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Hlx is a homeobox gene expressed in developing mouse intestinal and hepatic mesenchyme and is required for normal growth of these organs. Nominally Hlx-expressing cell lineages are present in [Hlx.sup.-/-] embryos, which allowed isolation of mesenchymal cell lines in which Hlx could be differential expressed to allow identification of downstream targets. All cell lines expressed the Hlx knockout allele as well as various combinations of transforming growth factor-[[Beta].sub.1], hepatocyte growth factor, and laminin-[Alpha]4. Hlx promoter segments actively drove reporter gene expression in these cells. They have been passaged [is greater than] 50 times since cloning with no change in growth or morphology. To add Hlx back to the cells, we prepared bicistronic retroviral vectors expressing FLAG-tagged Hlx (or no Hlx sequence as a control) along with green fluorescent protein (GFP). [Hlx.sup.-/-] cells were transduced with vectors with and without Hlx. GFP-positive cells were purified by fluorescence-activated cell sorting; both the control and the Hlx vectors transduced [is greater than] 70% of the cells. Total RNA isolated from the GFP-positive cells was used to prepare samples for analysis with Affymetrix GeneChips. We found the greatest change in expression was for Hlx itself (as expected). Genes showing altered expression (2x or greater) included participants in signaling pathways and extracellular matrix proteins. Further evaluation of the downstream targets identified will provide additional insights into digestive system 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., (Copyright 2001 Academic Press.)

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.

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.

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.

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.

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., (Copyright 1999 Academic Press.)

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., (Copyright 1999 Academic Press.)

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.

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.

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.

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.

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.

Spx1, a novel mouse homeobox gene, encodes a homeodomain characteristic of the paired-like class of homeobox genes and has been mapped to the distal end of the X chromosome. Northern blot hybridization of adult tissues detected high levels of a single Spx1 transcript in the testis. Further analysis by in situ hybridization revealed predominant Spx1 expression within the spermatogonia/preleptotene spermatocytes and round spermatids of spermatogenic stages IV-VII. These expression data suggest SPX1 may play a role in the regulation of spermatogenesis.

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.

Here we describe in detail both the expression of Hoxa-11 in the wild-type mouse uterus and the defects resulting in maternal reproductive failure of Hoxa-11 null female mice. The Hoxa-11 gene is expressed at peak levels in uterine stromal cells during metestrus. Hoxa-11 transcripts were induced beginning on Day 2 of gestation in the stromal cells underlying the uterine epithelium and appeared in the secondary decidual zone between Days 6 and 8 of gestation. At early gestational stages, stromal, decidual, and glandular cell development were deficient in Hoxa-11 null uteri in comparison to wild-type as assessed by histology and immunohistochemical localization of the decidual cell marker epitope, stage-specific embryonic antigen-3 (SSEA-3). Both steroid-induced uterine stromal and glandular cell proliferation as well as oil-induced stromal decidualization after induction of pseudopregnancy were deficient in mutant uteri. Moreover, both Western blotting and immunohistochemistry demonstrated that the burst of glandular leukemia inhibitory factor (LIF) found in normal pregnant uteri at Day 4.5 of gestation was absent in Hoxa-11-deficient uteri. The LIF burst was also not observed in the uteri of bilaterally ovariectomized, hormonally stimulated Hoxa-11 mutants. These results demonstrate that the Hoxa-11 gene is required for normal uterine stromal cell and glandular differentiation during pregnancy, as is the presence of the steroid-induced glandular LIF burst initiating embryo implantation.

We report the cloning, characterization, and targeting of an Sp1-related zinc finger transcription factor gene from the distal arm of mouse chromosome 12. This gene, previously identified in rats and humans and designated sp4, is homologous to the Drosophila buttonhead (btd) gene, which is expressed in the head region of developing flies. Similarly, in situ hybridizations show that sp4 is highly expressed in mouse embryos in the developing central nervous system (CNS). Expression of sp4 is seen as early as Day 9 of development, where transcripts are abundant in the posterior neuropore. Expression in later embryos is detected throughout the CNS as well as in other structures, including the nasal mucosa, the vomeronasal organ, the epithelium of the lung and intestinal tract, the testes, and the developing teeth. Northern blot analysis showed sp4 expression in the adult brain and other tissues. Gene targeting by homologous recombination was used to determine the role of sp4 during mouse development. Two-thirds of homozygous mutants die within the first few days after birth and those that survive are smaller than their wild-type littermates. While fertility of the female mutants appears normal, homozygous mutant males do not breed, despite having histologically intact testes containing mature sperm. sp4/sp4 mutant males fail to copulate, indicating that this gene is required for normal male reproductive behavior.