We are particularly interested in how the local tissue environment influences cell state, and reciprocally how cell state affects cellular responses to the environment. Modifying the cellular response to a signal allows signaling pathways to enact a different molecular response, and therefore can be reused during development or regeneration to impose patterning on the cells or tissues. This is the case in the mesenchymal/epithelial interactions used to form many different structures during development and in response to damage or disease. In this way, a signal may be used to initiate specification of a group of cells, but the results of the specification may be different in different settings. Most importantly, this view allows for distinct outcomes in the context of repair after injury as compared to development even when the same signaling molecule is utilized. Our overall goal is to further understand this process so that we may identify useful kidney therapies.

We are primarily focused on the development of the nephron, both the specification of cells to form the nephron, and the complex patterning that occurs to define the distinct physiological domains of the nephron tubules.

Kidney Development

We are investigating how patterning information is used in the formation of the physiologically segmented nephron using microarray analysis and genetic lineage tracing.  Prior work has established roles for the Notch signaling pathway in determining regions of the proximal nephron. We are building on this using transgenic mouse strains to lineage trace distal and middle nephron segments.

The Wnt signaling pathway is responsible for inducing the formation of new nephrons from the cap mesenchyme nephron progenitors. The subsequent morphogenesis of the renal vesicle to S-shaped body to mature nephron is regulated by reapplication of signaling pathways including Wnt, Notch and Bmp. We have designed a transgenic mouse strain to map the cell fate of distinct regions of the S-shaped body. This will help us to understand how the morphogenesis of new nephrons is controlled during development, and guide us in coaxing nephron progenitors, potentially created from patient derived iPS cells, to form new nephrons therapeutically.

Kidney Regeneration/Repair - a “Healing Niche”

Prior work has shown tubule repair after ischemia/reperfusion injury (IRI) results from dedifferentiation then redifferentiation of sub-lethally injured proximal tubule epithelial cells. Another part of this picture is that other cell types become involved in the repair, for example invading macrophages and adjacent undamaged regions of the nephron. We are trying to understand how all of these elements come the other in a healing niche to promote the repair of damaged nephron tubules. We have created transgenic mouse strains of facilitate our studies.

Projects

Lineage tracing distal nephron segments

Using a transgenic mouse that expresses Cre under the Sim1 promoter (Sim1cre), we have mapped the broad contribution of the Sim1 expression domain. Sim1 is expressed in the distal S-shaped body, but is not present in the preceding renal vesicle. By crossing to R26R-LacZ reporter line, was have been able to determine the fate of the distal S-shaped body cells marked by Sim1 expression.

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Sim1cre marks distal segment of the S-shaped body, and the collecting duct. Sim1cre/+;R26RlacZ/+ E15.5 kidney sections - X-gal staining, Nuclear Fast Red counter stain.

Labeling of the adult proximal tubule

We have generated a mouse strain that expresses Red Fluorescent Protein (RFP) in the adult proximal tubule. The construct uses the Slc34a1 gene promoter to drive expression of a nuclear localized TagRFPT. We want to learn more about the cell dynamics during tubule regeneration after acute kidney injury.

This work funded by the Harvard Stem Cell Institute Kidney Program.

Slc34a1-nTagRFPT-web.png

Slc34a1-nTagRFPT reporter expression in the adult proximal tubule. Nuclear TagRFPT (red) colocalizes with the proximal tubule marker LTL (green).

Expression profiling of the mouse nephron progenitors

Using the publicly available data from the GUDMAP consortium, I have been comparing adjacent cell populations in the cortex of the developing mouse kidney. Bioinformatic analysis was validated by whole mount in situ hybridization and we are currently mapping out the detailed expression patterns of each gene by section in situ hybridization.

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Above: Differential gene expression comparing Six2+ nephron progenitors with Foxd1+ cortical interstitium cells.

Six2 expressing cap condensate cells contribute exclusively to the developing nephron

In this work, I have made a transgenic mouse line that expresses a GFP::CRE-recombinase fusion gene in the Six2 gene expression domain, the cap condensate. This work formally demonstrated the fate of the cap condensate cell population was the epithelial nephron, and not other structures in the kidney. My labmate in Andy McMahon’s lab, Akio Kobayashi, made a Tamoxifen regulated GFP-Cre version in the Six2 locus and showed these cells self renew during development.  

Ectopic Notch signaling results in patterning defects in the developing nephron

Loss-of-function studies have shown Notch signaling to be required for the formation of proximal elements of the nephron, specifically the podocytes and the proximal tubule. In this study with Rafi Kopan, we further examined Notch signaling by creating Notch gain-of-function mutants by activating Notch in nephron progenitors. Our data shows Notch signaling is sufficient to pattern nephron progenitors to form the most proximal nephron tubule structures at the expense of distal structures in forming nephrons.