Lorine Wilkinson

CRIM1 Regulates the Rate of Processing and Delivery of Bone Morphogenetic Proteins to the Cell Surface

The Crim1 gene is predicted to encode a transmembrane protein containing six von Willebrand-like cysteine-rich repeats (CRRs) similar to those in the BMP-binding antagonist Chordin (Chrd). In this study, we verify that CRIM1 is a glycosylated, Type I transmembrane protein and demonstrate that the extracellular CRR-containing domain can also be secreted, presumably via processing at the membrane. We have previously demonstrated Crim1 expression at sites consistent with an interaction with bone morphogenetic proteins (BMPs). Here we show that CRIM1 can interact with both BMP4 and BMP7 via the CRR-containing portion of the protein and in so doing acts as an antagonist in three ways. CRIM1 binding of BMP4 and -7 occurs when these proteins are co-expressed within the Golgi compartment of the cell and leads to (i) a reduction in the production and processing of preprotein to mature BMP, (ii) tethering of pre-BMP to the cell surface, and (iii) an effective reduction in the secretion of mature BMP. Functional antagonism was verified by examining the effect of co-expression of CRIM1 and BMP4 on metanephric explant culture. The presence of CRIM1 reduced the effective BMP4 concentration of the media, thereby acting as a BMP4 antagonist. Hence, CRIM1 modulates BMP activity by affecting its processing and delivery to the cell surface.

Kidney development: two tales of tubulogenesis.

The mammalian kidney may well be one of the most complex organs of postnatal life. Each adult human kidney contains on average more than one million functional filtration units, the nephrons, residing within a specialized cellular interstitium. Each kidney also contains over 25 distinct cell types, each of which must be specifically aligned with respect to each other to ensure both normal development and ultimately, normal renal function. Despite this complexity, the development of the kidney can be simplistically described as the coordinate formation of two distinct sets of tubules. These tubules develop cooperatively with each other in time and space, yet represent two distinct but classical types of tubulogenesis. The first of these tubules, the ureteric bud, forms as an outgrowth of another epithelial tube, the nephric duct, and undergoes extensive branching morphogenesis to create the collecting system of the kidney. The second tubules are the nephrons themselves which arise via a mesenchyme-to-epithelial transition induced by the first set of tubules. These tubules never branch, but must elongate to become intricately patterned and functionally segmented tubules. The molecular drivers for these two tales of tubulogenesis include many gene families regulating tubulogenesis and branching morphogenesis in other organs; however, the individual players and codependent interrelationships between a branched and non-branched tubular network make organogenesis in the kidney unique. Here we review both what is known and remains to be understood in kidney tubulogenesis.

Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney

The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.

Crim1KST264/KST264 Mice Implicate Crim1 in the Regulation of Vascular Endothelial Growth Factor-A Activity during Glomerular Vascular Development

Crim1, a transmembrane cysteine-rich repeat-containing protein that is related to chordin, plays a role in the tethering of growth factors at the cell surface. Crim1 is expressed in the developing kidney; in parietal cells, podocytes, and mesangial cells of the glomerulus; and in pericytes that surround the arterial vasculature. A gene-trap mouse line with an insertion in the Crim1 gene (Crim1(KST264/KST264)) displayed perinatal lethality with defects in multiple organ systems. This study further analyzed the defects that are present within the kidneys of these mice. Crim1(KST264/KST264) mice displayed abnormal glomerular development, illustrated by enlarged capillary loops, podocyte effacement, and mesangiolysis. When outbred, homozygotes that reached birth displayed podocyte and glomerular endothelial cell defects and marked albuminuria. The podocytic co-expression of Crim1 with vascular endothelial growth factor-A (VEGF-A) suggested a role for Crim1 in the regulation of VEGF-A action. Crim1 and VEGF-A were shown to interact directly, providing evidence that cysteine-rich repeat-containing proteins can bind to non-TGF-beta superfamily ligands. Crim1(KST264/KST264) mice display a mislocalization of VEGF-A within the developing glomerulus, as assessed by immunogold electron microscopy and increased activation of VEGF receptor 2 (Flk1) in the glomerular endothelial cells, suggesting that Crim1 regulates the delivery of VEGF-A by the podocytes to the endothelial cells. This is the first in vivo demonstration of regulation of VEGF-A delivery and supports the hypothesis that Crim1 functions to regulate the release of growth factors from the cell of synthesis.

Mid‐ to late term hypoxia in the mouse alters placental morphology, glucocorticoid regulatory pathways and nutrient transporters in a sex‐specific manner

Maternal hypoxia is a common perturbation that may impair fetal development and programme sex specific disease outcomes in offspring. There is growing interest in the role of the placenta in mediating the effects of maternal hypoxia on fetal development, particularly in late gestation during maximal fetal growth. Multiple mechanisms have been proposed to play a role in hypoxia induced impairment of placental development. Here we investigated the role of glucocorticoids and glucose regulation. This study shows that fetal sex determines placental adaptations to maternal hypoxia: while maternal hypoxia increased maternal glucose and corticosterone levels in both sexes, placental adaptations to impaired maternal physiology were more evident in female fetuses, in which factors responsible for the regulation of glucocorticoids and nutrient transport were most severely affected by maternal hypoxia.

Crim1KST264/KST264 mice display a disruption of the Crim1 gene resulting in perinatal lethality with defects in multiple organ systems.

Crim1 is a transmembrane protein, containing six vWF‐C type cysteine‐rich repeats, that tethers growth factors to the cell surface. A mouse line, KST264, generated in a LacZ insertion mutagenesis gene‐trap screen, was examined to elucidate Crim1 function in development. We showed that Crim1KST264/KST264 mice were not null for Crim1 due to the production of a shortened protein isoform. These mice are likely to represent an effective hypomorph or a dominant‐negative for Crim1. Transgene expression recapitulated known Crim1 expression in lens, brain, and limb, but also revealed expression in the smooth muscle cells of the developing heart and renal vasculature, developing cartilage, mature ovary and detrusor of the bladder. Transgene expression was also observed in glomerular epithelial cells, podocytes, mesangial cells, and urothelium in the kidney. Crim1KST264/KST264 mice displayed perinatal lethality, syndactyly, eye, and kidney abnormalities. The severe and complex phenotype observed in Crim1KST264/KST264 mice highlights the importance of Crim1 in numerous aspects of organogenesis. Developmental Dynamics 236:502–511, 2007.

Molecular anatomy of the kidney: what have we learned from gene expression and functional genomics?

The discipline of paediatric nephrology encompasses the congenital nephritic syndromes, renal dysplasias, neonatal renal tumours, early onset cystic disease, tubulopathies and vesicoureteric reflux, all of which arise due to defects in normal kidney development. Indeed, congenital anomalies of the kidney and urinary tract (CAKUT) represent 20–30% of prenatal anomalies, occurring in 1 in 500 births. Developmental biologists have studied the anatomical and morphogenetic processes involved in kidney development for the last five decades. However, with the advent of transgenic mice, the sequencing of the genome, improvements in mutation detection and the advent of functional genomics, our understanding of the molecular basis of kidney development has grown significantly. Here we discuss how the advent of new genetic and genomics approaches has added to our understanding of kidney development and paediatric renal disease, as well as identifying areas in which we are still lacking knowledge.

Renal developmental defects resulting from in utero hypoxia are associated with suppression of ureteric β-catenin signaling

Gestational stressors, including glucocorticoids and protein restriction, can affect kidney development and hence final nephron number. Since hypoxia is a common insult during pregnancy, we studied the influence of oxygen tension on kidney development in models designed to represent a pathological hypoxic insult. In vivo mouse models of moderate, transient, midgestational (12% O₂, 48 h, 12.5 dpc) or severe, acute, early-gestational (5.5-7.5% O₂, 8 h, 9.5-10.5 dpc) hypoxia were developed. The embryo itself is known to mature under hypoxic conditions with embryonic tissue levels of oxygen estimated to be 5%-8% (physiological hypoxia) when the mother is exposed to ambient normoxia. Both in vivo models generated phenotypes seen in patients with congenital anomalies of the kidney and urinary tract (CAKUT). Severe, acute, early hypoxia resulted in duplex kidney, while moderate, transient, midgestational hypoxia permanently reduced ureteric branching and nephron formation. Both models displayed hypoxia-induced reductions in β-catenin signaling within the ureteric tree and suppression of the downstream target gene, Ccnd1. Thus, we show a link between gestational hypoxia and CAKUT, the phenotype of which varies with timing, duration, and severity of the hypoxic insult.

Distinct sites of renal fibrosis in Crim1 mutant mice arise from multiple cellular origins

Crim1 is a transmembrane protein that regulates the bioavailability of growth factors such as VEGFA. Crim1KST264/KST264 hypomorphic mice develop renal disease characterized by glomerular cysts and loss of endothelial integrity, progressing to peritubular and pericystic fibrosis. Peritubular capillary endothelial cells display morphological changes as well as detachment from the basement membrane. In this study, gene expression profiling of CD31+ endothelial cells isolated from Crim1KST264/KST264 kidneys showed up‐regulation of transcripts associated with fibrosis (Col3a1, Loxl1), endothelial dysfunction (Abp1, Dcn, Lcn2), biomarkers of renal damage (Lcn2, Havcr1/Kim1) as well as evidence for a TGFβ1/TNF‐associated inflammatory process. To determine whether the aberrant endothelium may in part contribute to the fibrogenic process, Tie2Cre‐DsRed lineage tracing was undertaken in Crim1KST264/KST264 mice. Approximately 31% of de novo αSMA+ myofibroblasts detected within the tubulointerstitium were Tie2+DsRed+. However, 5.3% were F4/80+DsRed+, indicating a small population of myofibroblasts of monocytic rather than endothelial origin. In contrast, only 12% of myofibroblasts located around glomerular cysts were Tie2+DsRed+, with 7.7% being monocyte‐derived (F4/80+DsRed+). Collectively, this model supports the involvement of endothelial cells/monocytes in fibrosis within the tubulointerstitium, but also the heterogeneity of the fibrotic process even within distinct regions of the same kidney. Copyright

Production of a Mouse Line with a Conditional Crim1 Mutant Allele

Crim1 is a developmentally expressed, transmembrane protein essential for normal embryonic development. We generated mice engineered to contain a Crim1 conditional null allele by flanking exons three and four of Crim1 with unidirectional LoxP sites. After crossing Crim1+/FLOX mice with a CMV‐Cre line, a Crim1+/Δflox colony was established after germline transmission of the deleted allele. We then analyzed genomic DNA, mRNA transcripts, and protein expression from Crim1Δflox/Δflox null mice to confirm the nature of the genomic lesion. Crim1Δflox/Δflox mice displayed phenotypes similar to those previously described for a Crim1 gene‐trap mutant, Crim1KST264/KST264, including perinatal lethality, digit syndactyly, eye, and kidney abnormalities, with varying penetrance and severity. The production of a conditional mutant allele represents a valuable resource for the study of the tissue‐specific roles for Crim1, and for understanding the pleimorphic phenotypes associated with Crim1 mutation. genesis 50:711–716, 2012.