Retinoic acid rescues inner ear defects in Hoxa1 deficient mice.

Little is known about the genetic pathways involved in the early steps of inner ear morphogenesis. Hoxa1 is transiently expressed in the developing hindbrain; its targeted inactivation in mice results in severe abnormalities of the otic capsule and membranous labyrinth. Here we show that a single maternal administration of a low dose of the vitamin A metabolite retinoic acid is sufficient to compensate the requirement for Hoxa1 function. It rescues cochlear and vestibular defects in mutant fetuses without affecting the development of the wildtype fetuses. These results identify a temporal window of susceptibility to retinoids that is critical for mammalian inner ear specification, and provide the first evidence that a subteratogenic dose of vitamin A derivative can be effective in rescuing a congenital defect in the mammalian embryo.

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.

Genetic interactions of Hox genes in limb development: learning from compound mutants.

The recent generation of mice harboring multiple mutations in Hox genes has highlighted the role of these genes in controlling growth and patterning of the limb bud. The study of the phenotypes has not only revealed a complex network of genetic interactions among paralogous and non-paralogous Abdominal-B-related Hox genes but has also refined our understanding of their function.

Generation of a novel functional neuronal circuit in Hoxa1 mutant mice.

Early organization of the vertebrate brainstem is characterized by cellular segmentation into compartments, the rhombomeres, which follow a metameric pattern of neuronal development. Expression of the homeobox genes of the Hox family precedes rhombomere formation, and analysis of mouse Hox mutations revealed that they play an important role in the establishment of rhombomere-specific neuronal patterns. However, segmentation is a transient feature, and a dramatic reconfiguration of neurons and synapses takes place during fetal and postnatal stages. Thus, it is not clear whether the early rhombomeric pattern of Hox expression has any influence on the establishment of the neuronal circuitry of the mature brainstem. The Hoxa1 gene is the earliest Hox gene expressed in the developing hindbrain. Moreover, it is rapidly downregulated. Previous analysis of mouse Hoxa1(-/-) mutants has focused on early alterations of hindbrain segmentation and patterning. Here, we show that ectopic neuronal groups in the hindbrain of Hoxa1(-/-) mice establish a supernumerary neuronal circuit that escapes apoptosis and becomes functional postnatally. This system develops from mutant rhombomere 3 (r3)-r4 levels, includes an ectopic group of progenitors with r2 identity, and integrates the rhythm-generating network controlling respiration at birth. This is the first demonstration that changes in Hox expression patterns allow the selection of novel neuronal circuits regulating vital adaptive behaviors. The implications for the evolution of brainstem neural networks are discussed.

A Xenopus multifinger protein, Xfin, is expressed in specialized cell types and is localized in the cytoplasm.

Xfin is a member of a large family of Krüppel-type transcripts stored in the Xenopus egg, whose function is unknown. By using polyclonal antibodies raised against fusion proteins containing different portions of Xfin, we have identified the Xfin gene product and established its pattern of expression in some adult tissues and during oogenesis and embryogenesis. The corresponding mRNA localization has been studied by in situ hybridization on ovary and testis sections. The Xfin product is found in the cytoplasm, both during oogenesis and adulthood; in adult tissues, it is differentially expressed in a cell-type specific fashion. The expression of the protein in specialized cell types and its cytoplasmic localization may favour the hypothesis that it could be involved in cell differentiation events through protein-RNA interactions.

Segmentation and specification in the branchial region of the head: the role of the Hox selector genes.

Hox genes are segmentally expressed in the developing vertebrate hindbrain, neural crest cells and pharyngeal arches suggesting an important role in patterning these structures. Here we discuss the cellular and molecular mechanisms controlling segmentation and specification in the branchial region of the head. In addition, based on the recent phenotypical and molecular analysis of loss-of-function mutants in the mouse, we speculate that Hox genes may act like Drosophila selector genes in this system.

A Zn-finger protein, Xfin, is expressed during cone differentiation in the retina of the frog Xenopus laevis.

Xfin is a member of a Zn-finger multigene family that shares homology with the Drosophila segmentation gene, Krüppel. This paper reports on our identification and cellular localization, through the utilization of specific antibodies, of the expression product of Xfin in the Xenopus laevis retina, and its pattern of expression during the retinal developmental stages. By immunostaining sections of the retina, we show that the major staining is localized in the cytoplasm of the cones. The protein appears at an early differentiation stage of the cones, when they can not be univocally identified by morphological criteria, and is maintained up to the adult retina. The same antigenicity pattern is detectable in the retina of the anuran genus Bufo. The immunostaining data are confirmed by Western blot analysis on Xenopus eye protein extracts. Because of its cytoplasmic localization, and because of the ability of Zn-finger proteins to bind nucleic acids, we think that Xfin may be involved in the terminal differentiation of the cones through RNA-protein interactions.

Vitamin A prevents inner ear defects in mice with congenital homeobox gene deficiency.

For the past 75 years, vitamin A and its biologically active metabolites, the retinoids, have been the object of intense study in biology and medicine. A large body of evidence demonstrates that these nutrients are essential for normal development and survival of vertebrate embryos, including mammals. In fact, it has been known since the mid-1930s that vitamin A deficiency during pregnancy results in death of the fetus and congenital abnormalities. Similarly, excess dietary intake of vitamin A can also cause teratogenic responses. Among the main targets of both deficiency and excess retinoid-induced teratogenesis are the heart, limbs, craniofacial structures, central nervous system, and the inner ear. Specific malformations are induced in a stage- and dose-dependent manner. Thus, these studies indicate that precise levels and timing of action of vitamin A metabolites are required for normal patterning of embryonic structures. In addition, the discovery of the nuclear receptors for retinoic acid (RA) and other vitamin A derivatives provided a molecular basis to explain how distinct doses of these compounds elicit cell-specific responses via the direct transcriptional activation of a panel of target genes.

Homeobox gene mutations and brain-stem developmental disorders: learning from knockout mice.

Analysis of mice that carry targeted inactivations of Hox, Nkx and Phox2 homeobox genes revealed their involvement in regional patterning of brain-stem territories, in specification of neuronal identity, in establishment of appropriate patterns of connectivity and in control of neurotransmission. The specific abnormalities generated by such mutations may provide clues to the genetic basis and cellular mechanisms that are involved in human brain-stem developmental disorders.

Homeobox genes in embryogenesis and pathogenesis.

The homeobox, a 60-amino acid-encoding DNA sequence, originally discovered in the genome of the fruit fly Drosophila, was subsequently identified throughout the three kingdoms of multicellular organisms. Homeobox-containing genes encode DNA-binding proteins that regulate gene expression and control various aspects of morphogenesis and cell differentiation. In particular, the Hox family of clustered homeobox genes plays a fundamental role in the morphogenesis of the vertebrate embryo, providing cells with regional information along the main body axis. The nonclustered or divergent homeobox genes include a large number of genes scattered throughout the genome that, nevertheless, can be organized in distinct families based on their homologies and functional similarities. This review will provide the reader with a brief overview on some recent studies aimed at understanding the functional role of homeobox genes in normal mammalian development as well as their involvement in congenital malformations and oncogenesis.

Ectopic Hoxa2 induction after neural crest migration results in homeosis of jaw elements in Xenopus.

Hox genes are required to pattern neural crest (NC) derived craniofacial and visceral skeletal structures. However, the temporal requirement of Hox patterning activity is not known. Here, we use an inducible system to establish Hoxa2 activity at distinct NC migratory stages in Xenopus embryos. We uncover stage-specific effects of Hoxa2 gain-of-function suggesting a multistep patterning process for hindbrain NC. Most interestingly, we show that Hoxa2 induction at postmigratory stages results in mirror image homeotic transformation of a subset of jaw elements, normally devoid of Hox expression, towards hyoid morphology. This is the reverse phenotype to that observed in the Hoxa2 knockout. These data demonstrate that the skeletal pattern of rhombomeric mandibular crest is not committed before migration and further implicate Hoxa2 as a true selector of hyoid fate. Moreover, the demonstration that the expression of Hoxa2 alone is sufficient to transform the upper jaw and its joint selectively may have implications for the evolution of jaws.

Role of Hoxa-2 in axon pathfinding and rostral hindbrain patterning.

Segmentation plays an important role in neuronal diversification and organisation in the developing hindbrain. For instance, cranial nerve branchiomotor nuclei are organised segmentally within the basal plates of successive pairs of rhombomeres. To reach their targets, motor axons follow highly stereotyped pathways exiting the hindbrain only via specific exit points in the even-numbered rhombomeres. Hox genes are good candidates for controlling this pathfinding, since they are segmentally expressed and involved in rhombomeric patterning. Here we report that in Hoxa-2(-/-) embryos, the segmental identities of rhombomere (r) 2 and r3 are molecularly as well as anatomically altered. Cellular analysis by retrograde dye labelling reveals that r2 and r3 trigeminal motor axons turn caudally and exit the hindbrain from the r4 facial nerve exit point and not from their normal exit point in r2. Furthermore, dorsal r2-r3 patterning is affected, with loss of cochlear nuclei and enlargement of the lateral part of the cerebellum. These results point to a novel role for Hoxa-2 in the control of r2-r3 motor axon guidance, and also suggest that its absence may lead to homeotic changes in the alar plates of these rhombomeres.

A homeotic transformation is generated in the rostral branchial region of the head by disruption of Hoxa-2, which acts as a selector gene.

The Hoxa-2 gene was disrupted by homologous recombination. Homozygous mutant mice died at birth. Defects were found in the branchial region of the head, which corresponds to the Hoxa-2 rostral expression domain. While rhombomeric and neural crest cell (NCC) segmentation was not affected, mesenchymal NCC derivatives of the second arch were lacking, and second arch mesenchymal NCC identity was changed to first arch identity, resulting in homeotic transformation of second to first arch skeletal elements. These results reveal the existence of a skeletogenic ground pattern program common to at least the mesenchymal NCC that originated from rhombomeres 2 and 4. The appearance of an atavistic reptilian pterygoquadrate element in Hoxa-2 mutants suggests that this ground pattern is intermediate between reptiles and mammals. The ground pattern program appears to be modified in the mouse first arch by a Hox-independent process, whereas Hoxa-2 acts as a selector gene in the second arch.

Functional cooperation between the non-paralogous genes Hoxa-10 and Hoxd-11 in the developing forelimb and axial skeleton.

The Abdominal B-related Hoxa-10 gene displays similar expression patterns in the differentiating forelimbs and hindlimbs of the mouse, with preferential expression around the humeral and femoral cartilages and more diffuse expression in distal regions. We found that a targeted disruption of Hoxa-10 has almost no effect in the forelimbs, while it affects the proximal hindlimb skeleton. The alterations were located along the dorsolateral side of the femur (labium laterale), with an enlargement and distal shift of the third trochanter, a misshapen lateral knee sesamoid, a supernumerary 'ligament' connecting these structures and an occasional duplication of the femoral trochlea. Some Hoxa-10-/- mutant mice developed severe degenerative alterations of the knee articulation upon ageing. Viable Hoxa-10/Hoxd-11 double mutant mice were produced by genetic intercrosses. The compound mutation resulted in synergistic forelimb phenotypic alterations, consisting of: (i) an exacerbation of Hoxd-11-/- phenotypic traits in the carpal and digital region, e.g. more pronounced truncations of the ulna styloid, pyramidal and pisiform bones and of some metacarpal and phalangeal bones and (ii) marked alterations in a more proximal region which is nearly unaffected in Hoxd-11-/- single mutants; the entire radius and ulna were truncated and thickened, with deformations of the ulna proximal extremity. Thus, functional redundancy can occur even between non-paralogous Abdominal B-related Hox genes. The double Hoxa-10/Hoxd-11 mutation also conferred full penetrance to the sacral and caudal vertebrae transformations which are approximately 50% penetrant in Hoxd-11-/- single mutants, revealing that functional cooperation can also occur between non-paralogous Hox gene products in axial skeleton patterning.

Two dispersed highly repeated DNA families of Triturus vulgaris meridionalis (Amphibia, Urodela) are widely conserved among Salamandridae.

Two BamHI families of repeated sequences were characterized from the genome of the Italian smooth newt, Triturus vulgaris meridionalis (Amphibia, Urodela). The first family, which is divided into subfamilies, consists of tandemly arranged arrays whose basic repeat is around 398 bp long; these arrays are dispersed throughout the entire chromosome sets of the various species of Triturus tested. Moreover the family is widely conserved among Salamandridae, being detected by genomic DNA blotting of Notophthalmus viridescens, Taricha granulosa, Salamandrina terdigitata and Euproctus platycephalus. The second BamHI family is represented by a cloned sequence of 419 bp, which is dispersed in the chromosome set of several species of Triturus. The sequence is also conserved in S. terdigitata and in E. platycephalus but is not detectable in N. viridescens or T. granulosa. The cloned sequence is most probably only part of a longer unit interspersed within the Triturus genome.

Insertion of a targeting construct in a Hoxd-10 allele can influence the control of Hoxd-9 expression.

A neomycin resistance (neo) gene driven by the phosphoglycerokinase (PGK) promoter was inserted into the Hoxd-10 homeobox by homologous recombination in embryonic stem (ES) cells. Chimeric mice derived from ES cell-injected blastocysts died shortly after birth. Craniofacial and axial abnormalities were found in the skeleton of these chimeras, resembling some of the previously described Hox gene gain-of-function phenotypes. The spatial expression patterns of various Hoxd gene transcripts were analysed in chimeric mutant embryos by in situ hybridization. Two main observations were made: (1) a wide ectopic expression domain of the Hoxd-9 gene was found in the spinal cord of these embryos, and (2) the neo gene exhibited a specific Hox-like expression domain which extended far more rostrally than that of the Hoxd-10 gene, showing that, in the context of this mutation, the PGK promoter could be regulated as a Hox promoter. These results provide the first evidence that a targeted insertion into a Hox gene coding sequence, in the context of its own cluster, could result in misexpression of a neighbour gene of the complex.

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.

In vivo functional analysis of the Hoxa-1 3' retinoic acid response element (3'RARE).

Retinoids are essential for normal development and both deficiency and excess of retinoic acid (RA) are teratogenic. Retinoic acid response elements (RAREs) have been identified in Hox gene promoters suggesting that endogenous retinoids may be involved in the direct control of Hox gene patterning functions. In order to test this hypothesis, we have mutated the Hoxa-1 3'RARE using the Cre-loxP targeting strategy, and studied its functional role during mouse development. We find that this enhancer plays an important role in the early establishment of the Hoxa-1 anterior expression boundary in the neural plate. This early disturbance in Hoxa-1 activation results in rhombomere and cranial nerve abnormalities reminiscent of those obtained in the Hoxa-1 total knockout, although their severity and penetrance are lower, thus providing strong evidence for direct control of Hox gene function by retinoids during normal development. Interestingly, we also find that the Hoxa-1 expression response to RA treatment is not entirely controlled by the RARE, suggesting the existence of other retinoid-induced factors mediating the Hoxa-1 response to RA and/or the presence of additional RAREs. Interestingly, although the RARE is not required for the spatiotemporal control of colinear expression of the Hoxa genes, it is absolutely required for correct Hoxa-2 expression in rhombomere 5.

Hoxa1 and Krox-20 synergize to control the development of rhombomere 3.

The transcription factor genes Hoxa1 and Krox-20 have been shown to play important roles in vertebrate hindbrain segmentation. In this report, we present evidence for novel functions of these genes which co-operate in specifying cellular identity in rhombomere (r) 3. Although Hoxa1 has not been observed to be expressed rostrally to the prospective r3/r4 boundary, its inactivation results in (i) the appearance of patches of cells presenting an r2-like molecular identity within r3, (ii) early neuronal differentiation in r3, normally characteristic of even-numbered rhombomeres, and (iii) abnormal navigation of r3 motor axons, similar to that observed in even-numbered rhombomeres. These phenotypic manifestations become more severe in the context of the additional inactivation of one allele of the Krox-20 gene, demonstrating that Hoxa1 and Krox-20 synergize in a dosage-dependent manner to specify r3 identity and odd- versus even-numbered rhombomere characters. In addition, these data suggest that the control of the development of r3 may not be autonomous but dependent on interactions with Hoxa1-expressing cells.

The expression pattern of the mouse receptor tyrosine kinase gene MDK1 is conserved through evolution and requires Hoxa-2 for rhombomere-specific expression in mouse embryos.

Segmentation of the hindbrain has been conserved throughout the vertebrate species and results in the transient formation of rhombomeres, which are lineage-restricted compartments. Studies on the molecular mechanisms underlying the segmentation process have revealed that rhombomeric boundaries coincide with the expression limits of several evolutionary conserved genes such as the zinc-finger transcription factor Krox-20 and homeobox genes which are expressed in a specific spatial and temporal order and have been shown to be important regulators of segmental identity. In addition to Krox-20 and Hox genes, several members of the Eph subfamily of receptor protein tyrosine kinase (RTK) genes are also expressed in a segment-restricted manner in the hindbrain, suggesting that these receptors may act in concert with Hox genes to establish regional identity. In the cascade of regulatory interactions leading to segmental identity, Krox-20 appears to act "upstream" of Hox genes, but the identity of the "downstream" effectors has not yet been identified. We report here the isolation of the zebrafish orthologue of the mouse RTK gene MDK1 which belongs to the Eph receptor subfamily and show that the major expression domains of the mouse and the zebrafish genes have been conserved through evolution. Since the coincident spatial and temporal expression of Hoxa-2 and MDK1 in the mouse hindbrain suggested a possible regulatory link between them, we analyzed the expression of the MDK1 in Hoxa-2 null mutant embryos. A selective lack of MDK1 expression in rhombomere 3 of Hoxa-2 mutant hindbrains together with an overall altered expression pattern in the other rhombomeres was observed, thus demonstrating that MDK1 lies downstream of Hoxa-2 in the morphogenetic signaling cascade.