Functional specificity of the Hoxa13 homeobox.

To better define Abd-B type homeodomain function, to test models that predict functional equivalence of all Hox genes and to initiate a search for the downstream targets of Hoxa13, we have performed a homeobox swap by replacing the homeobox of the Hoxa11 gene with that of the Hoxa13 gene. The Hoxa11 and Hoxa13 genes are contiguous Abd-B type genes located at the 5' end of the HoxA cluster. The modified Hoxa11 allele (A11(13hd)) showed near wild-type function in the development of the kidneys, axial skeleton and male reproductive tract, consistent with functional equivalence models. In the limbs and female reproductive tract, however, the A11(13hd) allele appeared to assume dominant Hoxa13 function. The uterus, in particular, showed a striking homeotic transformation towards cervix/vagina, where Hoxa13 is normally expressed. Gene chips were used to create a molecular portrait of this tissue conversion and revealed over 100 diagnostic gene expression changes. This work identifies candidate downstream targets of the Hoxa13 gene and demonstrates that even contiguous Abd-B homeoboxes have functional specificity.

Hoxa11 and Hoxd11 regulate branching morphogenesis of the ureteric bud in the developing kidney.

Hoxa11 and Hoxd11 are functionally redundant during kidney development. Mice with homozygous null mutation of either gene have normal kidneys, but double mutants have rudimentary, or in extreme cases, absent kidneys. We have examined the mechanism for renal growth failure in this mouse model and find defects in ureteric bud branching morphogenesis. The ureteric buds are either unbranched or have an atypical pattern characterized by lack of terminal branches in the midventral renal cortex. The mutant embryos show that Hoxa11 and Hoxd11 control development of a dorsoventral renal axis. By immunohistochemical analysis, Hoxa11 expression is restricted to the early metanephric mesenchyme, which induces ureteric bud formation and branching. It is not found in the ureteric bud. This suggests that the branching defect had been caused by failure of mesenchyme to epithelium signaling. In situ hybridizations with Wnt7b, a marker of the metanephric kidney, show that the branching defect was not simply the result of homeotic transformation of metanephros to mesonephros. Absent Bf2 and Gdnf expression in the midventral mesenchyme, findings that could by themselves account for branching defects, shows that Hoxa11 and Hoxd11 are necessary for normal gene expression in the ventral mesenchyme. Attenuation of normal gene expression along with the absence of a detectable proliferative or apoptotic change in the mutants show that one function of Hoxa11 and Hoxd11 in the developing renal mesenchyme is to regulate differentiation necessary for mesenchymal-epithelial reciprocal inductive interactions.

Novel cell lines promote the discovery of genes involved in early heart development.

Clonal cell lines representing early cardiomyocytes would provide valuable reagents for the dissection of the genetic program of early cardiogenesis. Here we describe the establishment and characterization of cell lines from the hearts of transgenic mice and embryos with SV40 large T antigen expressed in the heart-forming region. Ultrastructure analysis by transmission electron microscopy showed the primitive, precontractile nature of the resulting cells, with the absence of myofilaments, Z lines, and intercalated disks. Immunohistochemistry, RT-PCR, Northern blots, and oligonucleotide microarrays were used to determine the expression levels of thousands of genes in the 1H and ECL-2 cell lines. The resulting gene-expression profiles showed the transcription of early cardiomyocyte genes such as Nkx2.5, GATA4, Tbx5, dHAND, cardiac troponin C, and SM22-alpha. Furthermore, many genes not previously implicated in early cardiac development were expressed. Two of these genes, Hic-5, a possible negative regulator of muscle differentiation, and the transcription enhancing factor TEF-5 were selected and shown by in situ hybridizations to be expressed in the early developing heart. The results show that the 1H and ECL-2 cell lines can be used to discover novel genes expressed in the early cardiomyocyte.

Two CCAAT boxes in a novel inverted repeat motif are required for Hlx homeobox gene expression.

Hlx is a homeobox transcription factor gene required for normal intestinal and hepatic growth in development. We previously found high sequence identity and 17 conserved consensus cis-regulatory/transcription factor binding elements in the mouse and human Hlx 5' regions. A 594 bp sequence in the Hlx 5' region possessing the same activity in driving luciferase expression as larger Hlx 5' sequences had three segments each necessary but not sufficient for luciferase expression in NIH 3T3 cells (which express Hlx). Nine of the conserved putative regulatory elements are positioned within these segments, including two CCAAT boxes on opposite strands within a conserved 44 bp inverted repeat sequence. To test the hypothesis that these elements are required for promoter activity, we compared the reporter expression activity of segments containing mutations of these elements with activity of the parent Hlx promoter sequence. We found that mutation of either CCAAT box or a conserved AP-2 site resulted in a significant decrease in promoter activity. Restoration of the inverted repeat with complementary mutations of both CCAAT boxes did not restore activity. Further, mutation of other portions of the inverted repeat did not affect promoter activity. Mutation of other elements had no effect on promoter activity.

Structural and functional characterization of the mouse Hlx homeobox gene.

Hlx is a mesenchymally expressed homeobox transcription factor gene that is essential for normal intestinal and hepatic development in the mouse. Here we report further characterization of the mouse Hlx gene, including an additional 3.7 kb of 5' sequence as well as the sequence of the three introns. Comparison of the sequence of the mouse Hlx gene 5' to the coding region with that of the human gene revealed multiple regions of high conservation. Neither the mouse nor the human gene contained a TATA box, and ribonuclease protection studies defined heterogeneous transcription start sites for the mouse gene. A number of consensus transcription factor binding sites were conserved between the mouse and human Hlx genes both within and outside of the highly conserved regions. Reporter constructs containing 4.2 or 1.4 kb of mouse 5' sequence showed active expression in cell lines that express Hlx. Further characterization of the mouse Hlx gene will provide insight into the developmental regulation of the mouse digestive system.

Genetic control of dorsal-ventral identity in the telencephalon: opposing roles for Pax6 and Gsh2.

We have examined the genetic mechanisms that regulate dorsal-ventral identity in the embryonic mouse telencephalon and, in particular, the specification of progenitors in the cerebral cortex and striatum. The respective roles of Pax6 and Gsh2 in cortical and striatal development were studied in single and double loss-of-function mouse mutants. Gsh2 gene function was found to be essential to maintain the molecular identity of early striatal progenitors and in its absence the ventral telencephalic regulatory genes Mash1 and Dlx are lost from most of the striatal germinal zone. In their place, the dorsal regulators, Pax6, neurogenin 1 and neurogenin 2 are found ectopically. Conversely, Pax6 is required to maintain the correct molecular identity of cortical progenitors. In its absence, neurogenins are lost from the cortical germinal zone and Gsh2, Mash1 and Dlx genes are found ectopically. These reciprocal alterations in cortical and striatal progenitor specification lead to the abnormal development of the cortex and striatum observed in Pax6 (small eye) and Gsh2 mutants, respectively. In support of this, double homozygous mutants for Pax6 and Gsh2 exhibit significant improvements in both cortical and striatal development compared with their respective single mutants. Taken together, these results demonstrate that Pax6 and Gsh2 govern cortical and striatal development by regulating genetically opposing programs that control the expression of each other as well as the regionally expressed developmental regulators Mash1, the neurogenins and Dlx genes in telencephalic progenitors.

Characterization of Hoxa-10/Hoxa-11 transheterozygotes reveals functional redundancy and regulatory interactions.

Hox genes show related sequences and overlapping expression domains that often reflect functional redundancy as well as a common evolutionary origin. To accurately define their functions, it has become necessary to compare phenotypes of mice with single and multiple Hox gene mutations. Here, we focus on two Abd-B-type genes, Hoxa-10 and Hoxa-11, which are coexpressed in developing vertebrae, limbs, and reproductive tracts. To assess possible functional redundancy between these two genes, Hoxa-10/Hoxa-11 transheterozygotes were produced by genetic intercrosses and analyzed. This compound mutation resulted in synergistic defects in transheterozygous limbs and reproductive tracts, but not in vertebrae. In the forelimb, distal radial/ulnar thickening and pisiform/triangular carpal fusion were observed in 35 and 21% of transheterozygotes, respectively, but were effectively absent in Hoxa-10 and Hoxa-11 +/- forelimbs. In the hindlimb, distal tibial/fibular thickening and loss of tibial/fibular fusion were observed in >80% of transheterozygotes but in no Hoxa-10 or Hoxa-11 +/- hindlimbs, and all transheterozygotes displayed reduced medial patellar sesamoids, compared to modest incidences in Hoxa-10 and Hoxa-11 +/- mutants. Furthermore, while the reproductive tracts of Hoxa-10 and Hoxa-11 single heterozygous mutants of both sexes were primarily unaffected, male transheterozygotes displayed cryptorchidism and abnormal tortuosity of the ductus deferens, and female transheterozygotes exhibited abnormal uterotubal junctions and narrowing of the uterus. In addition we observed that the targeted mutations of Hoxa-10 and Hoxa-11 each affected the expression of the other gene in the developing prevertebra and reproductive tracts. These results provide a measure of the functional redundancy of Hoxa-10 and Hoxa-11 and a deeper understanding of the phenotypes resulting in the single mutants and help elucidate the regulatory interactions between these two genes.

Molecular motors: the driving force behind mammalian left-right development.

The molecular motors dynein and kinesin are large protein complexes that convert the energy generated by ATP hydrolysis into directional movement along the microtubule cytoskeleton. They are required for a myriad of cellular processes, including mitotic spindle movement, axonal and vesicular transport, and ciliary beating. Recently, it has been shown that, in addition, they have a unique role during embryonic patterning: they are required to orient and establish the left-right axis in early vertebrate development.

Targeted deletion of the ATP binding domain of left-right dynein confirms its role in specifying development of left-right asymmetries.

Vertebrates develop distinct asymmetries along the left-right axis, which are consistently aligned with the anteroposterior and dorsoventral axes. The mechanisms that direct this handed development of left-right asymmetries have been elusive, but recent studies of mutations that affect left-right development have shed light on the molecules involved. One molecule implicated in left-right specification is left-right dynein (LRD), a microtubule-based motor protein. In the LRD protein of the inversus viscerum (iv) mouse, there is a single amino acid difference at a conserved position, and the lrd gene is one of many genes deleted in the legless (lgl) mutation. Both iv and lgl mice display randomized left-right development. Here we extend the analysis of the lrd gene at the levels of sequence, expression and function. The complete coding sequence of the lrd gene confirms its classification as an axonemal, or ciliary, dynein. Expression of lrd in the node at embryonic day 7.5 is shown to be symmetric. At embryonic day 8.0, however, a striking asymmetric expression pattern is observed in all three germ layers of the developing headfold, suggesting roles in both the establishment and maintenance of left-right asymmetries. At later times, expression of lrd is also observed in the developing floorplate, gut and limbs. These results suggest function for LRD protein in both ciliated and non-ciliated cells, despite its sequence classification as axonemal. In addition, a targeted mutation of lrd was generated that deletes the part of the protein required for ATP binding, and hence motor function. The resulting left-right phenotype, randomization of laterality, is identical to that of iv and lgl mutants. Gross defects in ciliary structure were not observed in lrd/lrd mutants. Strikingly, however, the monocilia on mutant embryonic node cells were immotile. These results prove the identity of the iv and lrd genes. Further, they argue that LRD motor function, and resulting nodal monocilia movement, are required for normal left-right development.

Characterization of npas3, a novel basic helix-loop-helix PAS gene expressed in the developing mouse nervous system.

Here we describe the cloning and expression pattern of a new bHLH-PAS domain gene, Npas3. Npas3 shares 50.2% amino acid sequence identity with Npas1 and a lesser similarity with other members of the bHLH-PAS domain family of transcription factors. Northern blot analysis detected Npas3 mRNA between 11.5 and 17.5 d.p.c. in embryonic development and exclusively in the adult brain. Whole-mount and section in situ hybridization assays revealed expression of Npas3 between 9.5 and 11.5 d.p.c. in the developing neural tube. In addition, Npas3 mRNA was expressed throughout the neuroepithelium of the developing central nervous system between 10. 5 and 12.5 d.p.c. Interestingly, at 14.5 d.p.c., the expression of Npas3 mRNA became restricted to the neopallial layer of the cortex. At 12.5 d.p.c., Npas3 mRNA was evident in nonneural tissues such as the developing dermis and mesenchyme surrounding the otic and nasal placodes. Expression was also detected in the developing cardiac valves, limb and developing kidney.

Novel strategy yields candidate Gsh-1 homeobox gene targets using hypothalamus progenitor cell lines.

We describe the successful application of a strategy that potentially provides for an efficient and universal screen for downstream gene targets. We used the promoter of the Gsh-1 homeobox gene to drive expression of the SV40 T-antigen gene in transgenic mice. We have previously shown that the Gsh-1 homeobox gene is expressed in discrete domains of the ganglionic eminences, diencephalon, and hindbrain during brain development. Gsh-1-SV40 T transgenic mice showed cellular hyperplasia in regions of the brain coincident with Gsh-1 expression. The Gsh-1-SV40 T transgene was introduced, by breeding, into Gsh-1 homozygous mutant mice, and Gsh-1 -/- cell lines were made. Clonal cell lines were generated and analyzed by Northern blot hybridizations and Affymetrix GeneChip probe arrays to determine gene expression profiles. The results indicate that the cell lines remain representative of early developmental stages. Further, immunocytochemistry showed uniformly high levels of nestin expression, typical of central nervous system progenitor cells, and the absence of terminal differentiation markers of neuronal cells. One clonal cell line, No. 14, was then stably transfected with a tet-inducible Gsh-1 expression construct and subcloned. The starting clone 14, together with the uninduced and induced subclones, provided cell populations with varying levels of Gsh-1 expression. Differential display and Affymetrix GeneChip probe arrays were then used to identify transcript differences that represent candidate Gsh-1 target genes. Of particular interest, the drm and gas1 genes, which repress cell proliferation, were observed to be activated in Gsh-1-expressing cells. These observations support models predicting that homeobox genes function in the regulation of cell proliferation.

prx-1 functions cooperatively with another paired-related homeobox gene, prx-2, to maintain cell fates within the craniofacial mesenchyme.

The paired-related homeobox gene, prx-1, is expressed in the postmigratory cranial mesenchyme of all facial prominences and is required for the formation of proximal first arch derivatives. We introduced lacZ into the prx-1 locus to study the developmental fate of cells destined to express prx-1 in the prx-1 mutant background. lacZ was normally expressed in prx-1(neo); prx-1(lacZ )mutant craniofacial mesenchyme up until 11.5 d.p.c. At later time points, lacZ expression was lost from structures that are defective in the prx-1(neo) mutant mice. A related gene, prx-2, demonstrated overlapping expression with prx-1. To test the idea that prx-1 and prx-2 perform redundant functions, we generated prx-1(neo;)prx-2 compound mutant mice. Double mutant mice had novel phenotypes in which the rostral aspect of the mandible was defective, the mandibular incisor arrested as a single, bud-stage tooth germ and Meckel's cartilage was absent. Expression of two markers for tooth development, pax9 and patched, were downregulated. Using a transgene that marks a subset of prx-1-expressing cells in the craniofacial mesenchyme, we showed that cells within the hyoid arch take on the properties of the first branchial arch. These data suggest that prx-1 and prx-2 coordinately regulate gene expression in cells that contribute to the distal aspects of the mandibular arch mesenchyme and that prx-1 and prx-2 play a role in the maintenance of cell fate within the craniofacial mesenchyme.

Paired-related homeobox genes cooperate in handplate and hindlimb zeugopod morphogenesis.

The closely related homeobox genes prx-1 and prx-2 are expressed in lateral plate and limb bud mesoderm, but targeted inactivation of these genes failed to demonstrate a limb phenotype. Here we report that mice carrying compound mutations in prx-1 and prx-2 have severe limb deformities. In the forelimb autopod, pre- and postaxial polydactyly were found most commonly, but also syndactyly, oligodactyly, and abnormal digit placement affecting posterior elements were observed. In the hindlimb, preaxial polydactyly with variable expressivity was seen in all cases. Extreme distal digit duplications were seen in both the fore- and hindlimbs. prx-1; prx-2 double-mutant mice also displayed extreme shortening and impaired ossification of the hindlimb zeugopods. Integrity of the forelimb apical ectodermal ridge was abnormal as determined by expression of FGF8 and BMP4. Expression of msx-1 and msx-2, markers for BMP signaling pathways, was absent in regions of the posterior handplates, while expression of Shh and patched was unaffected. The mutant phenotypes were dosage dependent, since prx-1 -/-; prx-2 +/- mice also displayed severe limb abnormalities. These data suggest that prx-1 and prx-2 cooperatively regulate handplate and hindlimb zeugopod morphogenesis through BMP-mediated signaling pathways.

Evolutionary conservation and tissue-specific processing of Hoxa 11 antisense transcripts.

We previously described the existence of abundant, processed, polyadenylated murine Hoxa 11 antisense transcripts. Of particular interest, in the developing limbs the antisense transcripts were observed to be present in a pattern complementary to that of the sense transcripts, suggesting a possible regulatory function (Hsieh-Li et al. 1995). We have analyzed the human HOXA 11 genomic locus, showing strong evolutionary conservation of regions potentially encoding antisense transcripts. Human HOXA 11 fetal kidney antisense cDNAs were identified and sequenced, demonstrating the evolutionary conservation of Hoxa 11 antisense transcription. As for the mouse, the human antisense RNAs were polyadenylated and showed several alternative processing patterns, but shared the sequences of a common 3' exon. The evolutionary conservation of the opposite strand transcripts strongly suggests function. A significantly long open reading frame was observed, but mouse-human comparisons argued against true coding function. Murine kidney Hoxa 11 antisense transcription and processing was also examined, revealing tissue-specific differences between limb and kidney. A novel procedure, designated Race in Circles, was devised and used to define mouse limb antisense transcription start sites. Furthermore, comparisons of human, mouse, and chicken sense transcript Hoxa 11 homeobox nucleotide sequences and their respective encoded homeodomains indicate a very strong selective pressure in vertebrates against mutations that result in coding changes. Given the significant differences in amino acid sequences of the homeodomains of different Hox genes, this observation argues for individual homeodomain functional specificity.

Handed asymmetry in the mouse: understanding how things go right (or left) by studying how they go wrong.

All vertebrates have characteristic asymmetries along the left/right axis. The positioning of asymmetric visceral organs is highly conserved evolutionarily and disruptions in left/right patterning can lead to severe morphological defects, demonstrating the importance of regulation of left/right developmental asymmetries. Our understanding of vertebrate left/right pattern formation has been advanced by studying several mouse mutations which disrupt this process. These mutant mice have served as tools to help us to unravel the genetic pathways of left/right development. The identification and analysis of genes with asymmetric expression patterns has allowed us to begin to understand the mechanisms which regulate left/right development.

Altered forebrain and hindbrain development in mice mutant for the Gsh-2 homeobox gene.

The patterning of the mammalian brain is orchestrated by a large battery of regulatory genes. Here we examine the developmental function of the Gsh-2 nonclustered homeobox gene. Whole-mount and serial section in situ hybridizations have been used to better define Gsh-2 expression domains within the developing forebrain, midbrain, and hindbrain. Gsh-2 transcripts are shown to be particularly abundant in the hindbrain and within the developing ganglionic eminences of the forebrain. In addition, mice carrying a targeted mutation of Gsh-2 have been generated and characterized. Homozygous mutants uniformly failed to survive more than 1 day following birth. At the physiologic level the mutants experienced apnea and reduced levels of hemoglobin oxygenation. Histologically, the mutant brains had striking alterations of discrete components. In the forebrain the lateral ganglionic eminence was reduced in size. In the hindbrain, the area postrema, an important cardiorespiratory chemosensory center, was absent. The contiguous nucleus tractus solitarius, involved in integrating sensory input to maintain homeostasis, was also severely malformed in mutants. Immunohistochemistry was used to examine the mutant brains for alterations in the distribution of markers specific for serotonergic and cholinergic neurons. In addition, in situ hybridizations were used to define expression patterns of the Dlx 2 and Nkx 2.1 homeobox genes in Gsh-2 mutant mice. The mutant lateral ganglionic eminences showed an abnormal absence of Dlx 2 expression. These results better define the genetic program of development of the mammalian brain, support neuromeric models of brain development, and further suggest similar patterning function for homeobox genes in phylogenetically diverse organisms.

Multistep control of pituitary organogenesis.

Lhx3 and Lhx4 (Gsh4), two closely related LIM homeobox genes, determine formation of the pituitary gland in mice. Rathke's pouch is formed in two steps-first as a rudiment and later as a definitive pouch. Lhx3 and Lhx4 have redundant control over formation of the definitive pouch. Lhx3 controls a subsequent step of pituitary fate commitment. Thereafter, Lhx3 and Lhx4 together regulate proliferation and differentiation of pituitary-specific cell lineages. Thus, Lhx3 and Lhx4 dictate pituitary organ identity by controlling developmental decisions at multiple stages of organogenesis.

Mutation of an axonemal dynein affects left-right asymmetry in inversus viscerum mice.

The development of characteristic visceral asymmetries along the left-right (LR) axis in an initially bilaterally symmetrical embryo is an essential feature of vertebrate patterning. The allelic mouse mutations inversus viscerum (iv) and legless (lgl) produce LR inversion, or situs inversus, in half of live-born homozygotes. This suggests that the iv gene product drives correct LR determination, and in its absence this process is randomized. These mutations provide tools for studying the development of LR-handed asymmetry and provide mouse models of human lateralization defects. At the molecular level, the normally LR asymmetric expression patterns of nodal and lefty are randomized in iv/iv embryos, suggesting that iv functions early in the genetic hierarchy of LR specification. Here we report the positional cloning of an axonemal dynein heavy-chain gene, left/right-dynein (lrd), that is mutated in both lgl and iv. lrd is expressed in the node of the embryo at embryonic day 7.5, consistent with its having a role in LR development. Our findings indicate that dynein, a microtubule-based motor, is involved in the determination of LR-handed asymmetry and provide insight into the early molecular mechanisms of this process.

The Evi1 proto-oncogene is required at midgestation for neural, heart, and paraxial mesenchyme development.

The ecotropic viral integration site-1 (Evi1) locus was initially identified as a common site of retroviral integration in myeloid tumors of the AKXD-23 recombinant inbred mouse strain. The full-length Evi1 transcript encodes a putative transcription factor, containing ten zinc finger motifs found within two domains of the protein. To determine the biological function of the Evi1 proto-oncogene, the full-length, but not an alternately spliced, transcript was disrupted using targeted mutagenesis in embryonic stem cells. Evi1 homozygous mutant embryos die at approximately 10.5 days post coitum. Mutants were distinguished at 10.5 days post coitum by widespread hypocellularity, hemorrhaging, and disruption in the development of paraxial mesenchyme. In addition, defects in the heart, somites, and cranial ganglia were detected and the peripheral nervous system failed to develop. These results correlated with whole-mount in situ hybridization analyses of embryos which showed expression of the Evi1 proto-oncogene in embryonic mesoderm and neural crest-derived cells associated with the peripheral nervous system. These data suggest that Evi1 has important roles in general cell proliferation, vascularization, and cell-specific developmental signaling, at midgestation.