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Martin Blum

Publications and source records attributed to Martin Blum.

8 recordsLinked to original sources
PubMed2007

Cilia-driven leftward flow determines laterality in Xenopus.

Determination of the vertebrate left-right body axis during embryogenesis results in asymmetric development and placement of most inner organs. Although the asymmetric Nodal cascade is conserved in all vertebrates, the mechanism of symmetry breakage has remained controversial. In mammalian and fish embryos, a cilia-driven leftward flow of extracellular fluid is required for initiation of the Nodal cascade. This flow is localized at the posterior notochord ("node") and Kupffer's vesicle, respectively. In frog and chick embryos, however, molecular asymmetries are required earlier, from cleavage stages through gastrulation. The validity of a cilia-based mechanism for all vertebrates therefore has been questioned. Here we show that a cilia-driven leftward flow precedes asymmetric nodal expression in the frog Xenopus. Motile monocilia emerged on the gastrocoel roof plate during neurulation and lengthened and polarized from an initially central position to the posterior pole of cells. Concomitantly, a robust leftward fluid flow developed from stage 15 onward, significantly before asymmetric nodal transcription started in the left-lateral-plate mesoderm at stage 19. Injection of 1.5% methylcellulose into the archenteron prevented leftward flow and resulted in laterality defects, demonstrating that the flow itself was required for asymmetric gene expression and organ placement.

Animals↗
PubMed2006

Three types of cilia including a novel 9+4 axoneme on the notochordal plate of the rabbit embryo.

Motile monocilia play a pivotal role in left-right axis determination in mouse and zebrafish embryos. Cilia with 9+0 axonemes localize to the distal indentation of the mouse egg cylinder ("node"), while Kupffer's vesicle cilia in zebrafish show 9+2 arrangements. Here we studied cilia in a prototype mammalian embryo, the rabbit, which develops via a flat blastodisc. Transcription of ciliary marker genes Foxj1, Rfx3, lrd, polaris, and Kif3a initiated in Hensen's node and persisted in the nascent notochord. Cilia emerged on cells leaving Hensen's node anteriorly to form the notochordal plate. Cilia lengthened to about 5 mum and polarized from an initially central position to the posterior pole of cells. Electron-microscopic analysis revealed 9+0 and 9+2 cilia and a novel 9+4 axoneme intermingled in a salt-and-pepper-like fashion. Our data suggest that despite a highly conserved ciliogenic program, which initiates in the organizer, axonemal structures may vary widely within the vertebrates.

Animals↗
PubMed2005

Lack of a laterality phenotype in Pkd1 knock-out embryos correlates with absence of polycystin-1 in nodal cilia.

The invariant asymmetric placement of thoracic and abdominal organs in the vertebrates is controlled by the left-asymmetric activity of the Nodal signaling cascade during embryogenesis. In the mouse embryo asymmetric induction of nodal is thought to be dependent on functional monocilia on the ventral node cells and on the Pkd2 gene, which encodes the calcium channel polycystin-2 (PC2). In humans mutations in PKD2 and PKD1 give rise to polycystic kidney disease. The PC1 and PC2 proteins are thought to function as part of a multifactorial complex. Localization of both proteins to the primary renal cilium suggested a function on cilia of the ventral node. Here we investigated Pkd1 knock-out embryos for laterality defects and found wild-type organ morphogenesis and normal expression of nodal and Pitx2. While PC2 localized to nodal cilia, no ciliary localization of PC1 was detected in mouse embryos. This finding was confirmed in an archetypical mammalian blastodisc, the rabbit embryo. Thus, absence of PC1 localization to cilia corresponded with a lack of laterality defects in Pkd1 knock-out embryos. Our results demonstrate a PC1-independent function of PC2 in left-right axis formation, and indirectly support a ciliary role of PC2 in this process.

Animals↗
PubMed2003

Overexpression of hepatitis B virus surface antigens including the preS1 region in a serum-free Chinese hamster ovary cell line.

Current hepatitis B virus (HBV) vaccines consist of preparations of recombinant HBV major surface antigen (sAg) and are protective in about 90-95% of vaccinated subjects. In improved vaccines, the frequency of nonresponders to the classical vaccine could be reduced by including additional epitopes from the preS-domains of the middle and large surface antigens. In this report, the development and characterization of a CHO cell line for HBsAg, expressing major, middle, and large antigens are described. Despite the previously reported retention of secreted proteins by the preS1 domain, cell lines could be amplified that secreted large amounts of the complete set of antigens. A producer line was established that expressed 1mg HBsAg per 100ml suspension culture per week during exponential growth. The productivity per cell increased further by at least threefold when the culture reached the stationary phase at high cell densities. In the production cell line, several hundred copies of the HBV vector were integrated at two adjacent sites into chromosome 2. The cell line was adapted to growth in a defined protein-free medium minimizing the risk of adventitious agents introduced by animal derived supplements. The cell line stably produced antigen over several months. In the candidate vaccine, both preS2 and preS1 domains were present at ratios similar to HBsAg from human sera. In summary, a production cell line for an improved HBV vaccine is presented with properties such as high productivity, long term stability of expression, and growth in protein-free media.

Animals↗
PubMed2002

FGF8 acts as a right determinant during establishment of the left-right axis in the rabbit.

BACKGROUND: FGF8 has been implicated in the transfer of left-right (L-R) asymmetry from the embryonic midline (node) to the lateral plate mesoderm (LPM). Surprisingly, opposite roles have been described in chick and mouse. In mouse, FGF8 is required for the left-asymmetric expression of nodal, lefty2, and Pitx2. In chick, FGF8 represses nodal and Pitx2 on the right side. This discrepancy could reflect evolutionary differences between birds and mammals. Alternatively, the right-asymmetric expression of fgf8, which is not found in mouse, at the chick node may be a prerequisite of right-sided function. Finally, chick (blastodisc) and mouse (egg cylinder) differ with respect to the topology of the early gastrula/neurula embryo. RESULTS: The rabbit blastodisc was investigated as an additional mammalian L-R model system. While nodal, lefty, and Pitx2 showed asymmetric expression in the left LPM, fgf8 and all other midline marker genes were symmetrically expressed at the node like in mouse. Left-sided application of FGF8 repressed the endogenous transcription of nodal as well as ectopic expression induced by the parallel administration of BMP4. Right-sided inhibition of FGF8 signaling induced bilateral marker gene expression, demonstrating that, in rabbit, FGF8 acts as a right determinant like in chick. CONCLUSIONS: These findings suggest that the anatomy of the early embryo (blastodisc versus egg cylinder) rather than taxonomical differences or asymmetry in expression constitutes an important determinant of FGF8 function in L-R axis formation. The rabbit may provide a useful model for early human embryogenesis, as human embryos develop via a blastodisc as well.

Animals↗
PubMed2002

The ion channel polycystin-2 is required for left-right axis determination in mice.

Generation of laterality depends on a pathway which involves the asymmetrically expressed genes nodal, Ebaf, Leftb, and Pitx2. In mouse, node monocilia are required upstream of the nodal cascade. In chick and frog, gap junctions are essential prior to node/organizer formation. It was hypothesized that differential activity of ion channels gives rise to unidirectional transfer through gap junctions, resulting in asymmetric gene expression. PKD2, which if mutated causes autosomal dominant polycystic kidney disease (ADPKD) in humans, encodes the calcium release channel polycystin-2. We have generated a knockout allele of Pkd2 in mouse. In addition to malformations described previously, homozygous mutant embryos showed right pulmonary isomerism, randomization of embryonic turning, heart looping, and abdominal situs. Leftb and nodal were not expressed in the left lateral plate mesoderm (LPM), and Ebaf was absent from floorplate. Pitx2 was bilaterally expressed in posterior LPM but absent anteriorly. Pkd2 was ubiquitously expressed at headfold and early somite stages, with higher levels in floorplate and notochord. The embryonic midline, however, was present, and normal levels of Foxa2 and shh were expressed, suggesting that polycystin-2 acts downstream or in parallel to shh and upstream of the nodal cascade.

Animals↗
PubMed2002

Low proliferative and high migratory activity in the area of Brachyury expressing mesoderm progenitor cells in the gastrulating rabbit embryo.

General mechanisms initiating the gastrulation process in early animal development are still elusive, not least because embryonic morphology differs widely among species. The rabbit embryo is revived here as a model to study vertebrate gastrulation, because its relatively simple morphology at the appropriate stages makes interspecific differences and similarities particularly obvious between mammals and birds. Three approaches that centre on mesoderm specification as a key event at the start of gastrulation were chosen. (1) A cDNA fragment encoding 212 amino acids of the rabbit Brachyury gene was cloned by RT-PCR and used as a molecular marker for mesoderm progenitors. Whole-mount in situ hybridisation revealed single Brachyury-expressing cells in the epiblast at 6.2 days post conception, i.e. several hours before the first ingressing mesoderm cells can be detected histologically. With the anterior marginal crescent as a landmark, these mesoderm progenitors are shown to lie in a posterior quadrant of the embryonic disc, which we call the posterior gastrula extension (PGE), for reasons established during the following functional analysis. (2) Vital dye (DiI) labelling in vitro suggests that epiblast cells arrive in the PGE from anterior parts of the embryonic disc and then move within this area in a complex pattern of posterior, centripetal and anterior directions to form the primitive streak. (3) BrdU labelling shows that proliferation is reduced in the PGE, while the remaining anterior part of the embryonic disc contains several areas of increased proliferation. These results reveal similarities with the chick with respect to Brachyury expression and cellular migration. They differ, however, in that local differences in proliferation are not seen in the pre-streak avian embryo. Rather, rabbit epiblast cells start mesoderm differentiation in a way similar to Drosophila, where a transient downregulation of proliferation initiates mesoderm differentiation and, hence, gastrulation.

Amino Acid Sequence↗