Loss of function of the Prx1 and Prx2 homeobox genes alters architecture of the great elastic arteries and ductus arteriosus

Prx1 (MHox) and Prx2 (S8) are non-clustered homeobox genes that are expressed in a complex, mostly mesenchyme-specific pattern throughout embryogenesis. The expression pattern and gene-targeted mice previously revealed a major role for Prx1 in skeletogenesis. In addition, specific and high expression of both Prx genes was reported in the developing cardiovascular system, predominantly in prospective connective tissues of the heart and in the great arteries and veins. We examined embryos of previously generated gene-targeted mice. Prx2-/- mutants were viable and did not show cardiovascular malformations. Intracardiac morphology of Prxl-/- and Prx1/Prx2-combined null mutants also appeared normal throughout development. However, the Prx1-/- and Prx1/Prx2 double-null mutants showed a vascular abnormality with an abnormal positioning and awkward curvature of the aortic arch in addition to a misdirected and elongated ductus arteriosus, and in two of seven combined mutants, an anomalous retro-oesophageal right subclavian artery. Generally, all great arteries appeared to run somewhat tortuously through the surrounding mesenchyme. The vascular histology and vessel wall thickness were normal in all mutants. Prx1-/- and Prx double-gene-targeted mice revealed similar spectra of vascular anomalies, but double mutants appeared to be more seriously affected. The current findings suggest that other genes may compensate for the lossof Prx in the heart, but, in contrast, our data support a role for Prx in the development of vascular and perivascular matrix. Received: 1 April 1999 / Accepted: 1 June 1999     

Opposing roles in neurite growth control by two seven-pass transmembrane cadherins

The growth of neurites (axon and dendrite) should be appropriately regulated by their interactions in the development of nervous systems where a myriad of neurons and their neurites are tightly packed. We show here that mammalian seven-pass transmembrane cadherins Celsr2 and Celsr3 are activated by their homophilic interactions and regulate neurite growth in an opposing manner. Both gene-silencing and coculture assay with rat neuron cultures showed that Celsr2 enhanced neurite growth, whereas Celsr3 suppressed it, and that their opposite functions were most likely the result of a difference of a single amino acid residue in the transmembrane domain. Together with calcium imaging and pharmacological analyses, our results suggest that Celsr2 and Celsr3 fulfill their functions through second messengers, and that differences in the activities of the homologs results in opposite effects in neurite growth regulation.     

Analysis of two distinct retinoic acid response elements in the homeobox gene Hoxb1 in transgenic mice.

Expression of vertebrate Hox genes is regulated by retinoids such as retinoic acid (RA) in cell culture and in early embryonic development. Retinoic acid response elements (RAREs) have been identified in Hox gene regulatory regions, suggesting that endogenous retinoids may be involved in the direct control of Hox gene patterning functions. Previously, two RAREs located 3' of the murine Hoxb1 gene, a DR(2) RARE and a DR(5) RARE, have been shown to regulate Hoxb1 mRNA expression in the neural epithelium and the foregut region, respectively; the foregut develops into the esophagus, liver, pancreas, lungs, and stomach. We have now examined the functional roles of these two types of 3' RAREs in regulating Hoxb1 expression at different stages of gestation, from embryonic day 7.5 to 13.5, in transgenic mice carrying specific RARE mutations. We demonstrate that the DR(5) RARE is required for the regulation of Hoxb-1 transgene region-specific expression in the gut and extraembryonic tissues, as well as for the RA-induced anteriorization of Hoxb-1 transgene expression in the gut. In contrast, expression of the Hoxb1 transgene in the neural epithelium requires only the DR(2) RARE. By in situ hybridization, we have identified a new site of Hoxb1 expression in the developing forelimbs at approximately day 12.5, and we show that, in transgenic embryos, expression in the forelimb buds requires that either the DR(2) or the DR(5) RARE is functional. Attainment of a high level of Hoxb1 transgene expression in other regions, such as in rhombomere 4 (r4) and in the somites, requires that both the DR(2) and DR(5) RAREs are functional. In addition, our transgenic data indicate that the Hoxb1 gene is expressed in other tissues such as the hernia gut, genital eminence, and lung. Our analysis shows that endogenous retinoids act through individual DR(2) and DR(5) RAREs to regulate Hoxb1 expression in different regions of the embryo and that functional redundancy between these DR(2) and DR(5) RAREs does not exist with respect to neural epithelium and the gut Hoxb1 expression.     

Characterization of two T. gondii CK1 isoforms

Previous affinity chromatography experiments have described the unexpected binding of an isoform of casein kinase I (CK1) from Leishmania mexicana, Trypanosoma cruzi, Plasmodium falciparum and Toxoplasma gondii to an immobilized cyclin-dependent kinase (CDK) inhibitor (purvalanol B). In order to further evaluate CK1 as a potential anti-parasitic target, two T. gondii CK1 genes were cloned by PCR using primers derived from a putative CK1 gene fragment identified from a T. gondii EST database. The genes are predicted to encode a smaller polypeptide of 38 kDa (TgCK1alpha) and larger 49 kDa isoform bearing a C-terminal extension (TgCK1beta). Enzymatically active recombinant FLAG-epitope tagged TgCK1alpha and TgCK1beta enzymes were immuno-precipitated from transiently transfected T. gondii parasites. While TgCK1alpha expression was found to be cytosolic, TgCK1beta was expressed predominantly at the plasma membrane. Deletion mapping showed that the C-terminal domain of TgCK1beta confers this membrane-association. Recombinant TgCK1alpha and TgCK1beta isoforms were also expressed in E. coli and biochemically characterized. A 38kDa native CK1 activity was partially purified from T. gondii tachyzoites by ion-exchange and hydrophobic interaction chromatography with biochemical and serological properties closely resembling those of recombinant TgCK1alpha. In contrast, we were not able to identify a native CK1 activity corresponding to the larger TgCK1beta 49 kDa isoform in tachyzoite lysates. Purvalanol B and the related compound aminopurvalanol A selectively inhibit TgCK1alpha, confirming the existence of potentially exploitable structural differences between host and parasite CK1 enzymes. Since the more cell-permeable aminopurvalanol also inhibits parasite growth, these results provide further impetus to investigate inhibitors of CK1 as anti-parasitic agents.     

Signaling pathways regulating the expression of Prx1 and Prx2 in the chick mandibular mesenchyme

Prx1 and Prx2 are members of the aristaless‐related homeobox genes shown to play redundant but essential roles in morphogenesis of the mandibular processes. To gain insight into the signaling pathways that regulate expression of Prx genes in the mandibular mesenchyme, we used the chick as a model system. We examined the patterns of gene expression in the face and the roles of signals derived from the epithelium on the expression of Prx genes in the mandibular mesenchyme. Our results demonstrated stage‐dependent roles of mandibular epithelium on the expression of Prx in the mandibular mesenchyme and provide evidence for positive roles of members of the fibroblast and hedgehog families derived from mandibular epithelium on the expression of Prx genes in the mandibular mesenchyme. Our studies suggest that endothelin‐1 signaling derived from the mesenchyme is involved in restricting the expression of Prx2 to the medial mandibular mesenchyme. Developmental Dynamics 237:3115–3127, 2008. © 2008 Wiley‐Liss, Inc.     

Prx1 and Prx2 cooperatively regulate the morphogenesis of the medial region of the mandibular process

Mice lacking both Prx1 and Prx2 display severe abnormalities in the mandible. Our analysis showed that complete loss of Prx gene products leads to growth abnormalities in the mandibular processes evident as early as embryonic day (E) 10.5 associated with changes in the survival of the mesenchyme in the medial region. Changes in the gene expression in the medial and lateral regions were related to gradual loss of a subpopulation of mesenchyme in the medial region expressing eHand. Our analysis also showed that Prx gene products are required for the initiation and maintenance of chondrogenesis and terminal differentiation of the chondrocytes in the caudal and rostral ends of Meckel's cartilage. The fusion of the mandibular processes in the Prx1/Prx2 double mutants is caused by accelerated ossification. These observations together show that, during mandibular morphogenesis, Prx gene products play multiple roles including the cell survival, the region‐specific terminal differentiation of Meckelian chondrocytes and osteogenesis. Developmental Dynamics 238: 2599–2613, 2009. © 2009 Wiley‐Liss, Inc.     

The Prx1 limb enhancers: Targeted gene expression in developing zebrafish pectoral fins

Limbs represent an excellent model to study the induction, growth, and patterning of several organs. A breakthrough to study gene function in various tissues has been the characterization of regulatory elements that allow tissue‐specific interference of gene function. The mouse Prx1 promoter has been used to generate limb‐specific mutants and overexpress genes in tetrapod limbs. Although zebrafish possess advantages that favor their use to study limb morphogenesis, there is no driver described suitable for specifically interfering with gene function in developing fins. We report the generation of zebrafish lines that express enhanced green fluorescent protein (EGFP) driven by the mouse Prx1 enhancer in developing pectoral fins. We also describe the expression pattern of the zebrafish prrx1 genes and identify three c onserved n on‐coding e lements (CNEs) that we use to generate fin‐specific EGFP reporter lines. Finally, we show that the mouse and zebrafish regulatory elements may be used to modify gene function in pectoral fins. Developmental Dynamics 240:1977–1988, 2011. © 2011 Wiley‐Liss, Inc.     

Hematopoietic immortalizing function of the NKL‐subclass homeobox gene TLX1

Translocations resulting in ectopic expression of the TLX1 homeobox gene (previously known as HOX11) are recurrent events in human T‐cell acute lymphoblastic leukemia (T‐ALL). Transduction of primary murine hematopoietic stem/progenitor cells with retroviral vectors expressing TLX1 readily yields immortalized hematopoietic progenitor cell lines. Understanding the processes involved in TLX1‐mediated cellular immortalization should yield insights into the growth and differentiation pathways altered by TLX1 during the development of T‐ALL. In recent clinical gene therapy trials, hematopoietic clonal dominance or T‐ALL‐like diseases have occurred as a direct consequence of insertional activation of the EVI1, PRDM16 or LMO2 proto‐oncogenes by the retroviral vectors used to deliver the therapeutic genes. Additionally, the generation of murine hematopoietic progenitor cell lines due to retroviral integrations into Evi1 or Prdm16 has also been recently reported. Here, we determined by linker‐mediated nested polymerase chain reaction the integration sites in eight TLX1‐immortalized hematopoietic cell lines. Notably, no common integration site was observed among the cell lines. Moreover, no insertions into the Evi1 or Prdm16 genes were identified although insertion near Lmo2 was observed in one instance. However, neither Lmo2 nor any of the other genes examined surrounding the integration sites showed differential vector‐influenced expression compared to the cell lines lacking such insertions. While we cannot exclude the possibility that insertional side effects transiently provided a selective growth/survival advantage to the hematopoietic progenitor populations, our results unequivocally rule out insertions into Evi1 and Prdm16 as being integral to the TLX1‐initiated immortalization process. © 2009 Wiley‐Liss, Inc.     

Expression of the Vax family homeobox genes suggests multiple roles in eye development

BACKGROUND: Homeobox gene products are thought to regulate target genes involved in the regional specification of tissues and organs. Several genes play roles in eye development. RESULTS: We isolated two new mouse homeobox genes, Vax1 and Vax2, expressed in retina primordium. The two genes share the same gene organization, consisting of three exons with predicted amino acid sequences identical in the homeodomain but diversified in other regions. At 9. 5 days post coitum, both Vax genes were expressed in optic vesicles. At 11.5-14.5 dpc, Vax1 became restricted to the optic stalk, while Vax2 was expressed in the ventral half of the neural retina. Mapping of Vax2 between D6Mit3 and D6Mit8 on chromosome 6 indicated a possible linkage with Emx1. CONCLUSIONS: The expression patterns of Vax genes suggest separate and specific involvement in eye development. Vax1 may contribute to differentiation of the neuroretina, pigmented epithelium and optic stalk, while Vax2 could function in the establishment of the dorso-ventral axis of the retina and the visual system. The chromosomal location of Vax2 suggests that Vax2-Emx1 and Vax1-Emx2 arose by chromosome duplication of the same ancestral gene.     

Roles of the Nkx3.1 homeobox gene in prostate organogenesis and carcinogenesis.

Although it is often presumed that the molecular pathways that underlie normal organogenesis are similar to those perturbed during carcinogenesis, few examples exist of tissue-specific regulatory genes that play central roles in both processes. In the case of the prostate gland, molecular genetic analyses have demonstrated that the Nkx3.1 homeobox gene plays an important role in normal differentiation of the prostatic epithelium and that its loss of function is an initiating event in prostate carcinogenesis. Thus, the Nkx3.1 homeobox gene provides a paradigm for understanding the relationship between normal differentiation and cancer, as well as a model for studying the roles of homeobox genes in these processes. Here, we review recent findings concerning the biological as well as biochemical function of this central regulator of prostate development and carcinogenesis.     

Bapx1 homeobox gene gain-of-function mice show preaxial polydactyly and activated Shh signaling in the developing limb.

To explore Bapx1 homeobox gene function in embryonic control of development, we employed a gain-of-function approach to complement our previous loss-of-function mutant analysis. We show that transgenic mice overexpressing Bapx1 are affected by skeletal defects including hindlimb preaxial polydactyly and tibial hypoplasia. Bapx1 overexpression generates limb anteroposterior patterning defects including induction of Shh signaling and ectopic activation of functions downstream of Shh signaling into the anterior region of the autopod. Moreover, Bapx1 overexpression stimulates formation of limb prechondrogenic condensations. We also show that Shh is reciprocally able to activate Bapx1 expression in mouse embryos as the orthologous hedgehog (hh) does with the bagpipe/Bapx1 gene in Drosophila. Our results indicate that Bapx1 can modulate appendicular skeletal formation, that the genetic hierarchy between Shh/hh and Bapx1/bagpipe has been conserved during evolution, and that in mouse embryos these two genes can influence one another in a genetically reciprocal manner. We conclude that it is reasonable to expect overexpression of Bapx1 in certain forms of polydactyly.