Biomedical subjects

R Krumlauf

Publications and source records attributed to R Krumlauf.

At least 19 recordsLinked to original sources

PubMed1999

The role of kreisler in segmentation during hindbrain development.

The mouse kreisler gene is expressed in rhombomeres (r) 5 and 6 during neural development and kreisler mutants have patterning defects in the hindbrain that are not fully understood. Here we analyzed this phenotype with a combination of genetic, molecular, and cellular marking techniques. Using Hox/lacZ transgenic mice as reporter lines and by analyzing Eph/ephrin expression, we have found that while r5 fails to form in these mice, r6 is present. This shows that kreisler has an early role in the formation of r5. We also observed patterning defects in r3 and r4 that are outside the normal domain of kreisler expression. In both heterozygous and homozygous kreisler embryos some r5 markers are induced in r3, suggesting that there is a partial change in r3 identity that is not dependent upon the loss of r5. To investigate the cellular character of r6 in kreisler embryos we performed heterotopic grafting experiments in the mouse hindbrain to monitor its mixing properties. Control experiments revealed that cells from even- or odd-numbered segments only mixed freely with themselves, but not with cells of opposite character. Transposition of cells from the r6 territory of kreisler mutants reveals that they adopt mature r6 characteristics, as they freely mix only with cells from even-numbered rhombomeres. Analysis of Phox2b expression shows that some aspects of later neurogenesis in r6 are altered, which may be associated with the additional roles of kreisler in regulating segmental identity. Together these results suggest that the formation of r6 has not been affected in kreisler mutants. This analysis has revealed phenotypic and mechanistic differences between kreisler and its zebrafish equivalent valentino. While valentino is believed to subdivide preexisting segmental units, in the mouse kreisler specifies a particular segment. The formation of r6 independent of r5 argues against a role of kreisler in prorhombomeric segmentation of the mouse hindbrain. We conclude that the mouse kreisler gene regulates multiple steps in segmental patterning involving both the formation of segments and their A-P identity.

Animals↗

PubMed1999

Identification of sonic hedgehog as a candidate gene responsible for the polydactylous mouse mutant Sasquatch.

The mouse mutants of the hemimelia-luxate group (lx, lu, lst, Dh, Xt, and the more recently identified Hx, Xpl and Rim4; [1] [2] [3] [4] [5]) have in common preaxial polydactyly and longbone abnormalities. Associated with the duplication of digits are changes in the regulation of development of the anterior limb bud resulting in ectopic expression of signalling components such as Sonic hedgehog (Shh) and fibroblast growth factor-4 (Fgf4), but little is known about the molecular causes of this misregulation. We generated, by a transgene insertion event, a new member of this group of mutants, Sasquatch (Ssq), which disrupted aspects of both anteroposterior (AP) and dorsoventral (DV) patterning. The mutant displayed preaxial polydactyly in the hindlimbs of heterozygous embryos, and in both hindlimbs and forelimbs of homozygotes. The Shh, Fgf4, Fgf8, Hoxd12 and Hoxd13 genes were all ectopically expressed in the anterior region of affected limb buds. The insertion site was found to lie close to the Shh locus. Furthermore, expression from the transgene reporter has come under the control of a regulatory element that directs a pattern mirroring the endogenous expression pattern of Shh in limbs. In abnormal limbs, both Shh and the reporter were ectopically induced in the anterior region, whereas in normal limbs the reporter and Shh were restricted to the zone of polarising activity (ZPA). These data strongly suggest that Ssq is caused by direct interference with the cis regulation of the Shh gene.

Animals↗

PubMed1999

Hoxa2 and Hoxb2 control dorsoventral patterns of neuronal development in the rostral hindbrain.

Little is known about how the generation of specific neuronal types at stereotypic positions within the hindbrain is linked to Hox gene-mediated patterning. Here, we show that during neurogenesis, Hox paralog group 2 genes control both anteroposterior (A-P) and dorsoventral (D-V) patterning. Hoxa2 and Hoxb2 differentially regulate, in a rhombomere-specific manner, the expression of several genes in broad D-V-restricted domains or narrower longitudinal columns of neuronal progenitors, immature neurons, and differentiating neuronal subtypes. Moreover, Hoxa2 and Hoxb2 can functionally synergize in controlling the development of ventral neuronal subtypes in rhombomere 3 (r3). Thus, in addition to their roles in A-P patterning, Hoxa2 and Hoxb2 have distinct and restricted functions along the D-V axis during neurogenesis, providing insights into how neuronal fates are assigned at stereotypic positions within the hindbrain.

Animals↗

PubMed1999

Patterning the ascidian nervous system: structure, expression and transgenic analysis of the CiHox3 gene.

Hox genes play a fundamental role in the establishment of chordate body plan, especially in the anteroposterior patterning of the nervous system. Particularly interesting are the anterior groups of Hox genes (Hox1-Hox4) since their expression is coupled to the control of regional identity in the anterior regions of the nervous system, where the highest structural diversity is observed. Ascidians, among chordates, are considered a good model to investigate evolution of Hox gene, organisation, regulation and function. We report here the cloning and the expression pattern of CiHox3, a Ciona intestinalis anterior Hox gene homologous to the paralogy group 3 genes. In situ hybridization at the larva stage revealed that CiHox3 expression was restricted to the visceral ganglion of the central nervous system. The presence of a sharp posterior boundary and the absence of transcript in mesodermal tissues are distinctive features of CiHox3 expression when compared to the paralogy group 3 in other chordates. We have investigated the regulatory elements underlying CiHox3 neural-specific expression and, using transgenic analysis, we were able to isolate an 80 bp enhancer responsible of CiHox3 activation in the central nervous system (CNS). A comparative study between mouse and Ciona Hox3 promoters demonstrated that divergent mechanisms are involved in the regulation of these genes in vertebrates and ascidians.

Amino Acid Sequence↗

PubMed1999

Conserved and distinct roles of kreisler in regulation of the paralogous Hoxa3 and Hoxb3 genes.

During anteroposterior patterning of the developing hindbrain, the anterior expression of 3' Hox genes maps to distinct rhombomeric boundaries and, in many cases, is upregulated in specific segments. Paralogous genes frequently have similar anterior boundaries of expression but it is not known if these are controlled by common mechanisms. The expression of the paralogous Hoxa3 and Hoxb3 genes extends from the posterior spinal cord up to the rhombomere (r) 4/5 boundary and both genes are upregulated specifically in r5. However, in this study, we have found that Hoxa3 expression is also upregulated in r6, showing that there are differences in segmental expression between paralogues. We have used transgenic analysis to investigate the mechanisms underlying the pattern of segmental expression of Hoxa3. We found that the intergenic region between Hoxa3 and Hoxa4 contains several enhancers, which summed together mediate a pattern of expression closely resembling that of the endogenous Hoxa3 gene. One enhancer specifically directs expression in r5 and r6, in a manner that reflects the upregulation of the endogenous gene in these segments. Deletion analysis localized this activity to a 600 bp fragment that was found to contain a single high-affinity binding site for the Maf bZIP protein Krml1, encoded by the kreisler gene. This site is necessary for enhancer activity and when multimerized it is sufficient to direct a kreisler-like pattern in transgenic embryos. Furthermore the r5/r6 enhancer activity is dependent upon endogenous kreisler and is activated by ectopic kreisler expression. This demonstrates that Hoxa3, along with its paralog Hoxb3, is a direct target of kreisler in the mouse hindbrain. Comparisons between the Krml1-binding sites in the Hoxa3 and Hoxb3 enhancers reveal that there are differences in both the number of binding sites and way that kreisler activity is integrated and restricted by these two control regions. Analysis of the individual sites revealed that they have different requirements for mediating r5/r6 and dorsal roof plate expression. Therefore, the restriction of Hoxb3 to r5 and Hoxa3 to r5 and r6, together with expression patterns of Hoxb3 in other vertebrate species suggests that these regulatory elements have a common origin but have later diverged during vertebrate evolution.

Amino Acid Sequence↗

PubMed1999

Regulation of Hoxa2 in cranial neural crest cells involves members of the AP-2 family.

Hoxa2 is expressed in cranial neural crest cells that migrate into the second branchial arch and is essential for proper patterning of neural-crest-derived structures in this region. We have used transgenic analysis to begin to address the regulatory mechanisms which underlie neural-crest-specific expression of Hoxa2. By performing a deletion analysis on an enhancer from the Hoxa2 gene that is capable of mediating expression in neural crest cells in a manner similar to the endogenous gene, we demonstrated that multiple cis-acting elements are required for neural-crest-specific activity. One of these elements consists of a sequence that binds to the three transcription factor AP-2 family members. Mutation or deletion of this site in the Hoxa2 enhancer abrogates reporter expression in cranial neural crest cells but not in the hindbrain. In both cell culture co-transfection assays and transgenic embryos AP-2 family members are able to trans-activate reporter expression, showing that this enhancer functions as an AP-2-responsive element in vivo. Reporter expression is not abolished in an AP-2(alpha) null mutant embryos, suggesting redundancy with other AP-2 family members for activation of the Hoxa2 enhancer. Other cis-elements identified in this study critical for neural-crest-specific expression include an element that influences levels of expression and a conserved sequence, which when multimerized directs expression in a broad subset of neural crest cells. These elements work together to co-ordinate and restrict neural crest expression to the second branchial arch and more posterior regions. Our findings have identified the cis-components that allow Hoxa2 to be regulated independently in rhombomeres and cranial neural crest cells.

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PubMed1998

Selectivity, sharing and competitive interactions in the regulation of Hoxb genes.

The clustered organisation of Hox complexes is highly conserved in vertebrates and the reasons for this are believed to be linked with the regulatory mechanisms governing their expression. In analysis of the Hoxb4-Hoxb6 region of the HoxB complex we identified enhancers which lie in the intergenic region between Hoxb4 and Hoxb5, and which are capable of mediating the correct boundaries of neural and mesodermal expression for Hoxb5. We examined their regulatory properties in the context of the local genomic region spanning the two genes by transgenic analysis, in which each promoter was independently marked with a different reporter, to monitor simultaneously the relative transcriptional read-outs from each gene. Our analysis revealed that within this intergenic region: (i) a limb and a neural enhancer selectively activate Hoxb4 as opposed to Hoxb5; (ii) a separate neural enhancer is able to activate both genes, but expression is dependent upon competition between the two promoters for the enhancer and is influenced by the local genomic context; (iii) mesodermal enhancer activities can be shared between the genes. We found similar types of regulatory interactions between Hoxb5 and Hoxb6. Together these results provide evidence for three separate general mechanisms: selectivity, competition and sharing, that control the balance of cis-regulatory interactions necessary for generating the proper spatial and temporal patterns of Hox gene expression. We suggest that these mechanisms are part of a regulatory basis for maintenance of Hox organisation.

Alkaline Phosphatase↗

PubMed1998

Initiation of rhombomeric Hoxb4 expression requires induction by somites and a retinoid pathway.

Anteroposterior (AP) patterning in the vertebrate hindbrain is dependent upon the establishment of segmental domains of Hox expression. We investigated the mechanism that governs the early expression of Hoxb4 and found that transient signaling from the paraxial mesoderm induces expression in the hindbrain. Induction involves a retinoid pathway requiring retinoic acid receptor (RAR) function within the neural plate. Characterization of a prerhombomeric enhancer from Hoxb4 reveals that a retinoic acid (RA) response element is an essential component of the early neural response to somite (s) signaling and can interpret positional information for setting the anterior boundary of expression. These data suggest a mechanism whereby, during normal hindbrain development, Hoxb4 expression is initiated by extrinsic signals and is subsequently maintained by Hox feedback circuits. This mechanism also accounts for the ectopic response of Hoxb4 in rhombomere (r) transpositions and after exposure to retinoids.

Animals↗

PubMed1998

Genetic interactions between Hoxa1 and Hoxb1 reveal new roles in regulation of early hindbrain patterning.

In the developing vertebrate hindbrain Hoxa1 and Hoxb1 play important roles in patterning segmental units (rhombomeres). In this study, genetic analysis of double mutants demonstrates that both Hoxa1 and Hoxb1 participate in the establishment and maintenance of Hoxb1 expression in rhombomere 4 through auto- and para-regulatory interactions. The generation of a targeted mutation in a Hoxb1 3' retinoic acid response element (RARE) shows that it is required for establishing early high levels of Hoxb1 expression in neural ectoderm. Double mutant analysis with this Hoxb1(3'RARE) allele and other targeted loss-of-function alleles from both Hoxa1 and Hoxb1 reveals synergy between these genes. In the absence of both genes, a territory appears in the region of r4, but the earliest r4 marker, the Eph tyrosine kinase receptor EphA2, fails to be activated. This suggests a failure to initiate rather than maintain the specification of r4 identity and defines new roles for both Hoxb1 and Hoxa1 in early patterning events in r4. Our genetic analysis shows that individual members of the vertebrate labial-related genes have multiple roles in different steps governing segmental processes in the developing hindbrain.

Animals↗

PubMed1998

Hoxa1 and Hoxb1 synergize in patterning the hindbrain, cranial nerves and second pharyngeal arch.

The analysis of Hoxa1 and Hoxb1 null mutants suggested that these genes are involved in distinct aspects of hindbrain segmentation and specification. Here we investigate the possible functional synergy of the two genes. The generation of Hoxa1(3'RARE)/Hoxb1(3'RARE) compound mutants resulted in mild facial motor nerve defects reminiscent of those present in the Hoxb1 null mutants. Strong genetic interactions between Hoxa1 and Hoxb1 were uncovered by introducing the Hoxb1(3'RARE) and Hoxb1 null mutations into the Hoxa1 null genetic background. Hoxa1(null)/Hoxb1(3'RARE) and Hoxa1(null)/Hoxb1(null )double homozygous embryos showed additional patterning defects in the r4-r6 region but maintained a molecularly distinct r4-like territory. Neurofilament staining and retrograde labelling of motor neurons indicated that Hoxa1 and Hoxb1 synergise in patterning the VIIth through XIth cranial nerves. The second arch expression of neural crest cell markers was abolished or dramatically reduced, suggesting a defect in this cell population. Strikingly, the second arch of the double mutant embryos involuted by 10.5 dpc and this resulted in loss of all second arch-derived elements and complete disruption of external and middle ear development. Additional defects, most notably the lack of tympanic ring, were found in first arch-derived elements, suggesting that interactions between first and second arch take place during development. Taken together, our results unveil an extensive functional synergy between Hoxa1 and Hoxb1 that was not anticipated from the phenotypes of the simple null mutants.

Animals↗

PubMed1997

Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1.

Correct regulation of the segment-restricted patterns of Hox gene expression is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb2 in rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxb1 will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxb1, Hoxa1, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Furthermore, three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments we conclude that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene.

Animals↗

PubMed1997

Segmental regulation of Hoxb-3 by kreisler.

Hox genes control regional identity during segmentation of the vertebrate hindbrain into rhombomeres. Here we use transgenic analysis to investigate the upstream mechanisms for regulation of Hoxb-3 in rhombomere(r)5. We identified enhancers from the mouse and chick genes sufficient for r5-restricted expression. Sequence comparisons revealed two blocks of similarity (of 19 and 45 base pairs), which each contain in vitro binding sites for the kreisler protein (Kmrl1), a Maf/b-Zip protein expressed in r5 and r6 (ref. 4). Both sites are required for r5 activity, suggesting that Hoxb-3 is a direct target of kreisler. Multimers of the 19-base-pair (bp) block recreate a Krml1-like pattern in r5/r6, but the 45-bp block mediates expression only in r5. Therefore elements within the 45-bp block restrict the response to Krml1. We identified additional sequences that contain an Ets-related activation site, required for both the activation and restriction to r5. These studies demonstrate that Krml1 directly activates expression of Hoxb-3 in r5 in combination with an Ets-related activation site, and suggest that kreisler plays a primary role in regulating segmental identity through Hox genes.

Animals↗

PubMed1997

Hox9 genes and vertebrate limb specification.

Development of paired appendages at appropriate levels along the primary body axis is a hallmark of the body plan of jawed vertebrates. Hox genes are good candidates for encoding position in lateral plate mesoderm along the body axis and thus for determining where limbs are formed. Local application of fibroblast growth factors (FGFs) to the anterior prospective flank of a chick embryo induces development of an ectopic wing, and FGF applied to posterior flank induces an ectopic leg. If particular combinations of Hox gene expression determine where wings and legs develop, then formation of additional limbs from flank should involve changes in Hox gene expression that reflect the type of limb induced. Here we show that the same population of flank cells can be induced to form either a wing or a leg, and that induction of these ectopic limbs is accompanied by specific changes in expression of three Hox genes in lateral plate mesoderm. This then reproduces, in the flank, expression patterns found at normal limb levels. Hox gene expression is reprogrammed in lateral plate mesoderm, but is unaffected in paraxial mesoderm. Independent regulation of Hox gene expression in lateral plate mesoderm may have been a key step in the evolution of paired appendages.

Animals↗

PubMed1997

Positive cross-regulation and enhancer sharing: two mechanisms for specifying overlapping Hox expression patterns.

Vertebrate Hox genes display nested and overlapping patterns of expression. During mouse hindbrain development, Hoxb3 and Hoxb4 share an expression domain caudal to the boundary between rhombomeres 6 and 7. Transgenic analysis reveals that an enhancer (CR3) is shared between both genes and specifies this domain of overlap. Both the position of CR3 within the complex and its sequence are conserved from fish to mammals, suggesting it has a common role in regulating the vertebrate HoxB complex. CR3 mediates transcriptional activation by multiple Hox genes, including Hoxb4, Hoxd4, and Hoxb5 but not Hoxb1. It also functions as a selective HOX response element in Drosophila, where activation depends on Deformed, Sex combs reduced, and Antennapedia but not labial. Taken together, these data show that a Deformed/Hoxb4 autoregulatory loop has been conserved between mouse and Drosophila. In addition, these studies reveal the existence of positive cross-regulation and enhancer sharing as two mechanisms for reinforcing the overlapping expression domains of vertebrate Hox genes. In contrast, Drosophila Hox genes do not appear to share enhancers and where they overlap in expression, negative cross-regulatory interactions are observed. Therefore, despite many well documented aspects of Hox structural and functional conservation, there are mechanistic differences in Hox complex regulation between arthropods and vertebrates.

Animals↗

PubMed1997

Elements both 5' and 3' to the murine Hoxd4 gene establish anterior borders of expression in mesoderm and neurectoderm.

In this report, we show that a lacZ reporter spanning 12.5 kb of murine Hoxd4 genomic DNA contains the major regulatory elements controlling Hoxd4 expression in the mouse embryo. Mutational analysis revealed multiple regulatory regions both 5' and 3' to the coding region. These include a 3' enhancer region required for expression in the central nervous system (CNS) and setting the anterior border in the paraxial mesoderm, and a 5' mesodermal enhancer that directs expression in paraxial and lateral plate mesoderm. A previously defined retinoic acid response element (RARE) is a component of the 5' mesodermal enhancer. Our results support a model in which retinoic acid receptors (RARs) and HOX proteins mediate the initiation and maintenance of Hoxd4 expression.

Animals↗

PubMed1997

Organization of the Fugu rubripes Hox clusters: evidence for continuing evolution of vertebrate Hox complexes.

The clustered organization of Hox genes provides a powerful opportunity to examine gene gain and loss in evolution because physical linkage is a key diagnostic feature which allows homology to be established unambiguously. Furthermore, Hox genes play a key role in determination of axial and appendicular skeletal morphology and may be a key component of the evolution of diverse metazoan body forms. Despite suggestions that changes in Hox gene number played a role in evolution of metazoan body plans, there has been a general lack of evidence for such variation amongst gnathostomes (or indeed any vertebrate) and it has therefore been widely assumed that differential regulation may be the key element in all vertebrate Hox evolution. We have studied the Hox gene clusters of a teleost fish, Fugu rubripes, to test the possibility that Hox organization may have varied since the origin of jawed vertebrates. We have identified four Hox complexes in Fugu and found an unprecedented degree of variation when compared with tetrapod clusters. Our data show that: Fugu clusters are widely variant with respect to length; at least nine genes have been lost; there is a new group-2 paralogue; and pseudo-gene remnants of group-1 and group-3 paralogues were found in the Hoxc complex, when compared with the present mammalian clusters. We show that gene loss after duplication of the prototypical vertebrate Hox clusters is a key feature of both tetrapod and fish evolution.

Amino Acid Sequence↗

PubMed1997

Switching the in vivo specificity of a minimal Hox-responsive element.

The homeodomain proteins encoded by the Hox complex genes do not bind DNA with high specificity. In vitro, Hox specificity can be increased by binding to DNA cooperatively with the homeodomain protein extradenticle or its vertebrate homologs, the pbx proteins (together, the PBC family). Here we show that a two basepair change in a Hox-PBC binding site switches the Hox-dependent expression pattern generated in vivo, from labial to Deformed. The change in vivo correlates with an altered Hox binding specificity in vitro. Further, we identify similar Deformed-PBC binding sites in the Deformed and Hoxb-4 genes and show that they generate Deformed or Hoxb-4 expression patterns in Drosophila and mouse embryos, respectively. These results suggest a model in which Hox-PBC binding sites play an instructive role in Hox specificity by promoting the formation of different Hox-PBC heterodimers in vivo. Thus, the choice of Hox partner, and therefore Hox target genes, depends on subtle differences between Hox-PBC binding sites.

Animals↗

PubMed1997

Cell movements, neuronal organisation and gene expression in hindbrains lacking morphological boundaries.

Rhombomeres are segmental units of the hindbrain that are separated from each other by a specialised zone of boundary cells. Retinoic acid application to a recently segmented hindbrain leads to disappearance of posterior rhombomere boundaries. Boundary loss is preceded by changes in segmental expression of Krox-20 and Cek-8 and followed by alterations in Hox gene expression. The characteristic morphology of boundary cells, their expression of follistatin and the periodic accumulation of axons normally associated with boundaries are all lost. In the absence of boundaries, we detect no change in anteroposterior dispersal of precursor cells and, in most cases, no substantial cell mixing between former rhombomeric units. This is consistent with the idea that lineage restriction can be maintained by processes other than a mechanical barrier composed of boundary cells. Much of the early organisation of the motor nuclei appears normal despite the loss of boundaries and altered Hox expression.

Animals↗