Hoxb13 up-regulates transglutaminase activity and drives terminal differentiation in an epidermal organotypic model

Hox genes act to differentiate and pattern embryonic structures by promoting the proliferation of specific cell types. An exception is Hoxb13, which functions as a proapoptotic and antiproliferative protein during development of the caudal spinal cord and tail vertebrae and has also been implicated in adult cutaneous wound repair. The adult epidermis, which expresses several Hox genes including Hoxb13, is continually renewed in a program of growth arrest, differentiation, and a specialized form of apoptosis (cornification). Yet little is known about the function(s) of these genes in skin. Based on its role during embryogenesis, Hoxb13 is an attractive candidate to be involved in the regulation of epidermal differentiation. Here, we demonstrate that Hoxb13 overexpression in an adult organotypic epidermal model recapitulates actions of Hoxb13 reported in embryonic development. Epidermal cell proliferation is decreased, apoptosis increased, and excessive terminal differentiation observed, as characterized by enhanced transglutaminase activity and excessive cornified envelope formation. Overexpression of Hoxb13 also produces abnormal phenotypes in the epidermal tissue that resemble certain pathological features of dysplastic skin diseases. Our results suggest that Hoxb13 functions to promote epidermal differentiation, a critical process for skin regeneration and for the maintenance of normal barrier function.     

Abnormalities of caudal pharyngeal pouch development in Pbx1 knockout mice mimic loss of Hox3 paralogs

Pbx1 is a TALE-class homeodomain protein that functions in part as a cofactor for Hox class homeodomain proteins. Previous analysis of the in vivo functions of Pbx1 by targeted mutagenesis in mice has revealed roles for this gene in skeletal patterning and development and in the organogenesis of multiple systems. Both RNA expression and protein localization studies have suggested a possible role for Pbx1 in pharyngeal region development. As several Hox mutants have distinct phenotypes in this region, we investigated the potential requirement for Pbx1 in the development of the pharyngeal arches and pouches and their organ derivatives. Pbx1 homozygous mutants exhibited delayed or absent formation of the caudal pharyngeal pouches, and disorganized patterning of the third pharyngeal pouch. Formation of the third pouch-derived thymus/parathyroid primordia was also affected, with absent or hypoplastic primordia, delayed expression of organ-specific differentiation markers, and reduced proliferation of thymic epithelium. The fourth pouch and the fourth pouch-derived ultimobranchial bodies were usually absent. These phenotypes are similar to those previously reported in Hoxa3(-/-) single mutants and Hoxa1(-/-);Hoxb1(-/-) or Hoxa3(+/-);Hoxb3(-/-);Hoxd3(-/-) compound mutants, suggesting that Pbx1 acts together with multiple Hox proteins in the development of the caudal pharyngeal region. However, some aspects of the Pbx1 mutant phenotype included specific defects that were less severe than those found in known Hox mutant mice, suggesting that some functions of Hox proteins in this region are Pbx1-independent.     

Auto/cross-regulation of Hoxb3 expression in posterior hindbrain and spinal cord

The complex and dynamic pattern of Hoxb3 expression in the developing hindbrain and the associated neural crest of mouse embryos is controlled by three separate cis-regulatory elements: element I (region A), element IIIa, and the r5 enhancer (element IVa). We have examined the cis-regulatory element IIIa by transgenic and mutational analysis to determine the upstream trans-acting factors and mechanisms that are involved in controlling the expression of the mouse Hoxb3 gene in the anterior spinal cord and hindbrain up to the r5/r6 boundary, as well as the associated neural crest which migrate to the third and posterior branchial arches and to the gut. By deletion analysis, we have identified the sequence requirements within a 482-bp element III482. Two Hox binding sites are identified in element III482 and we have shown that in vitro both Hoxb3 and Hoxb4 proteins can interact with these Hox binding sites, suggesting that auto/cross-regulation is required for establishing the expression of Hoxb3 in the neural tube domain. Interestingly, we have identified a novel GCCAGGC sequence motif within element III482, which is also required to direct gene expression to a subset of the expression domains except for rhombomere 6 and the associated neural crest migrating to the third and posterior branchial arches. Element III482 can direct a higher level of reporter gene expression in r6, which led us to investigate whether kreisler is involved in regulating Hoxb3 expression in r6 through this element. However, our transgenic and mutational analysis has demonstrated that, although kreisler binding sites are present, they are not required for the establishment or maintenance of reporter gene expression in r6. Our results have provided evidence that the expression of Hoxb3 in the neural tube up to the r5/r6 boundary is auto/cross-regulated by Hox genes and expression of Hoxb3 in r6 does not require kreisler.     

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.     

Nuclear re-organisation of the Hoxb complex during mouse embryonic development

The spatial and temporal co-linear expression of Hox genes during development is an exquisite example of programmed gene expression. The precise mechanisms underpinning this are not known. Analysis of Hoxb chromatin structure and nuclear organisation, during the differentiation of murine ES cells, has lent support to the idea that there is a progressive 'opening' of chromatin structure propagated through Hox clusters from 3'to 5', which contributes to the sequential activation of gene expression. Here, we show that similar events occur in vivo in at least two stages of development. The first changes in chromatin structure and nuclear organisation were detected during gastrulation in the Hoxb1-expressing posterior primitive streak region: Hoxb chromatin was decondensed and the Hoxb1 locus looped out from its chromosome territory, in contrast to non-expressing Hoxb9, which remained within the chromosome territory. At E9.5, when differential Hox expression along the anteroposterior axis is being established, we found concomitant changes in the organisation of Hoxb. Hoxb organisation differed between regions of the neural tube that had never expressed Hoxb [rhombomeres (r) 1 and 2], strongly expressed Hoxb1 but not b9 (r4), had downregulated Hoxb1 (r5), expressed Hoxb9 but not Hoxb1 (spinal cord), and expressed both genes (tail bud). We conclude that Hoxb chromatin decondensation and nuclear re-organisation is regulated in different parts of the developing embryo, and at different developmental stages. The differential nuclear organisation of Hoxb along the anteroposterior axis of the developing neural tube is coherent with co-linear Hox gene expression. In early development nuclear re-organisation is coupled to Hoxb expression, but does not anticipate it.     

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.     

Targeted inactivation of Hoxb8 affects survival of a spinal ganglion and causes aberrant limb reflexes

Hoxb8 mutant mice were generated by inserting the lacZ coding sequence in frame with the first exon of Hoxb8. These mice express a fusion protein with a functional beta-galactosidase activity instead of Hoxb8. Mutant embryos were analyzed for anatomical changes. The results indicate that Hoxb8 is not an indispensable regulator of A-P patterning in the forelimb, unlike suggested by our Hoxb8 gain of function experiments (Charité J, DeGraaff W, Shen S, Deschamps J. Cell 1994;78:589-601). The null mutant phenotypic traits include degeneration of the second spinal ganglion (C2), an abnormality opposite to the alteration in the gain of function transgenic mice. Subtle changes in the thoracic part of the vertebral column were observed as well. Adult homozygous mutants exhibit an abnormal clasping reflex of the limbs.     

Expression of Hoxa2 in rhombomere 4 is regulated by a conserved cross-regulatory mechanism dependent upon Hoxb1

The Hoxa2 gene is an important component of regulatory events during hindbrain segmentation and head development in vertebrates. In this study we have used sequenced comparisons of the Hoxa2 locus from 12 vertebrate species in combination with detailed regulatory analyses in mouse and chicken embryos to characterize the mechanistic basis for the regulation of Hoxa2 in rhombomere (r) 4. A highly conserved region in the Hoxa2 intron functions as an r4 enhancer. In vitro binding studies demonstrate that within the conserved region three bipartite Hox/Pbx binding sites (PH1-PH3) in combination with a single binding site for Pbx-Prep/Meis (PM) heterodimers co-operate to regulate enhancer activity in r4. Mutational analysis reveals that these sites are required for activity of the enhancer, suggesting that the r4 enhancer from Hoxa2 functions in vivo as a Hox-response module in combination with the Hox cofactors, Pbx and Prep/Meis. Furthermore, this r4 enhancer is capable of mediating a response to ectopic HOXB1 expression in the hindbrain. These findings reveal that Hoxa2 is a target gene of Hoxb1 and permit us to develop a gene regulatory network for r4, whereby Hoxa2, along with Hoxb1, Hoxb2 and Hoxa1, is integrated into a series of auto- and cross-regulatory loops between Hox genes. These data highlight the important role played by direct cross-talk between Hox genes in regulating hindbrain patterning.     

Fitness Assays Reveal Incomplete Functional Redundancy of the HoxA1 and HoxB1 Paralogs of Mice

Gene targeting techniques have led to the phenotypic characterization of numerous genes; however, many genes show minimal to no phenotypic consequences when disrupted, despite many having highly conserved sequences. The standard explanation for these findings is functional redundancy. A competing hypothesis is that these genes have important ecological functions in natural environments that are not needed under laboratory settings. Here we discriminate between these hypotheses by competing mice (Mus musculus) whose Hoxb1 gene has been replaced by Hoxa1, its highly conserved paralog, against matched wild-type controls in seminatural enclosures. This Hoxb1^A1 swap was reported as a genetic manipulation resulting in no discernible embryonic or physiological phenotype under standard laboratory tests. We observed a transient decline in first litter size for Hoxb1^A1 homozygous mice in breeding cages, but their fitness was consistently and more dramatically reduced when competing against controls within seminatural populations. Specifically, males homozygous for the Hoxb1^A1 swap acquired 10.6% fewer territories and the frequency of the Hoxb1^A1 allele decreased from 0.500 in population founders to 0.419 in their offspring. The decrease in Hoxb1^A1 frequency corresponded with a deficiency of both Hoxb1^A1 homozygous and heterozygous offspring. These data suggest that Hoxb1 and Hoxa1 are more phenotypically divergent than previously reported and support that sub- and/or neofunctionalization has occurred in these paralogous genes leading to a divergence of gene function and incomplete redundancy. Furthermore, this study highlights the importance of obtaining fitness measures of mutants in ecologically relevant conditions to better understand gene function and evolution.     

Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and posterior axis elongation

Mouse Cdx and Hox genes presumably evolved from genes on a common ancestor cluster involved in anteroposterior patterning. Drosophila caudal (cad) is involved in specifying the posterior end of the early embryo, and is essential for patterning tissues derived from the most caudal segment, the analia. Two of the three mouse Cdx paralogues, Cdx 1 and Cdx2, are expressed early in a Hox-like manner in the three germ layers. In the nascent paraxial mesoderm, both genes are expressed in cells contributing first to the most rostral, and then to progressively more caudal parts of the vertebral column. Later, expression regresses from the anterior sclerotomes, and is only maintained for Cdx1 in the dorsal part of the somites, and for both genes in the tail bud. Cdx1 null mutants show anterior homeosis of upper cervical and thoracic vertebrae. Cdx2-null embryos die before gastrulation, and Cdx2 heterozygotes display anterior transformations of lower cervical and thoracic vertebrae. We have analysed the genetic interactions between Cdx1 and Cdx2 in compound mutants. Combining mutant alleles for both genes gives rise to anterior homeotic transformations along a more extensive length of the vertebral column than do single mutations. The most severely affected Cdx1 null/Cdx2 heterozygous mice display a posterior shift of their cranio-cervical, cervico-thoracic, thoraco-lumbar, lumbo-sacral and sacro-caudal transitions. The effects of the mutations in Cdx1 and Cdx2 were co-operative in severity, and a more extensive posterior shift of the expression of three Hox genes was observed in double mutants. The alteration in Hox expression boundaries occurred early. We conclude that both Cdx genes cooperate at early stages in instructing the vertebral progenitors all along the axis, at least in part by setting the rostral expression boundaries of Hox genes. In addition, Cdx mutants transiently exhibit alterations in the extent of Hox expression domains in the spinal cord, reminding of the strong effects of overexpressing Cdx genes on Hox gene expression in the neurectoderm. Phenotypical alterations in the peripheral nervous system were observed at mid-gestation stages. Strikingly, the altered phenotype at caudal levels included a posterior truncation of the tail, mildly affecting Cdx2 heterozygotes, but more severely affecting Cdx1/Cdx2 double heterozygotes and Cdx1 null/Cdx2 heterozygotes. Mutations in Cdx1 and Cdx2 therefore also interfere with axis elongation in a cooperative way. The function of Cdx genes in morphogenetic processes during gastrulation and tail bud extension, and their relationship with the Hox genes are discussed in the light of available data in Amphioxus, C. elegans, Drosophila and mice.