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The genetic basis for the acquisition and retention of innovative morphological structures is of wide interest to evolutionary biologists. Mammals possess specific anatomical features that are not found in other vertebrates, such as external ears, placenta, and hair. It has been inferred that genetic changes in developmental regulatory pathways may contribute, in part, to the evolution of such innovations. In particular, duplicated developmental genes (paralogs) play an important role in generating variations in evolutionarily important morphological structures via deployment of novel cis-regulatory elements. The use of comparative genomics together with mouse transgenic methods provides an effective strategy for interrogating the regulatory landscape of duplicated loci of major developmental genes, as reported here for one of the Dlx gene clusters. The Distal-less (Dlx) genes comprise a homeobox-containing multigene family. The mammalian Dlx gene family consists of six members that are orthologous to the Drosophila Distal-less gene and which are organized into three convergently transcribed (tail-to-tail) bigene clusters; it is presumed that the ancestral bigene cluster was generated initially by tandem gene duplication followed by whole genome duplications (Stock et al., ‘96). Bigene cluster pairs are Dlx1 and Dlx2, Dlx6 and Dlx5, and Dlx41 and Dlx3 (Simeone et al., ‘94; McGuinness et al., ‘96; Nakamura et al., ‘96; Ellies et al., ‘97b; Zerucha and Ekker, 2000), each of which is originally linked to a HOX cluster (HOXD, HOXA, and HOXB, respectively). Dlx expression patterns and functional studies by targeted Dlx deletions provide strong evidence that these transcription factors play major roles in patterning of multiple developmental structures (Merlo et al., 2000; Quint et al., 2000; Zerucha and Ekker, 2000; Zerucha et al., 2000; Depew et al., 2002; Panganiban and Rubenstein, 2002). Though vertebrate Distal-less genes were originally described as being primarily “head” homeobox genes (Duboule, ‘94), they have been shown also to be involved in development of many other structures, that is, limbs, peripheral neurons, whiskers/hair follicles, otic vesicles, teeth, median fins in zebrafish, cement glands in Xenopus, keratinocytes in Xenopus and mice, placenta, genital tubercle and hematopoietic cells in humans (Dirksen et al., ‘93; Akimenko et al., ‘94; Morasso et al., ‘94, ‘99; Porteus et al., ‘94; Shimamoto et al., ‘97; Zerucha et al., ‘97; Park and Morasso, ‘99; Hwang et al., 2008). cis-Regulatory elements confer cell and tissue specificity with regard to the transcriptional machinery and have been implicated as being important for morphological evolution, particularly in the context of embryonic transcription factor genes (Shashikant et al., ‘98; Carroll, 2008; Cretekos et al., 2008; Levine, 2010). Since Dlx genes have been co-opted recurrently in vertebrate evolution by acquisition of novel expression sites, their cis-regulatory element functionality and evolutionary history are of great interest in terms of bauplan divergences in mammals. Delineation of the genomic organization and overlapping expression patterns of the Dlx genes provides a window into understanding the genomic control of development of the many structures described above. Expression patterns of linked Dlx genes have suggested that cis-regulatory elements controlling spatio-temporal expression exist within their respective intergenic regions (Ellies et al., ‘97a). Functional analyses of the intergenic region between paired Dlx genes showed that this region indeed harbors important cis-regulatory elements (Zerucha et al., 2000; Sumiyama et al., 2002; Ghanem et al., 2003). In the Dlx3–4 cluster there are several evolutionarily conserved elements within about 20 kb of the intergenic non-coding region of human and mouse. Moreover, one of these was shown to be a vital enhancer element for branchial arch expression in a Dlx3 specific manner (Sumiyama and Ruddle, 2003). Global alignments using genomic sequences from multiple species is an effective means to identify potentially functional elements in the non-coding regions. The ENCODE project report has shown that a total of 5% of the bases in the genome can be confidently identified as being under evolutionary constraint in mammals, and that approximately 60% of these bases are biologically functional on the basis of experimental assays (ENCODE, 2007). The purpose of the current study was to investigate the rate of change of molecular evolution of cis-elements possibly related to mammalian specific characters of the Dlx3–4 bigene cluster using an extended dataset of 12 diverse mammalian species, including a marsupial and a monotreme. Large-scale sequence comparisons and subsequent constrained motif extraction were conducted with the aim of identifying molecular evolutionary trends in the respective elements. The analyses show that conserved nucleotide elements in intergenic region evolve at markedly different rates and highlight varying patterns of evolutionary divergence among the species. In transgenic mice experiments, we demonstrated that I37-2 cis-regulatory element for branchial arches showed theria-specific conservation and enhancer activity, whereas that of the platypus showed no enhancer activity in branchial arches. The findings serve as guideposts for our ultimate goal to examine the nature in which the cis-regulatory modules in the Dlx bigene clusters act to bring about developmental and gross morphological changes in vertebrate evolution.

Transgenic mouse models are valuable resources for analyzing functions of genes involved in human diseases. Mouse models provide critical insights into biological processes, including in vivo visualization of vasculature critical to our understanding of the immune system. Generating transgenic mice requires the capture and modification of large-insert DNAs representing genes of interest. We have developed a methodology using a yeast-bacterial shuttle vector, pClasper, that enables the capture and modification of bacterial artificial chromosomes (BAC)-sized DNA inserts. Numerous improvements and technical advances in the original pClasper vector have allowed greater flexibility and utility in this system. Examples of such pClasper mediated gene modifications include: Claspette-mediated capture of large-insert genomic fragments from BACs-human polycystic kidney disease-1 (PKD1); modification of pClasperA clones by the RareGap method-PKD1 mutations; Claspette-mediated modification of pClasper clones-mouse albumin-1 gene; and, of most relevance to our interest in lymph node vasculature-Claspimer-mediated modification of pClasper clones-high endothelial venule and lymphatic vessel genes. Mice that have been generated with these methods include mice with fluorescent high endothelial venules.

Hec-6st is a highly specific high endothelial venule (HEV) gene that is crucial for regulating lymphocyte homing to lymph nodes (LN). The enzyme is also expressed in HEV-like vessels in tertiary lymphoid organs that form in chronic inflammation in autoimmunity, graft rejection, and microbial infection. Understanding the molecular nature of Hec-6st regulation is crucial for elucidating its function in development and disease. However, studies of HEV are limited because of the difficulties in isolating and maintaining the unique characteristics of these vessels in vitro . The novel pClasper yeast homologous recombination technique was used to isolate from a BAC clone a 60-kb DNA fragment that included the Hec-6st ( Chst4 ) gene with flanking sequences. Transgenic mice were generated with the β- galactosidase ( LacZ ) reporter gene inserted in-frame in the exon II of Hec-6st within the isolated BAC DNA fragment. LacZ was expressed specifically on HEV in LN, as indicated by its colocalization with peripheral node vascular addressin. LacZ was increased in nasal-associated lymphoid tissue during development and was reduced in LN and nasal-associated lymphoid tissue by LTβR-Ig (lymphotoxin-β receptor human Ig fusion protein) treatment in a manner identical to the endogenous gene. The transgene was expressed at high levels in lymphoid accumulations with characteristics of tertiary lymphoid organs in the salivary glands of aged mice. Thus, the Hec-6s-LacZ construct faithfully reproduces Hec-6st tissue-specific expression and can be used in further studies to drive expression of reporter or effector genes, which could visualize or inhibit HEV in autoimmunity.

Mouse naked cuticle 2 (mNkd2), the mammalian homolog of the Drosophila segment polarity gene naked cuticle (nkd), encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. The transcription factor, Hoxc8, a member of the homeobox gene family, is vital for growth and differentiation. Chromatin immunoprecipitation (ChIP) assay, an electrophoretic mobility shift assay (EMSA), and a reporter assay demonstrated that endogenous Hoxc8 protein binds directly to the enhancer region of the mNkd2 gene, implying a Hoxc8-dependent transcriptional activity. Introduction of exogenous Hoxc8 into NIH3T3 cell lines lacking wild-type Hoxc8 dramatically reduced expression of mNkd2 mRNA. If, as the results suggest, mNkd2 is a direct target of Hoxc8, it represents a novel mechanism by which Hoxc8 might cross-talk with the Wnt signaling pathway by regulating mNkd2.

Hox genes encode transcription factors that control spatial patterning during embryogenesis. To date, downstream targets of Hox genes have proven difficult to identify. Here, we describe studies designed to identify target genes under the control of the murine transcription factor Hoxc8 . We used a mouse 16,463 gene oligonucleotide microarray to identify mRNAs whose expression was altered by the overexpression of Hoxc8 in C57BL/6J mouse embryo fibroblasts (MEF) in cell culture ( in vitro ). We identified a total of 34 genes whose expression was changed by 2-fold or greater: 16 genes were up-regulated, and 18 genes were down-regulated. The majority of genes encoded proteins involved in critical biological processes, such as cell adhesion, migration, metabolism, apoptosis, and tumorigenesis. Two genes showed high levels of regulation: ( i ) secreted phosphoprotein 1 ( Spp1 ), also known as osteopontin ( OPN ), was down-regulated 4.8-fold, and ( ii ) frizzled homolog 2 ( Drosophila ) ( Fzd2 ) was up-regulated 4.4-fold. Chromatin immunoprecipitation (ChIP) analysis confirmed the direct interaction between the OPN promoter and Hoxc8 protein in vivo , supporting the view that OPN is a direct transcriptional target of Hoxc8 .

The hnRNP A/B type proteins are abundant nuclear factors that bind to Pol II transcripts and are involved in numerous RNA-related activities. To date most data on the hnRNP A/B family have been obtained with recombinant proteins and cell cultures. Further characterization can result from an examination of the impact of various modifications in intact functional loci; however, such characterization is hampered by the presence of numerous and widely dispersed hnRNP A/B-related sequences in the mammalian genome. We have found hnRNP A3, a poorly recognized member of the hnRNP A/B family, among candidate transcription factors that interact with the regulatory region of the Hoxc8 gene and screened the human and mouse genomes for genes that encode hnRNP A3. We demonstrate that the sequence reported previously as the human hnRNP A3 gene (Accession number S63912) and located on 10p11.1 belongs to a processed pseudogene of the functional intron-containing locus HNRPA3, which we have identified on 2q31.2. We have also identified its murine orthologs on mouse chromosome 2D and rat chromosome 3q23. Alternative splices were revealed at the N-terminus and in the middle of hnRNP A3. 14 and 28 additional loci in the human and mouse genome, respectively, were mapped and identified as hnRNP A3 processed pseudogenes. In addition, we have found and compared hnRNP A3 orthologous genes in Gallus gallus, Xenopus tropicalis, and Danio rerio. The present in silico analysis serves as a necessary step toward a further functional characterization of hnRNP A3.

Williams–Beuren syndrome (also known as Williams syndrome) is caused by a deletion of a 1.55- to 1.84-megabase region from chromosome band 7q11.23. GTF2IRD1 and GTF2I , located within this critical region, encode proteins of the TFII-I family with multiple helix–loop–helix domains known as I repeats. In the present work, we characterize a third member, GTF2IRD2 , which has sequence and structural similarity to the GTF2I and GTF2IRD1 paralogs. The ORF encodes a protein with several features characteristic of regulatory factors, including two I repeats, two leucine zippers, and a single Cys-2/His-2 zinc finger. The genomic organization of human, baboon, rat, and mouse genes is well conserved. Our exon-by-exon comparison has revealed that GTF2IRD2 is more closely related to GTF2I than to GTF2IRD1 and apparently is derived from the GTF2I sequence. The comparison of GTF2I and GTF2IRD2 genes revealed two distinct regions of homology, indicating that the helix–loop–helix domain structure of the GTF2IRD2 gene has been generated by two independent genomic duplications. We speculate that GTF2I is derived from GTF2IRD1 as a result of local duplication and the further evolution of its structure was associated with its functional specialization. Comparison of genomic sequences surrounding GTF2IRD2 genes in mice and humans allows refinement of the centromeric breakpoint position of the primate-specific inversion within the Williams–Beuren syndrome critical region.

The statistical analysis of phylogenetic footprints in the two known horn shark Hox clusters and the four mammalian clusters shows that the shark HoxN cluster is HoxD-like. This finding implies that the most recent common ancestor of jawed vertebrates had at least four Hox clusters, including those which are orthologous to the four mammalian Hox clusters.

The study of Hox clusters and genes provides insights into the evolution of genomic regulation of development. Derived ray-finned fishes (Actinopterygii, Teleostei) such as zebrafish and pufferfish possess duplicated Hox clusters that have undergone considerable sequence evolution. Whether these changes are associated with the duplication(s) that produced extra Hox clusters is unresolved because comparison with basal lineages is unavailable. We sequenced and analyzed the HoxA cluster of the bichir (Polypterus senegalus), a phylogenetically basal actinopterygian. Independent lines of evidence indicate that bichir has one HoxA cluster that is mosaic in its patterns of noncoding sequence conservation and gene retention relative to the HoxA clusters of human and shark, and the HoxAα and HoxAβ clusters of zebrafish, pufferfish, and striped bass. HoxA cluster noncoding sequences conserved between bichir and euteleosts indicate that novel cis-sequences were acquired in the stem actinopterygians and maintained after cluster duplication. Hence, in the earliest actinopterygians, evolution of the single HoxA cluster was already more dynamic than in human and shark. This tendency peaked among teleosts after HoxA cluster duplication.

In the transforming growth factor β (TGF-β) superfamily, activin A, TGF-β1, and bone morphogenic protein 4 (BMP-4) have various effects on hematopoiesis, including early mesodermo-hematogenesis. After these cytokines bind to their respective receptor, a regulatory Smad is phosphorylated and becomes associated with Smad4, the common Smad, and the resulting complex translocates to the nucleus to regulate transcription. DLX1 is the product of a member of thedistal-lesshomeobox gene family, which is known to have important roles in embryogenesis, particularly in craniofacial development, and in GABAergic neurogenesis. DLX1 has been reported to be temporally and spatially coexpressed with BMP-4 during embryogenesis in selected contexts. We report here that, in addition to the previously reported regions/cells, DLX1 is expressed in hematopoietic cells in a lineage-dependent manner and that DLX1 interacts with Smad4 through its homeodomain. We show that it blocks multiple signals from TGF-β superfamily cytokines such as activin A, TGF-β1, and BMP-4, including differentiation of a hematopoietic cell line by activin A. Taken together, these data suggest that DLX1 may function as a regulator of multiple signals from TGF-β superfamily members in broad biological contexts during blood production.

Hox genes play a key role in animal body plan development. These genes tend to occur in tightly linked clusters in the genome. Vertebrates and invertebrates differ in their Hox cluster number, with vertebrates having multiple clusters and invertebrates usually having only one. Recent evidence shows that vertebrate Hox clusters are structurally more constrained than invertebrate Hox clusters; they exclude transposable elements, do not undergo tandem duplications, and conserve their intergenic distances and gene order. These constraints are only relaxed after a cluster duplication. In contrast, invertebrate Hox clusters are structurally more plastic; tandem duplications are common, the linkage of Hox genes can change quickly, or they can lose their structural integrity completely. We propose that the constraints on vertebrate Hox cluster structure lead to an association between the retention of duplicated Hox clusters and adaptive radiations. After a duplication the constraints on Hox cluster structure are temporarily lifted, which opens a window of evolvability for the Hox clusters. If this window of evolvability coincides with an adaptive radiation, chances are that a modified Hox cluster becomes recruited in an evolutionary novelty and then both copies of duplicated Hox clusters are retained.

The non-coding intergenic regions of Hox genes are remarkably conserved among mammals. To determine the usefulness of this sequence for phylogenetic comparisons, we sequenced an 800-bp fragment of the Hoxc9-Hoxc8 intergenic region from several species belonging to different mammalian clades. Results obtained from the phylogenetic analysis are congruent with currently accepted mammalian phylogeny. Additionally, we found a TC mini satellite repeat polymorphism unique to felines. This polymorphism may serve as a useful marker to differentiate between mammalian species or as a genetic marker in feline matings. This study demonstrates usefulness of a comparative approach employing non-coding regions of Hox gene complexes.

The Dlx gene family controls developmental patterning principally in the pharyngeal and cranial regions. We review the structure and function of these genes in the vertebrates and relate these properties to their evolution. We particularly focus on the Dlx3-7 bigene cluster which we postulate to be more derived phylogenetically and functionally than the other two bigene clusters, Dlx1-2 and Dlx5-6. We stress the transcriptional control of the Dlx3-7 bigene cluster, and postulate its control by Dlx1-2.

TFII-I family proteins are characterized structurally by the presence of multiple reiterated I-repeats, each containing a putative helix–loop–helix domain. Functionally, they behave as multifunctional transcription factors that are activated by a variety of extracellular signals. In studying their subcellular localization, we noticed that these transcription factors frequently reside in subnuclear domains/dots. Because nuclear dots are believed often to harbor components of histone deacetylase enzymes (HDACs), we investigated whether TFII-I family proteins colocalize and interact with HDACs. Here, we show that TFII-I and its related member hMusTRD1/BEN physically and functionally interact with HDAC3. The TFII-I family proteins and HDAC3 also show nearly identical expression patterns in early mouse development. Consistent with our earlier observation that TFII-I family proteins also interact with PIASxβ, a member of the E3 ligase family involved in the small ubiquitin-like modifier (SUMO) pathway, we show further that PIASxβ physically and functionally interacts with HDAC3 and relieves the transcriptional repression exerted by HDAC3 upon TFII-I-mediated gene activation. These results suggest a complex interplay between two posttranslational pathways—histone modification and SUMOylation—brokered in part by TFII-I family proteins.

The duplication of Hox clusters and their maintenance in a lineage has a prominent but little understood role in chordate evolution. Here we examined how Hox cluster duplication may influence changes in cluster architecture and patterns of noncoding sequence evolution. We sequenced the entire duplicated HoxAa and HoxAb clusters of zebrafish ( Danio rerio ) and extended the 5′ (posterior) part of the HoxM (HoxA-like) cluster of horn shark ( Heterodontus francisci ) containing the hoxa11 and hoxa13 orthologs as well as intergenic and flanking noncoding sequences. The duplicated HoxA clusters in zebrafish each house considerably fewer genes and are dramatically shorter than the single HoxA clusters of human and horn shark. We compared the intergenic sequences of the HoxA clusters of human, horn shark, zebrafish (Aa, Ab), and striped bass and found extensive conservation of noncoding sequence motifs, i.e., phylogenetic footprints, between the human and horn shark, representing two of the three gnathostome lineages. These are putative cis-regulatory elements that may play a role in the regulation of the ancestral HoxA cluster. In contrast, homologous regions of the duplicated HoxAa and HoxAb clusters of zebrafish and the HoxA cluster of striped bass revealed a striking loss of conservation of these putative cis-regulatory sequences in the 3′ (anterior) segment of the cluster, where zebrafish only retains single representatives of group 1, 3, 4, and 5 (HoxAa) and group 2 (HoxAb) genes and in the 5′ part of the clusters, where zebrafish retains two copies of the group 13, 11, and 9 genes, i.e., AbdB-like genes. In analyzing patterns of cis-sequence evolution in the 5′ part of the clusters, we explicitly looked for evidence of complementary loss of conserved noncoding sequences, as predicted by the duplication-degeneration-complementation model in which genetic redundancy after gene duplication is resolved because of the fixation of complementary degenerative mutations. Our data did not yield evidence supporting this prediction. We conclude that changes in the pattern of cis-sequence conservation after Hox cluster duplication are more consistent with being the outcome of adaptive modification rather than passive mechanisms that erode redundancy created by the duplication event. These results support the view that genome duplications may provide a mechanism whereby master control genes undergo radical modifications conducive to major alterations in body plan. Such genomic revolutions may contribute significantly to the evolutionary process.

The sea lamprey Petromyzon marinus is among the most primitive of extant vertebrates. We are interested in the organization of its Hox gene clusters, because, as a close relative of the gnathostomes, this information would help to infer Hox cluster organization at the base of the gnathostome radiation. We have partially mapped the P. marinus Hox clusters using phage, cosmid, and P1 artificial chromosome libraries. Complete homeobox sequences were obtained for the 22 Hox genes recovered in the genomic library screens and analyzed for cognate group identity. We estimate that the clusters are somewhat larger than those of mammals (roughly 140 kbp vs. 105 kbp) but much smaller than the single Hox cluster of the cephalochordate amphioxus (at more than 260 kb). We never obtained more than three genes from any single cognate group from the genomic library screens, although it is unlikely that our screen was exhaustive, and therefore conclude that P. marinus has a total of either three or four Hox clusters. We also identify four highly conserved non-coding sequence motifs shared with higher vertebrates in a genomic comparison of Hox 10 genes.

The transcriptional regulation of the Hoxc8 gene is controlled during early mouse embryogenesis by an enhanceosome-like control region, termed the early enhancer (EE), located 3 kb upstream from the Hoxc8 translation start site. The EE is involved in establishing the posterior expression pattern of Hoxc8 at embryonic day (E) 8.5–9.0. Genetic and biochemical data have shown that nuclear factors interact with this region in a sequence-specific manner. We have used a yeast one-hybrid screen in a search for transcription factors that bind to EE motifs and have isolated a novel murine DNA-binding protein, termed BEN (binding factor for early enhancer). The ORF of BEN encodes a protein of 1072 amino acids and contains six helix–loop–helix domains, a hydrophobic leucine zipper-like motif, and a serine-rich repeat. The murine BEN gene is structurally similar to the human gene TFII-I in that both genes encode unique 95-amino acid long helix–loop/span–helix domains. The BEN gene produces several major transcripts (3.6, 4.4, and 5.9 kb) present in most adult tissues and shows discrete spatial and temporal domains of expression in areas of epithelial-mesenchymal interaction during mouse embryogenesis from E9.5 to E12.5. Several BEN-encoded polypeptides of different sizes ranging from 165 to 40 kDa were identified by Western blot analysis using BEN-specific polyclonal Abs. We propose, on the bases of sequence homology, that BEN is the mouse ortholog of the recently described human gene, WBSCR11, known also as GTF2IRD1, GTF3, Cream1, and MusTRD1. This gene is deleted hemizygously in individuals with Williams Syndrome, an autosomal dominant genetic condition characterized by complex physical, cognitive, and behavioral traits resulting from a perturbed developmental process.

The identification of cis-sequences responsible for spatiotemporal patterns of gene expression often requires the functional analysis of large genomic regions. In this study a 100-kb zebrafish Hoxa-11b-lacZ reporter gene was constructed and expressed in transgenic mice. PAC clone 10-O19, containing a portion of the zebrafish HoxA-b cluster, was captured into the yeast-bacterial shuttle vector, pPAC-ResQ, by recombinogenic targeting. A lacZ reporter gene was then inserted in-frame into exon 1 of the zfHoxa-11b locus by a second round of recombinogenic targeting. Expression of the zfHoxa-11b-lacZ reporter gene in 10.5 d.p.f. transgenic mouse embryos was observed only in the posterior portion of the A-P axis, in the paraxial mesoderm, neural tube, and somites. These findings demonstrate the utility of recombinogenic targeting for the modification and expression of large inserts captured from P1/PAC clones.

Control of cell identity during development is specified in large part by the unique expression patterns of multiple homeobox-containing ( Hox ) genes in specific segments of an embryo. Trithorax and Polycomb-group (Trx-G and Pc-G) proteins in Drosophila maintain Hox expression or repression, respectively. Mixed lineage leukemia (MLL) is frequently involved in chromosomal translocations associated with acute leukemia and is the one established mammalian homologue of Trx . Bmi-1 was first identified as a collaborator in c-myc -induced murine lymphomagenesis and is homologous to the Drosophila Pc-G member Posterior sex combs . Here, we note the axial-skeletal transformations and altered Hox expression patterns of Mll -deficient and Bmi-1 -deficient mice were normalized when both Mll and Bmi-1 were deleted, demonstrating their antagonistic role in determining segmental identity. Embryonic fibroblasts from Mll -deficient compared with Bmi-1 -deficient mice demonstrate reciprocal regulation of Hox genes as well as an integrated Hoxc8 -lacZ reporter construct. Reexpression of MLL was able to overcome repression, rescuing expression of Hoxc8 -lacZ in Mll -deficient cells. Consistent with this, MLL and BMI-I display discrete subnuclear colocalization. Although Drosophila Pc-G and Trx-G members have been shown to maintain a previously established transcriptional pattern, we demonstrate that MLL can also dynamically regulate a target Hox gene.

We have developed a method to clone genomic DNA selectively into a yeast–bacterial shuttle vector, pClasper, by recombinogenic targeting in yeast. A gene-specific pClasper targeting vector was constructed with small recombinogenic ends (500 bp) derived from flanking sequences of the genomic region to be cloned. Linearized, recombinogenic pClasper targeting vector and native genomic DNA were cotransformed into yeast. The gene of interest is selectively cloned by recombination between the recombinogenic ends in the targeting vector and homologous regions in the genomic DNA. Here we demonstrate direct cloning of a stably integrated Hoxc8-LacZ-Ura3 reporter gene construct from a mouse embryo fibroblast cell line and single-copy genes from total human genomic DNA. The frequency of capture of the recombinant insert was 0.05–3% of transformants. In contrast to previous reports, we were able to clone genomic DNA directly with a vector containing yeast autonomous replicating sequences. This approach provides a powerful method with which to clone and modify genes precisely for functional analysis.

This study reports the results of a comparative sequencing study in higher primates, focusing on the intergenic region located between HOXB6 and HOXB7. We have examined an 832 bp. region, encompassing a putative Lateral Plate Mesoderm (LPM) enhancer element in a variety of anthropoid apes. The interspecific comparisons reveal extensive substitutions occurring within this region, with a marked bias in favor of CT transitions within the enhancer element. The pattern of these substitutions suggests that the LPM enhancer region is subject to specific sequence and compositional constraints that are only revealed through comparative sequencing. These constraints produce an enhancer signature, the CpG microisland, which may be useful in identifying additional regulatory elements located within the HOX complexes. J. Exp. Zool. (Mol. Dev. Evol.) 285:170–176, 1999. © 1999 Wiley-Liss, Inc.

In this article, we consider the role of the Hox genes in chordate and vertebrate evolution from the viewpoints of molecular and developmental evolution. Models of Hox cluster duplication are considered with emphasis on a threefold duplication model. We also show that cluster duplication is consistent with a semiconservative model of duplication, where following duplication, one daughter cluster remains unmodified, while the other diverges and assumes a new architecture and presumably new functions. Evidence is reviewed, suggesting that Hox gene enhancers have played an important role in body plan evolution. Finally, we contrast the invertebrates and vertebrates in terms of genome and Hox cluster duplication which are present in the latter, but not the former. We question whether gene duplication has been important in vertebrates for the introduction of novel features such as limbs, a urogenital system, and specialized neuromuscular interactions.

In an effort to characterize the signal transduction mechanisms that operate to regulate homeodomain protein function, we have analyzed the phosphorylation state of two homeodomain proteins, Hoxb-6 and Hoxc-8, in vitro and in vivo. The baculovirus expression system was employed to demonstrate that Hoxb-6 is phosphorylated in Sf9 cells while Hoxc-8 is not. Using two-dimensional tryptic phosphopeptide mapping and purified protein kinases, we demonstrate that Hoxb-6 is phosphorylated in vitro by casein kinase II and cAMP-dependent protein kinase. The casein kinase II phosphorylation site was mapped to serine-214. Two-dimensional tryptic phosphopeptide mapping of immunoprecipitated Hoxb-6 from mouse embryonic spinal cords demonstrates that the same peptide phosphorylated in vitro and in Sf9 cells by casein kinase II is also phosphorylated in vivo. The conservation of this site in several homeodomain proteins from various species is discussed. J. Exp. Zool. (Mol. Dev. Evol.) 285:76–84, 1999. © 1999 Wiley-Liss, Inc.

Variations in regulatory regions of developmental control genes have been implicated in the divergence of axial morphologies. To find potentially significant changes in cis-regulatory regions, we compared nucleotide sequences and activities of mammalian Hoxc8 early enhancers. The nucleotide sequence of the early enhancer region is extremely conserved among mammalian clades, with five previously described cis-acting elements, A–E, being invariant. However, a 4-bp deletion within element C of the Hoxc8 early enhancer sequence is observed in baleen whales. When assayed in transgenic mouse embryos, a baleen whale enhancer (unlike other mammalian enhancers) directs expression of the reporter gene to more posterior regions of the neural tube but fails to direct expression to posterior mesoderm. We suggest that regulation of Hoxc8 in baleen whales differs from other mammalian species and may be associated with variation in axial morphology.

Hox genes are expressed in dynamic patterns during embryogenesis consistent with their role in axial specifications. To study the distribution of mouse Hoxc8, a homeodomain containing protein, we raised monoclonal antibodies against the least conserved portion of Hoxc8. Using these antibodies, we have examined early and mid-gestation embryos for the distribution of the protein. At the end of gastrulation Hoxc8 is expressed in the caudal portion of the embryo. In the neural tube, an early phase when all cells express Hoxc8 is distinguished from a late phase with predominant expression in differentiating neurons. A comparison of this expression pattern with that of a reporter gene under the control of the early Hoxc8 enhancer demarcates three separate regulatory components: (1) initiation and establishment; (2) maintenance; and (3) downregulation. We propose that Hoxc8 expression during embryogenesis is established in multiple phases. Possible regulatory mechanisms involved in generating such a complex domain of Hox gene expression are discussed.

The mammalian Hox clusters arose by duplication of a primordial cluster. The duplication of Hox clusters created redundancy within cognate groups, allowing for change in function over time. The lamprey, Petromyzon marinus, occupies an intermediate position within the chordates, both in terms of morphologic complexity and possibly cluster number. To determine the extent of divergence among Hox genes after duplication events within vertebrates, we analyzed Hox genes belonging to cognate group 8. Here we report characterization of the HoxQ8 gene, which shows conservation with mammalian genes in its amino-terminal, homeobox and hexapeptide sequences, and in the position of its splice sites. A β-galactosidase reporter gene was introduced in the HoxQ8 genomic region by targeted recombinational cloning using a yeast-bacteria shuttle vector, pClasper. These reporter gene constructs were tested for their ability to direct region-specific expression patterns in transgenic mouse embryos. Lamprey enhancers direct expression to posterior neural tube but not to mesoderm, suggesting conservation of neuronal enhancers. In the presence of the mouse heat shock promoter, lamprey enhancers could also direct expression to the posterior mesoderm suggesting that there has been some divergence in promoter function. Our results suggest that comparative studies on Hox gene structure and analysis of regulatory elements may provide insights into changes concomitant with Hox cluster duplications in the chordates. J. Exp. Zool. 280:73–85, 1998. © 1998 Wiley-Liss, Inc.

We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt geneNvHBox-5.The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouseDlx7upstream of the homeodomain that may account for some of this divergence. We have mapped humanDLX7distal to the 5′ end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters.

The vertebrate Dlx gene family consists of homeobox-containing transcription factors distributed in pairs on the same chromosomes as the Hox genes. To investigate the evolutionary history of Dlx genes, we have cloned five new zebrafish family members and have provided additional sequence information for two mouse genes. Phylogenetic analyses of Dlx gene sequences considered in the context of their chromosomal arrangements suggest that an initial tandem duplication produced a linked pair of Dlx genes after the divergence of chordates and arthropods but prior to the divergence of tunicates and vertebrates. This pair of Dlx genes was then duplicated in the chromosomal events that led to the four clusters of Hox genes characteristic of bony fish and tetrapods. It is possible that a pair of Dlx genes linked to the Hoxc cluster has been lost from mammals. We were unable to distinguish between independent duplication and retention of the ancestral state of bony vertebrates to explain the presence of a greater number of Dlx genes in zebrafish than mammals. Determination of the linkage relationship of these additional zebrafish Dlx genes to Hox clusters should help resolve this issue.

Polymerase chain reaction (PCR) was used to amplify portion of homeobox genes present in a human 11-week fetal brain cDNA library. One of these PCR products was determined by sequencing to be the Gastrulation and brain specific-2 gene (GBX2). Screening this human fetal brain cDNA library with probes specific for GBX2 led to the identification of a 2151-bp cDNA clone. The nucleotide sequence of the cDNA clone encodes for a protein of 347 amino acid residues. The amino acid sequence of the GBX2 homeodomain is identical (100%) to the that of homologous gene, Gbx2, expressed in the developing mouse embryo and virtually identical (97%) to a gene expressed in the developing chicken embryo, CHox7. The 5' end of the GBX2 gene contains a CpG island in the untranslated region and a trinucleotide (CCG)8 repeat in the coding region. The amino-terminal end of the GBX2 protein is proline-rich, with 30 proline residues in one stretch of 120 amino acids. A single 2.2-kb transcript was detected by Northern analysis in the developing human CNS as well as in other tissues. The human genomic clone for GBX2 was also isolated, characterized, and mapped to 2q36(d)-q37 by somatic cell hybrid analysis and fluorescence in situ hybridization. These studies provide a framework for designing future experiments that are needed to determine the functional significance of this gene in CNS development.

The dentition is a segmental system whose evolution and morphology bears analogy to the evolution of segmentation in the vertebral column and limb. Combinatorial expression of members of the large "Hox" class of homeobox regulatory genes has been shown to play an important role in positional specification in these skeletal systems. This raises the possibility that homeobox genes are also used for positional specification in the dentition, and several homeobox genes are known to be expressed in developing teeth. To identify additional dentally expressed homeobox genes, cDNA from from murine tooth germs at 9.5, 14.5, and 17.5 days gestational age was amplified by PCR using sets of degenerate primers to the homeodomains of 18 different classes of homeobox genes. Amplification products were cloned and sequenced and compared to known gene sequences. To date this approach has confirmed the presence of Msx1, Msx2, Dlx1, and Dlx2, and identified several other homeobox genes not previously known to be expressed in teeth: Dbx, MHox, and Mox2A, plus an a additional Dlx gene, Dlx7. The Msx and Dlx genes are the best current candidates for a combinatorial mechanism that controls the differentiation of structures within and between teeth, and perhaps also the evolution of those structures.

The functional analysis of genes frequently requires manipulation of large genomic regions embedded in yeast artificial chromosomes (YACs). We have designed a yeast-bacteria shuttle vector, pClasper, that can be used to clone specific regions of interest from YACs by homologous recombination. The important feature of pClasper is the presence of the mini-F factor replicon. This leads to a significant increase in the size of the plasmid inserts that can be maintained in bacteria after cloning by homologous recombination in yeast. The utility of this vector lies in its ability to maintain large fragments in bacteria and yeast, allowing for mutagenesis in yeast and simplified preparation of plasmid DNA in bacteria. Using PCR-generated recombinogenic fragments in pClasper we cloned a 27 kb region from a YAC containing the Hoxc cluster and a 130 kb region containing the entire Hoxb cluster. No rearrangements were seen when the recombinants in the shuttle vector were transferred to bacteria. We outline the potential uses of pClasper for functional studies of large genomic regions by transgenic and other analyses.

The mammalian Dlx3 and Dlx4 genes are configured as a bigene cluster, and their respective expression patterns are controlled temporally and spatially by cis-elements that largely reside within the intergenic region of the cluster. Previous work revealed that there are conspicuously conserved elements within the intergenic region of the Dlx3–4 bigene clusters of mouse and human. In this paper we have extended these analyses to include 12 additional mammalian taxa (including a marsupial and a monotreme) in order to better define the nature and molecular evolutionary trends of the coding and non-coding functional elements among morphologically divergent mammals. Dlx3–4 regions were fully sequenced from 12 divergent taxa of interest. We identified three theria-specific amino acid replacements in homeodomain of Dlx4 gene that functions in placenta. Sequence analyses of constrained nucleotide sites in the intergenic non-coding region showed that many of the intergenic conserved elements are highly conserved and have evolved slowly within the mammals. In contrast, a branchial arch/craniofacial enhancer I37-2 exhibited accelerated evolution at the branch between the monotreme and therian common ancestor despite being highly conserved among therian species. Functional analysis of I37-2 in transgenic mice has shown that the equivalent region of the platypus fails to drive transcriptional activity in branchial arches. These observations, taken together with our molecular evolutionary data, suggest that theria-specific episodic changes in the I37-2 element may have contributed to craniofacial innovation at the base of the mammalian lineage.

control of metazoan organisms. In this article, we review the distribution of this gene family in selected species of the major phyla of metazoans, and from these data evaluate the usefulness of this system for establishing phyletic affinities, and the potential of using developmental genes as a means of ob­ taining insights into evolutionary mecha nisms. We begin with a discussion of mammalian homeobox genes, and then review the other phyletic groups com­ paratively.

Two human clathrin light-chain genes have been defined. The gene (CLTA) encoding the LCa light chain maps to the long arm of chromosome 12 at 12q23-q24 and that encoding the LCb light chain (CLTB) maps to the long arm of chromosome 4 at 4q2-q3. Isolation and characterization of partial genomic clones encoding human LCa and LCb reveal the neuron-specific insertions of the LCa and LCb proteins to he encoded by discrete exons, thus proving that clathrin light chains undergo alternate mRNA splicing to generate tissue-specific protein isoforms. The insertion sequence of LCb is encoded by a single exon and that of LCa by two exons. The first of the two neuron-specific LCa exons is homologous to the corresponding LCb exon. An intronic sequence of the LCb gene with similarity to the second neuron-specific exon of the LCa gene has been identified.

The chromosomal positions of three genes that are selectively expressed in mouse testis cells have been identified. These genes include (i) TAZ83, which codes for an early- to mid-pachytene germ cell stage-expressed, cysteine-rich transmembrane protein (cyritestin) with homologies to various snake toxins and guinea pig sperm-egg fusion proteins; (ii) TNZ1, which is expressed in neonatal Leydig cells; and (iii) TAZ4, a testis-specific gene isolated by immunoscreening with antiserum raised against Sertoli cell membranes. Our experimental data, derived from chromosomal in situ hybridizations and RFLP studies of genetic backcrosses, indicate that (i) the TAZ83 (cyritestin) gene maps to chromosome 8, band A2, near the Plat locus; (ii) TNZ1 is located in the proximal region of chromosome 11; and (iii) TAZ4 is located at band D in the distal portion of chromosome 11, near the Hlr1 locus, with a related sequence, TAZ4-rs1, in the proximal part of chromosome 1.

The sensitivity of small transgenes to position effects on their expression suggests that they could serve as indicators of the chromatin properties at their integration site. In particular, they might be expected to provide information on the functional properties of mammalian heterochromatin. We have produced a transgenic line that carries a mouse Hprt minigene on the Y chromosome. In situ hybridization localized the transgene to the heterochromatic portion of the Y. Analysis of transgene expression by isoelectric focusing indicated that the transgene is expressed in a mosaic pattern, and expressing cells have different levels of transgene activity. These findings can be explained as a position effect variegation induced by Y heterochromatin. However, two other transgenes, located at autosomal sites, also showed mosaic activity. If the mosaic transgene expression is attributed to the influence of the chromatin at the insertion site, the Y heterochromatin would appear less potent than some autosomal regions at inducing variegation. An alternative explanation consistent with our results is that the mosaic expression is a semi-autonomous characteristic of these transgene loci. Transgene-expressing and non-expressing cells differed in their ability to grow and be cloned in vitro, indicating that cellular differentiation affected the chromatin structure of the transgene locus on the Y. Karyotype analysis of male mice with the Y-linked transgene and from control male mice carrying the human HPRT transgene, or the mouse Pgk-1 gene at autosomal sites, indicated that the transgene-carrying Y is prone to non-disjunction, generating cells with two (or more) or no Y chromosomes in equal proportion. Further studies will determine if the propensity of this Y chromosome to mitotic errors is also observed in vivo.

Homeobox cluster genes (Hox genes) are highly conserved and can be usefully employed to study phyletic relation ships and the process of evolution itself. A phylogenetic survey of Hox genes shows an increase in gene number in some more recently evolved forms, particularly in verte brates. The gene increase has occurred through a two-step process involving first, gene expansion to form a cluster, and second, cluster duplication to form multiple clusters. We also describe data that suggests that non-Hox genes may be preferrentially associated with the Hox clusters and raise the possibility that this association may have an adaptive biological function. Hox gene loss may also play a role in evolution. Hox gene loss is well substantiated in the vertebrates, and we identify additional possible instances of gene loss in the echinoderms and urochordates based on PCR surveys. We point out the possible adaptive role of gene loss in evolution, and urge the extension of gene mapping studies to relevant species as a means of its sub stantiation.

Evidence derived from sequence comparisons and the genomic organization of the murine Antennapedia-class homeobox gene clusters suggest that they arose from a primordial cluster through a process of gene duplication and divergence followed by cluster duplication. A large chromosomal domain surrounding the ancestral homeobox cluster also appears to have been duplicated and has remained relatively stable since the divergence of humans and rodents. To test the extent of the duplicated chromosomal domain, we have initiated physical mapping studies of the regions surrounding the four murine homeobox clusters using pulsed-field gel electrophoresis and yeast artificial chromosome cloning. In this study, we present a long-range restriction map of mouse chromosome 11 spanning 1500 kb in the region surrounding the Hox-b cluster. We have determined that the gene for the nerve growth factor receptor is tightly linked to the Hox-b complex and is located within 50 kb of the Hox-b 1 gene at the 3' end of the cluster. Four yeast artificial chromosomes have been isolated and characterized by the polymerase chain reaction, long-range restriction mapping, and Southern blotting. Two clones of 150 and 300 kb contain the entire Hox-b cluster and the nerve growth factor receptor gene. A 440-kb clone contains the 3' end of the Hox-b cluster, the nerve growth factor receptor gene, and extends downstream. A 210-kb clone contains the 5' end of the Hox-b cluster and extends upstream. These clones confirm the pulsed-field restriction map of uncloned mouse DNA and represent a contig of approximately 600 kb of cloned material from mouse chromosomes 11.

We have analyzed the relationships of 337 homeodomain and related sequences using a distance matrix approach. Based on our results, homeodomain sequences can be classified into at least 30 distinguishable classes. Sequences belonging to the same class are highly similar to each other but there are no obvious close relationships between the separate classes. Since many of these classes each contain sequences from quite diverged organisms, we conclude that these separate classes of homeodomain sequences were established early during evolution.

Mammalian genomes contain clusters of homeobox genes ( Hox-C, HOX-C ) which are structurally similar to the homeotic genes of the Drosophila HOM complex. One method for assessing the functional similarity of particular Drosophila HOM and mammalian Hox genes is to test the ability of Hox genes to induce homeotic phenotypes when expressed in developing Drosophila . Here we describe such functional tests using mouse HoxB9 (formerly Hox-2.5 ), whose closest structural relative in Drosophila is Abdominal-B . When expressed from a heat shock promoter, HoxB9 induces transformations of head towards more posterior identities in Drosophila larvae and adults. These transformations share some similarities with the phenotypic effects produces by ectopically expressed Abdominal-B , but are also similar to the transformations induced by Antennapedia and mouse HoxB6 ( Hox-2.2 ), suggesting that HoxB9 specifies a positional identity that is intermediate between Antennapedia and Abdominal-B .

The importance of the Hox/HOM class of homeobox genes in early anterior-posterior pattern formation and the conserved genomic organization of this gene family provides an interesting study in genome evolution. The Platyhelminthes (flatworms) are a basal metazoan group with a simple bilateral body plan. We used the polymerase chain reaction (PCR) to detect Hox/HOM-class homeobox genes from species representing two classes of flatworms. Seven planarian and five trematode Hox/HOM-class homeoboxes were found. The sequences of the genes are consistent with the presence of one Hox/HOM-type cluster in the flatworms. Further analysis of this putative cluster may be expected to provide outgroup information for studying the evolution of the Hox/HOM clusters in the higher metazoa.

In this report we investigate the role of homeobox genes of the Antennapedia class in adult erythropoiesis, the process by which erythrocytes are formed in the bone marrow. We initially identified Hox genes expressed in mouse erythroleukemia cells by a PCR technique using degenerate primers capable of detecting most of the known genes in Hox clusters 1-4. Four different transcripts, Hox-3.3, -3.5, -3.6, and -4.3 were identified. An antisense oligonucleotide directed against HOX3C (human homologue of Hox-3.3) was used to study the effect of the colony formation by human erythroid progenitor cells. The oligonucleotide inhibited the formation of colony-forming unit erythroid-derived colonies but did not affect the size or degree of hemoglobinization. The more primitive erythroid burst-forming unit colonies or myeloid colonies were not affected. These results show that Hox-3.3 is involved in an early step in the proliferation of the erythroid colony-forming unit subset of progenitor cells.

With the increasing accumulation of data on the presence of the HOM/HOX class of homeobox genes in the animal kingdom, and with new comparative analyses of these data, strong evolutionary conservation is apparent. It is clear that HOM/HOX genes and their roles in pattern formation were established early during the evolution of major phyla. The functional indications that this system is utilized in quite diverged organisms attest to the fundamental roles of homeobox genes in organismal development.

Transgenic mouse models are valuable resources for analyzing functions of genes involved in human diseases. Mouse models provide critical insights into biological processes, including in vivo visualization of vasculature critical to our understanding of the immune system. Generating transgenic mice requires the capture and modification of large-insert DNAs representing genes of interest. We have developed a methodology using a yeast-bacterial shuttle vector, pClasper, that enables the capture and modification of bacterial artificial chromosomes (BAC)-sized DNA inserts. Numerous improvements and technical advances in the original pClasper vector have allowed greater flexibility and utility in this system. Examples of such pClasper mediated gene modifications include: Claspette-mediated capture of large-insert genomic fragments from BACs-human polycystic kidney disease-1 (PKD1); modification of pClasperA clones by the RareGap method-PKD1 mutations; Claspette-mediated modification of pClasper clones-mouse albumin-1 gene; and, of most relevance to our interest in lymph node vasculature-Claspimer-mediated modification of pClasper clones-high endothelial venule and lymphatic vessel genes. Mice that have been generated with these methods include mice with fluorescent high endothelial venules.

Lymphotoxin (LT) is crucial for the regulation of HEV adhesion molecules MAdCAM-1 and PNAd and a sulfotransferase, GlcNAc6ST-2 (gene symbol Chst4); here called HEC-6ST. Following immunization, some HEVs express markers of both HEVs (PNAd) and LVs (LYVE-1). In order to evaluate this process in real time, we have developed mice transgenic for a construct that consists of an HEV specific gene driving a green fluorescent reporter gene (eGFP). These mice express the reporter gene in HEVs in concurrence with the endogenous gene and PNAd. Additional mice transgenic for lymphatic vessel reporter constructs are in development. These will provide material for in vivo imaging and allow us to evaluate the regulation and interaction of HEVs and LVs.

Homeobox DNA polymorphisms (restriction fragment length polymorphisms, RFLPs) were first found in 13 individuals, and then in 121 subterranean mole rats of the Spalax ehrenbergi superspecies in Israel. Spalacidae are adapted morphologically to life underground apparently since Oligocene times, 30 million years ago. The superspecies S. ehrenbergi comprises four chromosomal species (2n = 52, 54, 58, and 60) displaying active speciation and dynamic adaptive radiation into four climatic regimes. A Hox-1.1 and -1.5 polymorphism was found with EcoRI, where three of four individuals of the species 2n = 60 were lacking the diagnostic locus-specific band present in all other nine individuals tested in the first stage. Next we found monomorphism in 121 animals in Hox-2.1 digested with five six-base recognition endonucleases (EcoRI, HindIII, BstEII, KpnI, BamHI, and one four-base enzyme, TaqI). However, we found in the same 121 animals from 13 populations of the four species in Hox-3.1 species-specific TaqI polymorphism in two populations of 2n = 54, and HindIII polymorphism in five populations, three of 2n = 54 and two of 2n = 58. The homeobox polymorphisms found display significant correlations with the burrow microclimates of mole rats. Furthermore, they are significantly correlated primarily with diverse morphological variables, and secondarily with protein (allozyme) and DNA polymorphisms, but very little with physiological and behavioral variables. We suggest that in subterranean mole rats, the variation in microclimatic factors, particularly in more unpredictable xeric regions, may maintain genetic polymorphisms by diversifying selection in the developmental control homeobox genes Hox-1.1, -1.5, and Hox-3.1, affecting regulatory morphogenetic processes in evolution. By contrast, the Hox-2.1 monomorphism suggests strong purifying selection for conservation across the superspecies over long periods of evolutionary times. © 1992 Wiley-Liss, Inc.