Emmanuelle Lesieur

Analysis of early nephron patterning reveals a role for distal RV proliferation in fusion to the ureteric tip via a cap mesenchyme-derived connecting segment.

While nephron formation is known to be initiated by a mesenchyme-to-epithelial transition of the cap mesenchyme to form a renal vesicle (RV), the subsequent patterning of the nephron and fusion with the ureteric component of the kidney to form a patent contiguous uriniferous tubule has not been fully characterized. Using dual section in situ hybridization (SISH)/immunohistochemistry (IHC) we have revealed distinct distal/proximal patterning of Notch, BMP and Wnt pathway components within the RV stage nephron. Quantitation of mitoses and Cyclin D1 expression indicated that cell proliferation was higher in the distal RV, reflecting the differential developmental programs of the proximal and distal populations. A small number of RV genes were also expressed in the early connecting segment of the nephron. Dual ISH/IHC combined with serial section immunofluorescence and 3D reconstruction revealed that fusion occurs between the late RV and adjacent ureteric tip via a process that involves loss of the intervening ureteric epithelial basement membrane and insertion of cells expressing RV markers into the ureteric tip. Using Six2-eGFPCre x R26R-lacZ mice, we demonstrate that these cells are derived from the cap mesenchyme and not the ureteric epithelium. Hence, both nephron patterning and patency are evident at the late renal vesicle stage.

Identification of anchor genes during kidney development defines ontological relationships, molecular subcompartments and regulatory pathways

The development of the mammalian kidney is well conserved from mouse to man. Despite considerable temporal and spatial data on gene expression in mammalian kidney development, primarily in rodent species, there is a paucity of genes whose expression is absolutely specific to a given anatomical compartment and/or developmental stage, defined here as ‘anchor’ genes. We previously generated an atlas of gene expression in the developing mouse kidney using microarray analysis of anatomical compartments collected via laser capture microdissection. Here, this data is further analysed to identify anchor genes via stringent bioinformatic filtering followed by high resolution section in situ hybridisation performed on 200 transcripts selected as specific to one of 11 anatomical compartments within the midgestation mouse kidney. A total of 37 anchor genes were identified across 6 compartments with the early proximal tubule being the compartment richest in anchor genes. Analysis of minimal and evolutionarily conserved promoter regions of this set of 25 anchor genes identified enrichment of transcription factor binding sites for Hnf4a and Hnf1b, RbpJ (Notch signalling), PPARγ:RxRA and COUP-TF family transcription factors. This was reinforced by GO analyses which also identified these anchor genes as targets in processes including epithelial proliferation and proximal tubular function. As well as defining anchor genes, this large scale validation of gene expression identified a further 92 compartment-enriched genes able to subcompartmentalise key processes during murine renal organogenesis spatially or ontologically. This included a cohort of 13 ureteric epithelial genes revealing previously unappreciated compartmentalisation of the collecting duct system and a series of early tubule genes suggesting that segmentation into proximal tubule, loop of Henle and distal tubule does not occur until the onset of glomerular vascularisation. Overall, this study serves to illuminate previously ill-defined stages of patterning and will enable further refinement of the lineage relationships within mammalian kidney development.

A Synthetic Resilin Is Largely Unstructured

Proresilin is the precursor protein for resilin, an extremely elastic, hydrated, cross-linked insoluble protein found in insects. We investigated the secondary-structure distribution in solution of a synthetic proresilin (AN16), based on 16 units of the consensus proresilin repeat from Anopheles gambiae. Raman spectroscopy was used to verify that the secondary-structure distributions in cross-linked AN16 resilin and in AN16 proresilin are similar, and hence that solution techniques (such as NMR and circular dichroism) may be used to gain information about the structure of the cross-linked solid. The synthetic proresilin AN16 is an intrinsically unstructured protein, displaying under native conditions many of the characteristics normally observed in denatured proteins. There are no apparent alpha-helical or beta-sheet features in the NMR spectra, and the majority of backbone protons and carbons exhibit chemical shifts characteristic of random-coil configurations. Relatively few peaks are observed in the nuclear Overhauser effect spectra, indicating that overall the protein is dynamic and unstructured. The radius of gyration of AN16 corresponds to the value expected for a denatured protein of similar chain length. This high degree of disorder is also consistent with observed circular dichroism and Raman spectra. The temperature dependences of the NH proton chemical shifts were also measured. Most values were indicative of protons exposed to water, although smaller dependences were observed for glycine and alanine within the Tyr-Gly-Ala-Pro sequence conserved in all resilins found to date, which is the site of dityrosine cross-link formation. This result implies that these residues are involved in hydrogen bonds, possibly to enable efficient self-association and subsequent cross-linking. The beta-spiral model for elastic proteins, where the protein is itself shaped like a spring, is not supported by the results for AN16. Both the random-network elastomer model and the sliding beta-turn model are consistent with the data. The results indicate a flat energy landscape for AN16, with very little energy required to switch between conformations. This ease of switching is likely to lead to the extremely low energy loss on deformation of resilin.

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.

Three-dimensional visualization of testis cord morphogenesis, a novel tubulogenic mechanism in development

Testis cords are specialized tubes essential for generation and export of sperm, yet the mechanisms directing their formation, and the regulation of their position, size, shape, and number remain unclear. Here, we use a novel fluorescence‐based three‐dimensional modeling approach to show that cords initially form as a network of irregular cell clusters that are subsequently remodeled to form regular parallel loops, joined by a flattened plexus at the mesonephric side. Variation in cord number and structure demonstrates that cord specification is not stereotypic, although cord alignment and diameter becomes relatively consistent, implicating compensatory growth mechanisms. Branched, fused, and internalized cords were commonly observed. We conclude that the tubule‐like structure of testis cords arise through a novel form of morphogenesis consisting of coalescence, partitioning, and remodeling. The methods we describe are applicable to investigating defects in testis cord development in mouse models, and more broadly, studying morphogenesis of other tissues. Developmental Dynamics 238:1033–1041, 2009.

Identification of Novel Markers of Mouse Fetal Ovary Development

In contrast to the developing testis, molecular pathways driving fetal ovarian development have been difficult to characterise. To date no single master regulator of ovarian development has been identified that would be considered the female equivalent of Sry. Using a genomic approach we identified a number of novel protein-coding as well as non-coding genes that were detectable at higher levels in the ovary compared to testis during early mouse gonad development. We were able to cluster these ovarian genes into different temporal expression categories. Of note, Lrrc34 and AK015184 were detected in XX but not XY germ cells before the onset of sex-specific germ cell differentiation marked by entry into meiosis in an ovary and mitotic arrest in a testis. We also defined distinct spatial expression domains of somatic cell genes in the developing ovary. Our data expands the set of markers of early mouse ovary differentiation and identifies a classification of early ovarian genes, thus providing additional avenues with which to dissect this process.

Expression of metanephric nephron‐patterning genes in differentiating mesonephric tubules

The metanephros is the functional organ in adult amniotes while the mesonephros degenerates. However, parallel tubulogenetic events are thought to exist between mesonephros and metanephros. Mesonephric tubules are retained in males and differentiate into efferent ducts of the male reproductive tract. By examining the murine mesonephric expression of markers of distinct stages and regions of metanephric nephrons during tubule formation and patterning, we provide further evidence to support this common morphogenetic mechanism. Renal vesicle, early proximal and distal tubule, loop of Henle, and renal corpuscle genes were expressed by mesonephric tubules. Vip, Slc6a20b, and Slc18a1 were male‐specific. In contrast, mining of the GUDMAP database identified candidate late mesonephros‐specific genes, 10 of which were restricted to the male. Among the male‐specific genes are candidates for regulating ion/fluid balance within the efferent ducts, thereby regulating sperm maturation and genes marking tubule‐associated neurons potentially critical for normal male reproductive tract function. Developmental Dynamics 240:1600–1612, 2011.

Arap3 is dysregulated in a mouse model of hypotrichosis-lymphedema-telangiectasia and regulates lymphatic vascular development

Mutations in SOX18, VEGFC and Vascular Endothelial Growth Factor 3 underlie the hereditary lymphatic disorders hypotrichosis-lymphedema-telangiectasia (HLT), Milroy-like lymphedema and Milroy disease, respectively. Genes responsible for hereditary lymphedema are key regulators of lymphatic vascular development in the embryo. To identify novel modulators of lymphangiogenesis, we used a mouse model of HLT (Ragged Opossum) and performed gene expression profiling of aberrant dermal lymphatic vessels. Expression studies and functional analysis in zebrafish and mice revealed one candidate, ArfGAP with RhoGAP domain, Ankyrin repeat and PH domain 3 (ARAP3), which is down-regulated in HLT mouse lymphatic vessels and necessary for lymphatic vascular development in mice and zebrafish. We position this known regulator of cell behaviour during migration as a mediator of the cellular response to Vegfc signalling in lymphatic endothelial cells in vitro and in vivo. Our data refine common mechanisms that are likely to contribute during both development and the pathogenesis of lymphatic vascular disorders.

Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice

Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics. DOI: http://dx.doi.org/10.7554/eLife.21221.001

SoxF factors induce Notch1 expression via direct transcriptional regulation during early arterial development

Arterial specification and differentiation are influenced by a number of regulatory pathways. While it is known that the Vegfa-Notch cascade plays a central role, the transcriptional hierarchy controlling arterial specification has not been fully delineated. To elucidate the direct transcriptional regulators of Notch receptor expression in arterial endothelial cells, we used histone signatures, DNaseI hypersensitivity and ChIP-seq data to identify enhancers for the human NOTCH1 and zebrafish notch1b genes. These enhancers were able to direct arterial endothelial cell-restricted expression in transgenic models. Genetic disruption of SoxF binding sites established a clear requirement for members of this group of transcription factors (SOX7, SOX17 and SOX18) to drive the activity of these enhancers in vivo. Endogenous deletion of the notch1b enhancer led to a significant loss of arterial connections to the dorsal aorta in Notch pathway-deficient zebrafish. Loss of SoxF function revealed that these factors are necessary for NOTCH1 and notch1b enhancer activity and for correct endogenous transcription of these genes. These findings position SoxF transcription factors directly upstream of Notch receptor expression during the acquisition of arterial identity in vertebrates. Summary: Identification of novel enhancers in vertebrate Notch1 genes places SoxF transcription factors directly upstream of Notch signalling in controlling arterial specification and differentiation.