An activating mutation in the PDGF receptor-beta causes abnormal morphology in the mouse placenta.

An oncogenic D842V mutation in the platelet-derived growth factor (PDGF) alpha-receptor (Pdgfra) has recently been described in patients with gastrointestinal stromal tumors. In order to test if the same mutation would confer oncogenic properties to the homologous PDGF beta-receptor (Pdgfrb), the corresponding aspartic acid residue at position 849 of Pdgfrb was changed into valine (D849V) using a knock-in strategy. This mutation turned out to be dominantly lethal and caused death even in chimeras (from 345 transferred chimeric blastocysts, no living coat chimeras were detected). Experiments employing mouse embryonic fibroblasts (MEFs) indicated hyperactivity of the mutant receptor. The mutant receptor was phosphorylated in a ligand-independent manner and, in contrast to wild-type MEFs, mutant cells proliferated even in the absence of ligand. Knockout experiments have previously indicated a role for Pdgfrb in placental development. We therefore analyzed wild-type and Pdgfrb D849V chimeric placentas from different gestational stages. No differences were detected at embryonic days 11.5 and 13.5 (n=4). At embryonic day 17.5, however, chimeric placentas (n=3/4) displayed abnormalities both in the labyrinth and in the chorionic plate. The changes included hyper-proliferation of alpha-smooth muscle actin and platelet/endothelial cell adhesion molecule-1 positive cells in the labyrinth and cells in the chorionic plate. In addition, the fetal blood vessel compartment of the labyrinth was completely disorganized.

Platelet-derived growth factor receptor-beta constitutive activity promotes angiogenesis in vivo and in vitro.

OBJECTIVE: Knockout studies have demonstrated crucial roles for the platelet-derived growth factor-B and its cognate receptor, platelet-derived growth factor receptor-beta (PDGFR-beta), in blood vessel maturation, that is, the coverage of newly formed vessels with mural cells/pericytes. This study describes the consequences of a constitutively activating mutation of the PDGFR-beta (Pdgfrb(D849V)) introduced into embryonic stem cells with respect to vasculogenesis/angiogenesis in vitro and in vivo. METHODS AND RESULTS: Embryonic stem cells were induced to either form teratomas in vivo or embryoid bodies, an in vitro model for mouse embryogenesis. Western blotting studies on embryoid bodies showed that expression of a single allele of the mutant Pdgfrb led to increased levels of PDGFR-beta tyrosine phosphorylation and augmented downstream signal transduction. This was accompanied by enhanced vascular development, followed by exaggerated angiogenic sprouting with abundant pericyte coating as shown by immunohistochemistry/immunofluorescence. Pdgfrb(D849V/+) embryoid bodies were characterized by increased expression of vascular endothelial growth factor (VEGF)-A and VEGF receptor-2; neutralizing antibodies against VEGF-A/VEGF receptor-2 blocked vasculogenesis and angiogenesis in mutant embryoid bodies. Moreover, Pdgfrb(D849V/+) embryonic stem cell-derived teratomas in nude mice were more densely vascularized than wild-type teratomas. CONCLUSIONS: Increased PDGFR-beta kinase activity is associated with elevated expression of VEGF-A and VEGF receptor-2, acting directly on endothelial cells and resulting in increased vessel formation.

A novel Krüppel-Associated Box identified in a panel of mammalian zinc finger proteins.

Krüppel-related zinc finger proteins probably constitute the largest individual family of transcription factors in mammals. These proteins often carry a potent repressor domain called the Krüppel Associated Box (KRAB), which is known to effectively repress transcription through interaction with transcriptional intermediary factor 1beta (TIF1beta). Here we report the isolation and characterization of a novel human KRAB A zinc finger protein, HZF12. The gene encoding HZF12 is located on Chromosome (Chr) 19p13.11-p12, and a 4.4-kb transcript from this gene is expressed in a variety of adult and fetal tissues. Two additional, larger transcripts are expressed in testis only. Interestingly, the KRAB A domain of HZF12 is followed by a 21-amino acid domain, encoded by a separate exon. This domain, which we designate KRAB C, was also identified in more than 25 additional human, mouse, and rat KRAB zinc finger proteins. On the basis of results from a previous study, we conclude that this novel KRAB domain strengthens the interaction with TIF1beta, thereby improving the ability of these KRAB zinc finger proteins to recruit TIF1beta to specific sites.

MZF6D, a novel KRAB zinc-finger gene expressed exclusively in meiotic male germ cells.

Spermatogenesis takes place in the seminiferous tubule in the testes and culminates in the production of spermatozoa (male gametes). Here we report the identification of a novel mouse zinc-finger gene, MZF6D, which is selectively expressed in meiotic spermatocytes. The MZF6D protein contains an N-terminally located repressor domain, a KRAB domain, followed by at least seven successive Krüppel zinc-finger motifs. The KRAB domain of MZF6D, which consists of a KRAB A box and the newly identified KRAB C box, has previously been shown to interact with TIF1beta, which is the common corepressor of all KRAB zinc-finger proteins. Northern blot analysis shows that the expression of MZF6D is restricted to testes. This was confirmed by RT-PCR analysis of a panel of mouse tissues. In situ hybridization of sections from adult mouse testes localizes the expression to meiotic spermatocytes, suggesting a specific role for MZF6D in the regulation of spermatogenesis.

KRAB zinc finger proteins: an analysis of the molecular mechanisms governing their increase in numbers and complexity during evolution.

Krüppel-related zinc finger proteins, with 564 members in the human genome, probably constitute the largest individual family of transcription factors in mammals. Approximately 30% of these proteins carry a potent repressor domain called the Krüppel associated box (KRAB). Depending on the structure of the KRAB domain, these proteins have been further divided into three subfamilies (A + B, A + b, and A only). In addition, some KRAB zinc finger proteins contain another conserved motif called SCAN. To study their molecular evolution, an extensive comparative analysis of a large panel of KRAB zinc finger genes was performed. The results show that both the KRAB A + b and the KRAB A subfamilies have their origin in a single member or a few closely related members of the KRAB A + B family. The KRAB A + B family is also the most prevalent among the KRAB zinc finger genes. Furthermore, we show that internal duplications of individual zinc finger motifs or blocks of several zinc finger motifs have occurred quite frequently within this gene family. However, zinc finger motifs are also frequently lost from the open reading frame, either by functional inactivation by point mutations or by the introduction of a stop codon. The introduction of a stop codon causes the exclusion of part of the zinc finger region from the coding region and the formation of graveyards of degenerate zinc finger motifs in the 3'-untranslated region of these genes. Earlier reports have shown that duplications of zinc finger genes commonly occur throughout evolution. We show that there is a relatively low degree of sequence conservation of the zinc finger motifs after these duplications. In many cases this may cause altered binding specificities of the transcription factors encoded by these genes. The repetitive nature of the zinc finger region and the structural flexibility within the zinc finger motif make these proteins highly adaptable. These factors may have been of major importance for their massive expansion in both number and complexity during metazoan evolution.