CFAP45, a heterotaxy and congenital heart disease gene, affects cilia stability.

Congenital heart disease (CHD) is the most common and lethal birth defect, affecting 1.3 million individuals worldwide. During early embryogenesis, errors in Left-Right (LR) patterning called Heterotaxy (Htx) can lead to severe CHD. Many of the genetic underpinnings of Htx/CHD remain unknown. In analyzing a family with Htx/CHD using whole-exome sequencing, we identified a homozygous recessive missense mutation in CFAP45 in two affected siblings. CFAP45 belongs to the coiled-coil domain-containing protein family, and its role in development is emerging. When we depleted Cfap45 in frog embryos, we detected abnormalities in cardiac looping and global markers of LR patterning, recapitulating the patient's heterotaxy phenotype. In vertebrates, laterality is broken at the Left-Right Organizer (LRO) by motile monocilia that generate leftward fluid flow. When we analyzed the LRO in embryos depleted of Cfap45, we discovered "bulges" within the cilia of these monociliated cells. In addition, epidermal multiciliated cells lost cilia with Cfap45 depletion. Via live confocal imaging, we found that Cfap45 localizes in a punctate but static position within the ciliary axoneme, and depletion leads to loss of cilia stability and eventual detachment from the cell's apical surface. This work demonstrates that in Xenopus, Cfap45 is required to sustain cilia stability in multiciliated and monociliated cells, providing a plausible mechanism for its role in heterotaxy and congenital heart disease.

Impaired tubulogenesis of cyst-derived cells from autosomal dominant polycystic kidneys.

Under appropriate growth factor or hormonal influence, renal epithelial cells cultured in collagen gels form branching tubular elements, reminiscent of metanephric tubulogenesis. This study evaluates the phenotypic characteristics of normal human renal epithelial cells (NK) and epithelial cells from cysts of autosomal dominant polycystic kidneys (ADPKD) grown in collagen gels under the influence of the growth factors (GFs) epidermal (EGF), transforming (TGF-alpha), hepatocyte (HGF) and fibroblast (FGF). All GFs induced cell proliferation with the formation of cell aggregates in both group of cells, however, NK cells exhibited proliferation at a much higher rate compared to ADPKD. All GFs induced formation of branching tubular elements with cell-polarity characteristics in NK cells. Such organized tubular elements were essentially absent in ADPKD cell cultures. Both NK and ADPKD cells expressed cell adhesion and matrix macromolecules. Expression of heparan sulfate-proteoglycan was diminished but enhanced for fibronectin in ADPKD cells. Receptor expression for EGF and FGF was similar. These findings indicate an impairment in tubulogenesis of ADPKD cells, perhaps related to the aberrant morphogenetic cell aggregation. Alternatively, this differentiation arrest may relate to abnormal biosynthesis of secretory matrix glycoproteins rather than those expressed on the plasmalemma.

Fractal geometry in rat chimeras demonstrates that a repetitive cell division program may generate liver parenchyma.

In the development of mammalian organs, a rapid and robust expansion of the parenchymal compartment must occur following allocation of organ primordia. This expansion must be regulated so that sufficient tissue mass is generated for further organization into functional tissues. The discovery that mosaic patches in the liver of rat chimeras are fractal (a geometric form with characteristic complexity) suggests a possible information storage scheme for programs of parenchyma generation. Since fractal objects are produced by the repetitive application of specific rules, it is possible that such a mechanism is responsible for the generation of organ parenchyma. The model cell division program for the generation of organ parenchyma considered here is to choose a cell at random to divide and place the daughter cell in a randomly chosen adjacent position displacing other cells which might occupy the chosen position. The completion of the division creates a new population of cells representing the input conditions for the next division. When this is repeated over and over in a tissue comprising two genetically distinguishable populations of cells, analysis of the geometry of the mosaic pattern obtained should fulfill specific predictions. If cell division occurred in this manner, the complexity of patch boundaries (patches are contiguous aggregates of cells of the same marker lineage in tissue from a chimera) should be independent of the proportion of the two parental cell lineages which make up the chimera's tissue. However, the complexity of the entire patch pattern should be dependent on this proportion. The complexity of the spatial distribution of the patches within a chimera's tissue should also be dependent on the proportion of the two parental lineages. We have measured the complexity of patch boundaries (surface fractal dimension), the complexity of entire fields of patches (mass fractal dimension), and the complexity of the spatial distribution of patches (fractal fragmentation) in rat liver from chimeras. We have established that the surface fractal dimension does not change as the proportion of the two parental lineages in the chimera's tissue changes, that there is a simple relationship between the complexity of entire patches and this proportion, and that the patches are fractally fragmented. These results are consistent with the hypothesis that repetitive application of this simple cell division program accounts for the generation of liver parenchyma.

Insertional mutation of a gene involved in growth regulation of the early mouse embryo.

A transgenic mouse strain derived from embryonic stem (ES) cells infected with multiple copies of a retroviral vector carries a recessive insertional mutation resulting in prenatal lethality. A detailed histological analysis of developing embryos has shown that the mutation results in hyperplasia of both embryonic and extraembryonic ectoderm and failure of mesoderm formation in the egg cylinder stage embryo. The number of cells in each lineage of normal and mutant embryos was estimated using stereological analysis of serial sections taken from implantation sites. We observed a 2-fold increase in the number of embryonic ectoderm cells in mutant embryos at 7.5 days postcoitum (dpc). In addition, we found that mutant embryonic ectoderm cells are only 0.6 times as large as normal cells. The number of extraembryonic ectoderm cells in mutant embryos at 7.5 dpc is also increased, by almost 4-fold. Mutant extraembryonic ectoderm cells are also smaller than normal, being only two-thirds the size of wild-type cells. The mutant phenotype suggests that the gene identified by this insertional mutation plays an important role in the growth control of early embryonic lineages.