26 research outputs found
Genetic Drivers of Kidney Defects in the DiGeorge Syndrome
Background The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. Methods We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. Results We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5Ă—10(-14)). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. Conclusions We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.)
Involvement of FGF and BMP family proteins and VEGF in early human kidney development
The spatial and temporal pattern of the
appearance of the fibroblast growth factor proteins
(FGF-8 and FGF-10), the bone morphogenetic proteins
(BMP-2/4 subfamily and BMP-7) and the vascular
endothelial growth factor protein (VEGF) was
investigated in the human mesonephros and metanephros
of the 5-9 week-old conceptuses. In the mesonephros,
both FGF’s and BMP’s were found in all structures and
their expression slightly decreased in the early fetal
period. VEGF positivity appeared in all mesonephric
structures, and increased in the fetal period coincidently
with formation of the mesonephric blood vessel network.
In the metanephros, FGF-8 first appeared only in the
metanephric mesenchyme, but from the 7th week on, its
reactivity increased and spread to other metanephric
structures. FGF-10 positive cells appeared in all
metanephric structures already in the 5th week, and
slightly intensified with progression of development.
Cell survival and nephrogenesis in the permanent kidney
might be associated with the appearance of both growth
factors. Both BMP-2/4 and BMP-7 displayed a similar
pattern of reactivity in all metanephric structures, and
their reactivity intensified with advancing development.
Alterations in their pattern of appearance might lead to
the formation of small and dysplastic kidneys. Already in the earliest developmental stages, VEGF protein
appeared in all metanephric structures. At later stages,
VEGF showed more intense reaction in the collecting
system than in the differentiating nephrons and
interstitium. Due to VEGF involvement in
vasculogenesis and angiogenesis, abnormal VEGF
appearance might lead to impaired formation of the
blood vessel network in the human permanent kidney
Involvement of pro-apoptotic and anti-apoptotic factors in the early development of the human pituitary gland
The spatial and temporal pattern of
appearance of pro-apoptotic caspase-3 and p53 proteins,
and anti-apoptotic bcl-2 protein was investigated in the
developing pituitary gland of 6 human embryos 5-8-
weeks old, using morphological and immunohistochemical
techniques. Their dynamic appearance
was analyzed in the Rathke's pouch (future
adenohypophysis), mesenchyme, and in the developing
neurohypophysis. In the 5th and 6th week, caspase-3
positive cells appeared in the Rathke's pouch (5%) and
stalk (11%), in the mesenchyme, but not in the
neurohypophysis. In the 6th and 7th week, apoptotic
cells were more numerous in the caudal part of the
Rathke's pouch due to its separation from the oral
epithelium. Pro-apoptotic p53 protein was detected in all
parts of the pituitary gland throughout the investigated
period. Nuclear condensations characterized cells
positive to caspase-3 and p53 proteins. Apoptotic cells
displayed condensations of nuclear chromatin on an
ultrastructural level as well. While caspase-3 dependent
pathway of cell death participated in morphogenesis of
the adenohypophysis and associated connective tissue,
p53-mediated apoptosis most likely participates in
morphogenesis of all parts of the gland, including neurohypophysis. The anti-apoptotic bcl-2 protein was
also detected in all parts of the developing gland. With
advancing development, the positivity to bcl-2 protein
increased in the cells of the adenohypophysis, while it
decreased in the neurohypophysis. Bcl-2 protein
probably prevented cell death in all parts of the gland
and enhanced cell differentiation. The described pattern
of appearance of the investigated pro-apoptotic and antiapoptotic
factors might be important for normal morphogenesis and function of the pituitary gland
Role of skeletal muscle in lung development
Skeletal (striated) muscle is one of the four basic tissue types, together with the epithelium, connective and nervous tissues. Lungs, on the other hand, develop from the foregut and among various cell types contain smooth, but not skeletal muscle. Therefore, during earlier stages of development, it is unlikely that skeletal muscle and lung depend on each other. However, during the later stages of development, respiratory muscle, primarily the diaphragm and the intercostal muscles, execute so called fetal breathing-like movements (FBMs), that are essential for lung growth and cell differentiation. In fact, the absence of FBMs results in pulmonary hypoplasia, the most common cause of death in the first week of human neonatal life. Most knowledge on this topic arises from in vivo experiments on larger animals and from various in vitro experiments. In the current era of mouse mutagenesis and functional genomics, it was our goal to develop a mouse model for pulmonary hypoplasia. We employed various genetically engineered mice lacking different groups of respiratory muscles or lacking all the skeletal muscle and established the criteria for pulmonary hypoplasia in mice, and therefore established a mouse model for this disease. We followed up this discovery with systematic subtractive microarray analysis approach and revealed novel functions in lung development and disease for several molecules. We believe that our approach combines elements of both in vivo and in vitro approaches and allows us to study the function of a series of molecules in the context of lung development and disease and, simultaneously, in the context of lung’s dependence on skeletal muscle-executed FBMs
Connexin Signaling in the Juxtaglomerular Apparatus (JGA) of Developing, Postnatal Healthy and Nephrotic Human Kidneys
Our study analyzed the expression pattern of different connexins (Cxs) and renin positive cells in the juxtaglomerular apparatus (JGA) of developing, postnatal healthy human kidneys and in nephrotic syndrome of the Finnish type (CNF), by using double immunofluorescence, electron microscopy and statistical measuring. The JGA contained several cell types connected by Cxs, and consisting of macula densa, extraglomerular mesangium (EM) and juxtaglomerular cells (JC), which release renin involved in renin-angiotensin- aldosteron system (RAS) of arterial blood pressure control. During JGA development, strong Cx40 expression gradually decreased, while expression of Cx37, Cx43 and Cx45 increased, postnatally showing more equalized expression patterning. In parallel, initially dispersed renin cells localized to JGA, and greatly increased expression in postnatal kidneys. In CNF kidneys, increased levels of Cx43, Cx37 and Cx45 co-localized with accumulations of renin cells in JGA. Additionally, they reappeared in extraglomerular mesangial cells, indicating association between return to embryonic Cxs patterning and pathologically changed kidney tissue. Based on the described Cxs and renin expression patterning, we suggest involvement of Cx40 primarily in the formation of JGA in developing kidneys, while Cx37, Cx43 and Cx45 might participate in JGA signal transfer important for postnatal maintenance of kidney function and blood pressure control
Connexin 37, 40, 43 and Pannexin 1 Expression in the Gastric Mucosa of Patients with Systemic Sclerosis
Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Although its pathogenesis is not fully understood, connexins (Cxs) and pannexins (Panx) could be involved in the process of fibrosis. We analyzed the protein expression of Cx37, Cx40, Cx43, and Panx1 in the gastric mucosa of patients with SSc and healthy volunteers, using immunofluorescence staining. Protein levels of Cx37 were slightly increased, while the levels of Cx40 were significantly decreased in the lamina propria of the gastric mucosa of SSc patients compared to the controls. The changes were proportional to SSc severity, with the most prominent changes found in patients with severe diffuse cutaneous SSc. No differences in Cx43 or Panx1 levels were found between the analyzed groups of samples. The lack of changes in Cx43 expression, which has been previously associated with fibrosis, could be due to the weak expression of Cx43 in the gastric mucosa in general. Further studies on full-thickness gastric biopsies containing muscle layers and animal SSc models are needed to fully elucidate the role of Cxs and Panxs in SSc-associated fibrosis
CRKL, AIFM3, AIF, BCL2, and UBASH3A during Human Kidney Development
We aimed to investigate the spatio-temporal expression of possible CAKUT candidate genes CRKL, AIFM3, and UBASH3A, as well as AIF and BCL2 during human kidney development. Human fetal kidney tissue was stained with antibodies and analyzed by fluorescence microscopy and RT-PCR. Quantification of positive cells was assessed by calculation of area percentage and counting cells in nephron structures. Results showed statistically significant differences in the temporal expression patterns of the examined markers, depending on the investigated developmental stage. Limited but strong expression of CRKL was seen in developing kidneys, with increasing expression up to the period where the majority of nephrons are formed. Results also lead us to conclude that AIFM3 and AIF are important for promoting cell survival, but only AIFM3 is considered a CAKUT candidate gene due to the lack of AIF in nephron developmental structures. Our findings imply great importance of AIFM3 in energy production in nephrogenesis and tubular maturation. UBASH3A raw scores showed greater immunoreactivity in developing structures than mature ones which would point to a meaningful role in nephrogenesis. The fact that mRNA and proteins of CRKL, UBASH3A, and AIFM3 were detected in all phases of kidney development implies their role as renal development control genes
Role of skeletal muscle in mandible development
As a continuation of the previous study on
palate development (Rot and Kablar, 2013), here we
explore the relationship between the secondary cartilage
mandibular condyles (parts of the temporomandibular
joint) and the contributions (mechanical and secretory)
from the adjacent skeletal musculature. Previous
analysis of Myf5-/-
:MyoD-/- mouse fetuses lacking
skeletal muscle demonstrated the importance of muscle
contraction and static loading in mouse skeletogenesis.
Among abnormal skeletal features, micrognathia
(mandibular hypoplasia) was detected: small, bent and
posteriorly displaced mandible. As an example of
Waddingtonian epigenetics, we suggest that muscle, in
addition to acting via mechanochemical signal
transduction pathways, networks and promoters, also
exerts secretory stimuli on skeleton. Our goal is to
identify candidate molecules at that muscle-mandible
interface. By employing Systematic Subtractive
Microarray Analysis approach, we compared gene
expression between mandibles of amyogenic and wild
type mouse fetuses and we identified up- and downregulated genes. This step was followed by a
bioinformatics approach and consultation of webaccessible mouse databases. We searched for individual
tissue-specific gene expression and distribution, and for
the functional effects of mutations in a particular gene.
The database search tools allowed us to generate a set of
candidate genes with involvement in mandibular
development: Cacna1s, Ckm, Des, Mir300, Myog and
Tnnc1. We also performed mouse-to-human translational
experiments and found analogies. In the light of our
findings we discuss various players in mandibular
morphogenesis and make an argument for the need to
consider mandibular development as a consequence of
reciprocal epigenetic interactions of both skeletal and
non-skeletal compartments