Biology subjects

Gerlach, G. F.

Publications and source records attributed to Gerlach, G. F..

4 recordsLinked to original sources

bioRxiv2024

The tricellular junctional protein Ildr2 helps maintain glomerular podocyte architecture

Podocytes are highly specialized epithelial cells of the glomerular filtration barrier essential for maintaining proper kidney function. Their intricate cellular projections, called foot processes, wrap around the capillary endothelium, interdigitate, and connect to one another via slit diaphragm intercellular junctions to form a sieve-like barrier. Compromise to this unique podocyte architecture can lead to altered glomerular function and eventual kidney disease. We previously identified Immunoglobulin-like domain containing receptor 2 (Ildr2) as a component of the podocyte foot process through proteomic analyses. Ildr2 is a tricellular tight junction constituent and localizes to distinct puncta in podocytes that likely represent these specialized junctions. However, the significance of tricellular tight junctions and Ildr2 to podocyte integrity is unknown. To this end, we generated a conditional knockout mouse with podocyte-specific deletion of Ildr2 (Podocin-Cretg/+;Ildr2fl/flor Ildr2PodKO). Kidneys from Ildr2PodKO adult animals show disruptions to podocyte foot process architecture including effacement and the unique formation of electron dense strands connecting subsets of processes. Additionally, the glomerular basement membrane was significantly thicker in conditional knockouts and histological analyses revealed glomerular collagen and glycoprotein accumulation. Surprisingly, Ildr2PodKO animals displayed no significant albuminuria. When Ildr2PodKO mice were challenged with a stress to kidney function, immunohistochemistry revealed podocyte loss from a subset of glomeruli with concomitant detection of podocytes in urine, although function was still preserved. Collectively, our data provide novel insights into the function of Ildr2 and suggest tricellular junctions help preserve podocyte architecture but are likely not necessary to maintain proper filtration in no or low stress physiological states.

physiology↗

bioRxiv2022

Mapping of the podocin proximity-dependent proteome reveals novels components of the kidney podocyte foot process

The unique architecture of glomerular podocytes is integral to kidney filtration. Interdigitating foot processes extend from the podocyte cell body, wrap around fenestrated capillaries, and form specialized junctional complexes termed slit diaphragms to create a molecular sieve. However, the full complement of proteins which maintain foot process integrity, and how this localized proteome changes with disease, remains to be elucidated. Proximity-dependent biotin identification (BioID) enables the identification of spatially localized proteomes. To this end, we developed a novel in vivo BioID knock-in mouse model. We utilized the slit diaphragm protein podocin (Nphs2) to create a podocin-BioID fusion. Podocin-BioID localizes to the slit diaphragm and biotin injection leads to podocyte-specific protein biotinylation. We isolated the biotinylated proteins and performed mass spectrometry to identify proximal interactors. Gene ontology analysis of 54 proteins specifically enriched in our podocin-BioID sample revealed cell junctions, actin binding, and cytoskeleton organization as top terms. Known foot process components were identified and we further uncovered two novel proteins: the tricellular junctional protein Ildr2 and the CDC42 and N-WASP interactor Fnbp1l. We confirmed Ildr2 and Fnbp1l are expressed by podocytes and partially colocalize with podocin. Finally, we investigated how this proteome changes with age and uncovered a significant increase in Ildr2. This was confirmed by immunofluorescence on human kidney samples and suggests altered junctional composition may preserve podocyte integrity. Together, these assays have led to new insights into podocyte biology and supports the efficacy of utilizing BioID in vivo to interrogate spatially localized proteomes in health, aging, and disease.

developmental biology↗

bioRxiv2020

osr1 maintains renal progenitors and regulates podocyte development by promoting wnt2ba through antagonism of hand2

Knowledge about the genetic pathways that control renal cell lineage development is essential to better understand the basis of congenital malformations of the kidney and design regenerative medicine therapies. The embryonic zebrafish kidney, or pronephros, contains two nephrons that are conserved with humans. Recently, the transcription factors Osr1 and Hand2 were found to exert antagonistic influences to balance kidney specification (Perens et al., 2016). Here, we performed a forward genetic screen in zebrafish to identify nephrogenesis regulators, where whole genome sequencing of the novel oceanside (ocn) mutant revealed a nonsense mutation in osr1. ocn mutants evince severe pronephros defects including abrogation of podocytes and proximal tubule cells. Our studies reveal that osr1 is not needed to specify renal progenitors, but rather required to maintain their survival. Additionally, osr1 is requisite for expression of the canonical Wnt ligand wnt2ba, where wnt2ba is expressed in the intermediate mesoderm (IM) and later restricts to podocytes. Deficiency of wnt2ba reduced podocyte progenitors, where overexpression of wnt2ba was sufficient to rescue the podocyte lineage as well as osr1 loss of function. Finally, we demonstrate that reciprocal antagonism between osr1 and hand2 mediates podocyte development specifically by controlling wnt2ba expression in the IM. Together, our data show that Osr1 is essential for a sequence of temporal functions that mediate the survival and lineage decisions of IM progenitors, and subsequently the maintenance of podocytes and proximal tubule epithelium in the embryonic nephron.

developmental biology↗

bioRxiv2018

Tfap2a is a novel gatekeeper of differentiation in renal progenitors during kidney development

Renal functional units known as nephrons undergo patterning events during development that create a segmental array of cellular populations with discrete physiological tasks. Knowledge about the terminal differentiation programs of each nephron segment has central importance for understanding kidney disease and to advance regenerative medicine, as mammalian nephrons grown in organoid cultures from pluripotent cells fail to terminally differentiate. Here, from a novel forward genetic screen using zebrafish we report the discovery that transcription factor AP-2 alpha (tfap2a) coordinates a gene regulatory network that controls the progression of nephron distal segment progenitors into the differentiated state. Overexpression of tfap2a rescued differentiation in mutants and caused ectopic expression of distal segment markers in wild-type nephrons, indicating tfap2a is sufficient to instigate the distal segment differentiation program. tfap2a/2b deficiency exacerbated distal nephron segment differentiation defects, revealing functional redundancy where tfap2a has a dominant role upstream of its family member. With further genetic studies, we assembled a blueprint of the tfap2a gene regulatory network during nephrogenesis. We demonstrate that tfap2a acts downstream of Iroquois homeobox 3b, a conserved distal lineage transcription factor. tfap2a controls a circuit consisting of irx1a, tfap2b, and genes encoding solute transporters that dictate the specialized metabolic functions of the distal nephron segments, and we show for the first time that this regulatory node is distinct from the pathway circuits controlling aspects such as apical-basal polarity and ciliogenesis during the differentiation process. Thus, our studies reveal new insights into the genetic control of differentiation, where tfap2a regulates the suite of segment transporter traits. These findings have relevance for understanding renal birth defects, as well as efforts to recapitulate nephrogenesis in vivo to make functional units that can facilitate organoid applications such as drug discovery and regenerative therapies.\n\nSummary StatementHere, we report for the first time that transcription factor AP-2 alpha (tfap2a) controls the progression from nephron progenitor into the fully differentiated state. This fundamentally deepens our knowledge about the genetic control of kidney development.

developmental biology↗