Hoxb13 mutations cause overgrowth of caudal spinal cord and tail vertebrae

To address the expression and function of Hoxb13, the 5' most Hox gene in the HoxB cluster, we have generated mice with loss-of-function and beta-galactosidase reporter insertion alleles of this gene. Mice homozygous for Hoxb13 loss-of-function mutations show overgrowth in all major structures derived from the tail bud, including the developing secondary neural tube (SNT), the caudal spinal ganglia, and the caudal vertebrae. Using the beta-galactosidase reporter allele of Hoxb13, also a loss-of-function allele, we found that the expression patterns of Hoxb13 in the developing spinal cord and caudal mesoderm are closely associated with overgrowth phenotypes in the tails of homozygous mutant animals. These phenotypes can be explained by the observed increased cell proliferation and decreased levels of apoptosis within the tail of homozygous mutant mice. This analysis of Hoxb13 function suggests that this 5' Hox gene may act as an inhibitor of neuronal cell proliferation, an activator of apoptotic pathways in the SNT, and as a general repressor of growth in the caudal vertebrae.     

Neuronal defects in the hindbrain of Hoxa1, Hoxb1 and Hoxb2 mutants reflect regulatory interactions among these Hox genes

Hox genes are instrumental in assigning segmental identity in the developing hindbrain. Auto-, cross- and para-regulatory interactions help establish and maintain their expression. To understand to what extent such regulatory interactions shape neuronal patterning in the hindbrain, we analysed neurogenesis, neuronal differentiation and motoneuron migration in Hoxa1, Hoxb1 and Hoxb2 mutant mice. This comparison revealed that neurogenesis and differentiation of specific neuronal subpopulations in r4 was impaired in a similar fashion in all three mutants, but with different degrees of severity. In the Hoxb1 mutants, neurons derived from the presumptive r4 territory were re-specified towards an r2-like identity. Motoneurons derived from that territory resembled trigeminal motoneurons in both their migration patterns and the expression of molecular markers. Both migrating motoneurons and the resident territory underwent changes consistent with a switch from an r4 to r2 identity. Abnormally migrating motoneurons initially formed ectopic nuclei that were subsequently cleared. Their survival could be prolonged through the introduction of a block in the apoptotic pathway. The Hoxa1 mutant phenotype is consistent with a partial misspecification of the presumptive r4 territory that results from partial Hoxb1 activation. The Hoxb2 mutant phenotype is a hypomorph of the Hoxb1 mutant phenotype, consistent with the overlapping roles of these genes in facial motoneuron specification. Therefore, we have delineated the functional requirements in hindbrain neuronal patterning that follow the establishment of the genetic regulatory hierarchy between Hoxa1, Hoxb1 and Hoxb2.     

Axial skeletal patterning in mice lacking all paralogous group 8 Hox genes

We present a detailed study of the genetic basis of mesodermal axial patterning by paralogous group 8 Hox genes in the mouse. The phenotype of Hoxd8 loss-of-function mutants is presented, and compared with that of Hoxb8- and Hoxc8-null mice. Our analysis of single mutants reveals common features for the Hoxc8 and Hoxd8 genes in patterning lower thoracic and lumbar vertebrae. In the Hoxb8 mutant, more anterior axial regions are affected. The three paralogous Hox genes are expressed up to similar rostral boundaries in the mesoderm, but at levels that strongly vary with the axial position. We find that the axial region affected in each of the single mutants mostly corresponds to the area with the highest level of gene expression. However, analysis of double and triple mutants reveals that lower expression of the other two paralogous genes also plays a patterning role when the mainly expressed gene is defective. We therefore conclude that paralogous group 8 Hox genes are involved in patterning quite an extensive anteroposterior (AP) axial region. Phenotypes of double and triple mutants reveal that Hoxb8, Hoxc8 and Hoxd8 have redundant functions at upper thoracic and sacral levels, including positioning of the hindlimbs. Interestingly, loss of functional Hoxb8 alleles partially rescues the phenotype of Hoxc8- and Hoxc8/Hoxd8-null mutants at lower thoracic and lumbar levels. This suggests that Hoxb8 affects patterning at these axial positions differently from the other paralogous gene products. We conclude that paralogous Hox genes can have a unique role in patterning specific axial regions in addition to their redundant function at other AP levels.     

Mice mutant for both Hoxa1 and Hoxb1 show extensive remodeling of the hindbrain and defects in craniofacial development.

The analysis of mice mutant for both Hoxa1 and Hoxb1 suggests that these two genes function together to pattern the hindbrain. Separately, mutations in Hoxa1 and Hoxb1 have profoundly different effects on hindbrain development. Hoxa1 mutations disrupt the rhombomeric organization of the hindbrain, whereas Hoxb1 mutations do not alter the rhombomeric pattern, but instead influence the fate of cells originating in rhombomere 4. We suggest that these differences are not the consequences of different functional roles for these gene products, but rather reflect differences in the kinetics of Hoxa1 and Hoxb1 gene expression. In strong support of the idea that Hoxa1 and Hoxb1 have overlapping functions, Hoxa1/Hoxb1 double mutant homozygotes exhibit a plethora of defects either not seen, or seen only in a very mild form, in mice mutant for only Hoxa1 or Hoxb1. Examples include: the loss of both rhombomeres 4 and 5, the selective loss of the 2(nd) branchial arch, and the loss of most, but not all, 2(nd) branchial arch-derived tissues. We suggest that the early role for both of these genes in hindbrain development is specification of rhombomere identities and that the aberrant development of the hindbrain in Hoxa1/Hoxb1 double mutants proceeds through two phases, the misspecification of rhombomeres within the hindbrain, followed subsequently by size regulation of the misspecified hindbrain through induction of apoptosis.     

Getting a GRIP on liprins

Excessive grooming behaviors, cleansing rituals, and self-mutilation are important features of a range of neuropsychiatric diseases including obsessive compulsive (OC)-spectrum disorders. In this issue of Neuron, Greer and Capecchi (2002) report that Hoxb8 mutant mice exhibit this behavioral phenotype. These Hoxb8 mutants will be valuable in exploring the genetics and pathophysiology of OC-spectrum disorders as well as strategies for their treatment.     

Medaka unextended-fin mutants suggest a role for Hoxb8a in cell migration and osteoblast differentiation during appendage formation

Hoxb8 has been suggestively implicated in the formation of the zone of polarizing activity (ZPA) in the limb bud. However, as hoxb8-/- mice did not show any defects in their limb development, the role of Hoxb8 during limb development has not been fully elucidated. Here, we report the identification of the medaka hoxb8a mutant, unextended-fin (ufi), in which all the fin tissues were malformed. Since the abnormal phenotype was observed in the caudal fin, the ufi phenotype suggests that the medaka Hoxb8a has a fundamental role in the formation of appendages protruding from the trunk. Our analyses revealed that the expression of wnt5a, a regulator of cell migration that signals through the non-canonical Wnt/Ca2+ pathway, was down-regulated in the ufi fin-folds. In fact, we found that the proximal-distal cell migration was impaired in ufi mutants and that the defect could be reversed by the injection of a Wnt5a protein. Moreover, we show herein that the numbers of proliferating cells and osteoblastic cells were increased in the ufi mutants. According to these results, we propose that the medaka Hoxb8a protein functions in the outgrowth of appendages through the regulation of cell migration and osteoblast differentiation.     

Hoxb13 is required for normal differentiation and secretory function of the ventral prostate

The murine prostate is a structure that is made up of four distinct lobes; the dorsal and lateral prostates (often grouped together as the dorsolateral prostate), the anterior (coagulating gland) and the ventral prostate. Previous work has implicated Hox genes in the development of these structures, but how each lobe acquires unique identities for specific functions has not been addressed. In this study, the ventral prostate-specific function of Hoxb13 is described. Mice lacking Hoxb13 function show normal numbers of duct tips, but mice mutant for both Hoxb13 and Hoxd13 exhibit severe hypoplasia of the duct tips, revealing a role for Hoxb13 in ventral prostate morphogenesis. Additionally, a ventral lobe-specific defect was identified in Hoxb13 mutants wherein the epithelium is composed of simple cuboidal cells rather than of tall columnar cells. Ventral prostate ducts appear devoid of contents and do not express the ventral prostate-specific secretory proteins p12, a kazal-type protease inhibitor and p25, a spermine binding protein. These defects are not due to reduction of Nkx3.1 expression or to a global effect on androgen receptor signaling. These results suggest a specific role for Hoxb13 in a differentiation pathway that gives the ventral prostate epithelium a unique identity, as well as a more general role in ventral prostate morphogenesis that is redundant with other Hox13 paralogs.     

Roles of Hoxa1 and Hoxa2 in patterning the early hindbrain of the mouse

Early in its development, the vertebrate hindbrain is transiently subdivided into a series of compartments called rhombomeres. Genes have been identified whose expression patterns distinguish these cellular compartments. Two of these genes, Hoxa1 and Hoxa2, have been shown to be required for proper patterning of the early mouse hindbrain and the associated neural crest. To determine the extent to which these two genes function together to pattern the hindbrain, we generated mice simultaneously mutant at both loci. The hindbrain patterning defects were analyzed in embryos individually mutant for Hoxa1 and Hoxa2 in greater detail and extended to embryos mutant for both genes. From these data a model is proposed to describe how Hoxa1, Hoxa2, Hoxb1, Krox20 (Egr2) and kreisler function together to pattern the early mouse hindbrain. Critical to the model is the demonstration that Hoxa1 activity is required to set the anterior limit of Hoxb1 expression at the presumptive r3/4 rhombomere boundary. Failure to express Hoxb1 to this boundary in Hoxa1 mutant embryos initiates a cascade of gene misexpressions that result in misspecification of the hindbrain compartments from r2 through r5. Subsequent to misspecification of the hindbrain compartments, ectopic induction of apoptosis appears to be used to regulate the aberrant size of the misspecified rhombomeres.     

Hoxb2 and hoxb4 act together to specify ventral body wall formation

Three different alleles of the Hoxb4 locus were generated by gene targeting in mice. Two alleles contain insertions of a selectable marker in the first exon in either orientation, and, in the third, the selectable marker was removed, resulting in premature termination of the protein. Presence and orientation of the selectable marker correlated with the severity of the phenotype, indicating that the selectable marker induces cis effects on neighboring genes that influence the phenotype. Homozygous mutants of all alleles had cervical skeletal defects similar to those previously reported for Hoxb4 mutant mice. In the most severe allele, Hoxb4(PolII), homozygous mutants died either in utero at approximately E15.5 or immediately after birth, with a severe defect in ventral body wall formation. Analysis of embryos showed thinning of the primary ventral body wall in mutants relative to control animals at E11.5, before secondary body wall formation. Prior to this defect, both Alx3 and Alx4 were specifically down regulated in the most ventral part of the primary body wall in Hoxb4(PolII) mutants. Hoxb4(loxp) mutants in which the neo gene has been removed did not have body wall or sternum defects. In contrast, both the Hoxb4(PolII) and the previously described Hoxb2(PolII) alleles that have body wall defects have been shown to disrupt the expression of both Hoxb2 and Hoxb4 in cell types that contribute to body wall formation. Our results are consistent with a model in which defects in ventral body wall formation require the simultaneous loss of at least Hoxb2 and Hoxb4, and may involve Alx3 and Alx4.     

Hoxb8 is required for normal grooming behavior in mice.

Repertoires of grooming behaviors critical to survival are exhibited by most animal species, including humans. Genes that influence this complex behavior are unknown. We report that mice with disruptions of Hoxb8 show, with 100% penetrance, excessive grooming leading to hair removal and lesions. Additionally, these mice excessively groom normal cagemates. We have been unable to detect any skin or PNS abnormalities in Hoxb8 mutants. These observations suggest that the excessive, pathological grooming exhibited by these mice results from CNS abnormalities. Consistent with this interpretation, we demonstrate Hoxb8 expression in regions of the adult mouse CNS previously implicated in the control of grooming. The aberrant behavior observed in Hoxb8 mutants is not unlike that of humans suffering from the OC-spectrum disorder, trichotillomania. Interestingly, Hoxb8 is expressed in regions of the CNS known as the "OCD-circuit."     

A large targeted deletion of Hoxb1-Hoxb9 produces a series of single-segment anterior homeotic transformations

Hox genes regulate axial regional specification during animal embryonic development and are grouped into four clusters. The mouse HoxB cluster contains 10 genes, Hoxb1 to Hoxb9 and Hoxb13, which are transcribed in the same direction. We have generated a mouse strain with a targeted 90-kb deletion within the HoxB cluster from Hoxb1 to Hoxb9. Surprisingly, heterozygous mice show no detectable abnormalities. Homozygous mutant embryos survive to term and exhibit an ordered series of one-segment anterior homeotic transformations along the cervical and thoracic vertebral column and defects in sternum morphogenesis. Neurofilament staining indicates abnormalities in the IXth cranial nerve. Notably, simultaneous deletion of Hoxb1 to Hoxb9 resulted in the sum of phenotypes of single HoxB gene mutants. Although a higher penetrance is observed, no synergistic or new phenotypes were observed, except for the loss of ventral curvature at the cervicothoracic boundary of the vertebral column. Although Hoxb13, the most 5' gene, is separated from the rest by 70 kb, it has been suggested to be expressed with temporal and spatial colinearity. Here, we show that the expression pattern of Hoxb13 is not affected by the targeted deletion of the other 9 genes. Thus, Hoxb13 expression seems to be independent of the deleted region, suggesting that its expression pattern could be achieved independent of the colinear pattern of the cluster or by a regulatory element located 5' of Hoxb9.     

Retinoids regulate the anterior expression boundaries of 5' Hoxb genes in posterior hindbrain

We describe the regulatory interactions that cause anterior extension of the mouse 5' Hoxb expression domains from spinal cord levels to their definitive boundaries in the posterior hindbrain between embryonic day E10 and E11.5. This anterior expansion is retinoid dependent since it does not occur in mouse embryos deficient for the retinoic acid-synthesizing enzyme retinaldehyde dehydrogenase 2. A retinoic acid response element (RARE) was identified downstream of Hoxb5 and shown to be essential for expression of Hoxb5 and Hoxb8 reporter transgenes in the anterior neural tube. The spatio-temporal activity of this element overlaps with rostral extension of the expression domain of endogenous Hoxb5, Hoxb6 and Hoxb8 into the posterior hindbrain. The RARE and surrounding sequences are found at homologous positions in the human, mouse and zebrafish genome, which supports an evolutionarily conserved regulatory function.     

Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the mammalian Polycomb group of genes

During animal development, Hox genes are expressed in characteristic, spatially restricted patterns and specify regional identities along the anterior-posterior (A-P) axis. Polycomb group (PcG) proteins in Drosophila repress Hox expression and maintain the expression patterns during development. Mice deficient for homologues of the Drosophila PcG genes, such as M33, bmi1, mel18, rae28 and eed, show altered Hox expression patterns. In this study, we examined the time course of Hoxb3 expression during late gastrulation and early segmentation of rae28-deficient mice. Hoxb3 was expressed ectopically in pharyngeal arch and hindbrain from embryonic day (E) 9.5 and 10.5, respectively. The anterior boundary of ectopic expression in the hindbrain extended gradually in the rostral direction as development proceeded from E10.5 to E12.5. Expression of kreisler and Krox20, which function as positive regulators of Hoxb3 expression, was not affected in rae28-deficient embryos. Analysis of a neural crest marker, p75, in rae28-deficient mice revealed that the neural crest cells begin to ectopically express Hoxb3 after leaving the hindbrain. Our results suggest that rae28 is not required for the establishment but maintenance of Hoxb3 expression.