system is composed of two BRs with different optimal functional pH ranges and together they maintain light‐driven proton transport activity under pH > 4.0, which might contribute the survival of H. marismortui under the acidic pH of the Dead Sea.     

Synteny conservation of the Huntington's disease gene and surrounding loci on mouse Chromosome 5

The mouse homologs of the Huntington's disease (HD) gene and 17 other human Chromosome (Chr) 4 loci (including six previously unmapped) were localized by use of an interspecific cross. All loci mapped in a continuous linkage group on mouse Chr 5, distal to En2 and Il6, whose human counterparts are located on Chr y. The relative order of the loci on human Chr 4 and mouse Chr 5 was maintained, except for a break between D5H4S115E and Idua/rd, with relocation of the latter to the opposite end of the map. The mouse HD homolog (Hdh) mapped within a cluster of seven genes that were completely linked in our data set. In human these loci span a1.8 Mb stretch of human 4p 16.3 that has been entirely cloned. To date, there is no phenotypic correspondence between human and mouse mutations mapping to this region of synteny conservation.     

A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana

Summary Monogenic mutants of the early ecotype Landsberg erecta were selected on the basis of late flowering under long day (LD) conditions after treatment with ethyl methanesulphonate or irradiation. In addition to later flowering the number of rosette and cauline leaves is proportionally higher in all mutants, although the correlation coefficient between the two parameters is not the same for all genotypes. Forty-two independently induced mutants were found to represent mutations at 11 loci. The mutations were either recessive, intermediate (co locus) or almost completely dominant (fwa locus). The loci are located at distinct positions on four of the five Arabidopsis chromosomes. Recombinants carrying mutations at different loci flower later than or as late as the later parental mutant. This distinction led to the assignment of eight of the loci to three epistatic groups. In wild type, vernalization promotes flowering to a small extent. For mutants at the loci fca, fve, fy and fpa, vernalization has a large effect both under LD and short day (SD) conditions, whereas co, gi, fd and fwa mutants are almost completely insensitive to this treatment. SD induces later flowering except for mutants at the co and gi loci, which flower with the same number of leaves under LD and SD conditions. This differential response of the mutants to environmental factors and their subdivision into epistatic groups is discussed in relation to a causal model for floral initiation in Arabidopsis thaliana.     

Antisuppressors in yeast

Summary 1. Twenty-three mutations, mapping at eight different loci, were shown to prevent detectable suppression of ade2-1 and can1-100 by the ochre suppressor, ocSUPQ2. 2. Some reduce the efficiency of suppression of his5-2 and lys1-1 also. 3. Mutants from each of the eight loci prevented the lethal phenotype of ocSUPQ2 in [psi+] strains. They are, therefore, antisuppressor mutations. 4. One of the loci, asu3, is centromere-linked; two of the loci, asu2 and asu6, are linked to each other.     

IL-1 Receptor antagonist as a positional candidate gene in a murine model of allergic asthma

Interleukin-1 receptor antagonist (IL-1ra) is an inhibitor of the proinflammatory IL-1. The IL-1ra gene (Il1rn) maps near the allergen-induced bronchial hyper-responsiveness-1 locus, Abhr1, which we previously mapped to murine chromosome 2 using A/J (asthma susceptible) and C3H/HeJ (asthma resistant) mice. We evaluated the role of Il1rn in our mouse model by comparing its genomic sequence between A/J and C3H/HeJ mice as well as assessing strain-specific RNA and protein production in response to allergen. We identified no functional sequence variations in the Il1rn gene between A/J and C3H/HeJ mice. Il1rn mRNA and protein were induced by ovalbumin (OVA) exposure in both strains, but to a greater extent in A/J mice at the earlier time points. We examined other IL-1 family members (Il1a, Il1b, Il1f9, and Il1r2) and found OVA-induced expression increases at 6 h, yet only Il1b and Il1f9 had strain-specific differences. Of these, only Il1f9 is located within Abhr1, and we found several non-coding polymorphisms in the Il1f9 gene between A/J and C3H/HeJ mice. Our results exclude Il1rn as the gene for Abhr1 and indicate that Il1f9 warrants further investigation based on genetic and expression differences observed in our mouse model of allergic asthma.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Msx1 and Dlx5 function synergistically to regulate frontal bone development

The Msx and Dlx families of homeobox proteins are important regulators for embryogenesis. Loss of Msx1 in mice results in multiple developmental defects including craniofacial malformations. Although Dlx5 is widely expressed during embryonic development, targeted null mutation of Dlx5 mainly affects the development of craniofacial bones. Msx1 and Dlx5 show overlapping expression patterns during frontal bone development. To investigate the functional significance of Msx1/Dlx5 interaction in regulating frontal bone development, we generated Msx1 and Dlx5 double null mutant mice. In Msx1^?/?;Dlx5^?/? mice, the frontal bones defect was more severe than that of either Msx1^?/? or Dlx5^?/? mice. This aggravated frontal bone defect suggests that Msx1 and Dlx5 function synergistically to regulate osteogenesis. This synergistic effect of Msx1 and Dlx5 on the frontal bone represents a tissue specific mode of interaction of the Msx and Dlx genes. Furthermore, Dlx5 requires Msx1 for its expression in the context of frontal bone development. Our study shows that Msx1/Dlx5 interaction is crucial for osteogenic induction during frontal bone development. genesis 48:645–655, 2010. © 2010 Wiley‐Liss, Inc.     

Temperature-sensitive yellow mutants of Chlamydomonas reinhardtii

Summary We have isolated and genetically characterized 10 mutants of Chlamydomonas reinhardtii carrying single, mendelian, temperature-sensitive yellow mutations. The mutants have a yellow phenotype at the restrictive temperature (33°C), but have a wildtype phenotype at the permissive temperature (25°C). Based on complementation and recombination tests, the ten mutations include alleles of two previously described yellow loci (y-1 and y-6) and three new yellow loci (y-8, y-9, and y-10). At the restrictive temperature, y-8, y-9, and y-10 are physiologically similar to other yellow mutants. They accumulate small amounts of protochlorophyllide when grown under dim light, but synthesize normal amounts of chlorophyll when grown in the light. Linkage tests indicate that the three new mutations are not linked to each other. y-8 is linked to y-7 on linkage group III, and y-10 is linked to y-5 and y-6 on linkage group I. y-9 is located on linkage group II. We conclude that the control of light-independent protochlorophyllide reduction is complex, involving several genetic loci which are scattered in the genome and which code for gene products able to complement in trans. Temperature-sensitive alleles at several of the yellow loci suggest that the gene products made by these loci are proteins.     

N-Ethyl-N-nitrosourea-induced prenatally lethal mutations define at least two complementation groups within the embryonic ectoderm development (eed) locus in mouse Chromosome 7

Two loci [l(7)5Rn and l(7)6Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the albino (c) locus in Chromosome (Chr) 7. Both loci were found to map to the subregion of the Mod-2-sh-1 interval that contains the eed (embryonic ectoderm development) locus. eed has been defined by the inability of embryos homozygous for certain c deletions to develop beyond the early stages of gastrulation. Evidence for at least two loci necessary for normal prenatal development, rather than one locus, that map within the eed interval came from the observation that two prenatally lethal mutations, 3354SB [l(7)5Rn ^3354SB ] and 4234SB [l(7)6Rn ^4234SB ], could complement each other in trans, but could not each be complemented individually by c deletions known to include the eed locus. A somewhat leaky allele of l(7)5Rn [l(7)5Rn ^1989SB ] was also recovered, in which hemizygotes are often stillborn and homozygotes exhibit variable fitness and survival. The mapping of the loci defined by these mutations is likely to be useful for genetic, molecular, and phenotypic characterization of the eed region, and mutations at either locus (or both loci) may contribute to the eed phenotype.     

Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development

The homeobox-containing genes Msx1 and Msx2 are highly expressed in the limb field from the earliest stages of limb formation and, subsequently, in both the apical ectodermal ridge and underlying mesenchyme. However, mice homozygous for a null mutation in either Msx1 or Msx2 do not display abnormalities in limb development. By contrast, Msx1; Msx2 double mutants exhibit a severe limb phenotype. Our analysis indicates that these genes play a role in crucial processes during limb morphogenesis along all three axes. Double mutant limbs are shorter and lack anterior skeletal elements (radius/tibia, thumb/hallux). Gene expression analysis confirms that there is no formation of regions with anterior identity. This correlates with the absence of dorsoventral boundary specification in the anterior ectoderm, which precludes apical ectodermal ridge formation anteriorly. As a result, anterior mesenchyme is not maintained, leading to oligodactyly. Paradoxically, polydactyly is also frequent and appears to be associated with extended Fgf activity in the apical ectodermal ridge, which is maintained up to 14.5 dpc. This results in a major outgrowth of the mesenchyme anteriorly, which nevertheless maintains a posterior identity, and leads to formation of extra digits. These defects are interpreted in the context of an impairment of Bmp signalling.