Biology subjects

Fuhrmann, S.

Publications and source records attributed to Fuhrmann, S..

6 recordsLinked to original sources

bioRxiv2025

Direct comparison of constitutive Rax-Cre transgenic drivers that activate in the mouse embryonic eye field

PurposeMultiple mouse lines using constitutive or inducible Cre recombinase expression have taken advantage of the early optic field expression of the Rax/Rx gene and its subsequent, progressive restriction to retinal progenitors, retinal pigmented epithelium and the optic stalk. Among these, the constitutive transgenic lines, Rx3-Cre and Rax-Cre BAC Tg, are currently used by vision researchers. Here we directly compare their prenatal ocular and extra-ocular Cre activities. MethodsRx3-Cre or Rax-Cre BAC transgenic male mice were mated to the Cre-dependent lineage tracer Ai9/tdTomato. The resulting live and fixed tissue expression for each line was evaluated at multiple stages of development, from the optic vesicle stage onward. Both whole embryo and immunolabeled cryosectioned material were digitally imaged. ResultsRx3-Cre recapitulates endogenous RAX protein expression at the optic vesicle and optic cup stages, but there is also ectopic Cre activity in periocular mesenchyme (POM), within PITX2- and PECAM-1-expressing subpopulations. We also show that ectopic Cre activity can include germline expression. Besides a small ectopic domain in developing limbs, the Rax-Cre BAC mouse driver fully recapitulates endogenous RAX expression in the embryonic and postnatal eye. ConclusionsWe provide a systematic analysis of two Rax-Cre drivers during embryonic and postnatal eye development that are very useful to recapitulate severe congenital eye diseases. Data presented here strongly support the inclusion of lineage reporting and evaluation of littermates lacking a Cre transgene, whenever conditional targeting strategies are used in studies of optic vesicle-derived tissues.

developmental biology↗

bioRxiv2025

Dicer loss in Muller glia leads to a defined sequence of pathological events beginning with cone dysfunction

PurposeThe loss of Dicer in Muller glia (MG) results in severe photoreceptor degeneration as it occurs in retinitis pigmentosa or AMD. However, the sequence of events leading to this severe degenerative state is unknown. The aim of this study was to conduct a chronological functional and structural characterization of the pathological events in MG-specific Dicer-cKO mice in vivo and histologically. MethodsTo delete Dicer and mature microRNAs (miRNAs) in MG, two conditional Dicer1 knock-out mouse strains namely RlbpCre:Dicer-cKOMG and GlastCre:Dicer-cKOMG, were created. Optical coherence tomography (OCT), electroretinograms (ERGs) as well as histological analyses were conducted to investigate structural and functional changes up to six months after Dicer deletion. ResultsDicer/miRNA loss in MG leads to 1) impairments of the external limiting membrane (ELM) - retinal pigment epithelium (RPE), 2) cone photoreceptor dysfunction and 3) retinal remodeling and functional loss of the inner retina, 1, 3 and 6 months after Dicer loss, respectively, in both strains. Furthermore, in the Rlbp:Dicer-cKOMG strain, rod photoreceptor impairment was found 4 months after Dicer depletion (4) accompanied by alteration of RPE integrity (5). ConclusionsMG Dicer loss in the adult mouse retina impacts cone function prior to any measurable changes in rod function, suggesting a pivotal role for MG Dicer and miRNAs in supporting cone health. A partially impaired RPE however seems to accelerate rod degeneration and overall degenerative events.

neuroscience↗

bioRxiv2025

Diet gel-based oral drug delivery system for controlled dosing of small molecules for microglia depletion and inducible Cre recombination in mice

Small molecules like PLX5622 for microglia depletion and Tamoxifen for inducible Cre recombination are commonly used in mouse research. Traditional application methods, such as chow or oral gavage and injections, have limitations, including uncontrolled dosage and risk of injury. To address this issue, we have developed an alternative oral drug delivery system using a gel-based rodent maintenance diet that allows for controlled consumption and adjustment of dosage and is suitable for water-insoluble small molecules. We tested DietGel(R) 93M (93M) infused with PLX5622 (0.8 mg/g and 2.0 mg/g) in the Cx3cr1gfp/+ retinal microglia reporter mouse and Tamoxifen-infused 93M (0.3125 mg/g) in the Rlbp1-CreERT2;Rosaai14 mouse with an inducible tdTomato reporter in retinal Muller glia. Mice were single-caged and received daily batches of PLX5622-infused 93M over 14 days or Tamoxifen-infused 93M for one or three days followed by a 14-day observation period. Longitudinal scanning laser ophthalmoscopy in vivo and fixed tissue imaging were used to track GFP and tdTomato expression. Following evaluation of a suitable 93M consumption rate (g/d) to sustain body weight, the PLX5622-93M diet at both concentrations showed a 94% microglia depletion rate at 3 days and >99% after one and two weeks. The Tamoxifen-93M diet confirmed suitability for inducible Cre recombination, with significant treatment-time dependent efficacy and a positive correlation between total Tamoxifen dose and tdTomato expression. This study demonstrates that a diet gel-based drug delivery system offers a controllable and less invasive alternative to current drug application methods for PLX5622 and Tamoxifen.

cell biology↗

bioRxiv2024

Loss of Cdc42 causes abnormal optic cup morphogenesis and microphthalmia in mouse

Congenital ocular malformations originate from defective morphogenesis during early eye development and cause 25% of childhood blindness. Formation of the eye is a multi-step, dynamic process; it involves evagination of the optic vesicle, followed by distal and ventral invagination, leading to the formation of a two-layered optic cup with a transient optic fissure. These tissue folding events require extensive changes in cell shape and tissue growth mediated by cytoskeleton mechanics and intercellular adhesion. We hypothesized that the Rho GTPase Cdc42 may be an essential, convergent effector downstream of key regulatory factors required for ocular morphogenesis. CDC42 controls actin remodeling, apicobasal polarity, and junction assembly. Here we identify a novel essential function for Cdc42 during eye morphogenesis in mouse; in Cdc42 mutant eyes expansion of the ventral optic cup is arrested, resulting in microphthalmia and a wide coloboma. Our analyses show that Cdc42 is required for expression of the polarity effector proteins PRKCZ and PARD6, intercellular junction protein tight junction protein 1, {beta}-catenin, actin cytoskeleton F-actin, and contractile protein phospho myosin light chain 2. Expression of RPE fate determinants OTX2 and MITF, and formation of the RPE layer are severely affected in the temporal domain of the proximal optic cup. EdU incorporation is significantly downregulated. In addition, mitotic retinal progenitor cells mis-localized deeper, basal regions, likely contributing to decreased proliferation. We propose that morphogenesis of the ventral optic cup requires Cdc42 function for coordinated optic cup expansion and establishment of subretinal space, tissue tension, and differentiation of the ventral RPE layer.

developmental biology↗

bioRxiv2022

Porcn is essential for growth and invagination of the mammalian optic cup

Microphthalmia, anophthalmia, and coloboma (MAC) are congenital ocular malformations causing 25% of childhood blindness. The X-linked disorder Focal Dermal Hypoplasia is frequently associated with MAC and results from mutations in Porcn, a membrane bound O-acyl transferase required for palmitoylation of Wnts to activate multiple Wnt-dependent pathways. Wnt/{beta}-catenin signaling is suppressed in the anterior neural plate for initiation of eye formation and is subsequently required during differentiation of the retinal pigment epithelium (RPE). Non-canonical Wnts are critical for early eye formation in frog and zebrafish, however, it is unclear whether this also applies to mammals. We performed ubiquitous conditional inactivation of Porcn in mouse around the eye field stage. In PorcnCKO, optic vesicles (OV) arrest in growth and fail to form an optic cup. Ventral proliferation is significantly decreased in the mutant OV, with a concomitant increase in apoptotic cell death. While pan-ocular transcription factors such as PAX6, SIX3, LHX2, PAX2 are present, indicative of maintenance of OV identity, regional expression of VSX2, MITF, OTX2 and NR2F2 is downregulated. Failure of RPE differentiation in PorcnCKO is consistent with downregulation of the Wnt/{beta}-catenin effector LEF1, starting around 2.5 days after inactivation. This suggests that Porcn inactivation affects signaling later than a potential requirement for Wnts to promote eye field formation. Altogether, our data shows a novel requirement for Porcn in regulating growth and morphogenesis of the OV, likely by controlling proliferation and survival. In FDH patients with ocular manifestations, growth deficiency during early ocular morphogenesis may be the underlying cause for microphthalmia.

developmental biology↗

bioRxiv2020

Nf2 fine-tunes proliferation and tissue alignment during closure of the optic fissure in the embryonic mouse eye

Uveal coloboma represents one of the most common congenital ocular malformations accounting for up to 10% of childhood blindness (1~ in 5,000 live birth). Coloboma originates from defective fusion of the optic fissure (OF), a transient gap that forms during eye morphogenesis by asymmetric, ventral invagination. Genetic heterogeneity combined with the activity of developmentally regulated genes suggest multiple mechanisms regulating OF closure. The tumor suppressor and FERM domain protein neurofibromin 2 (NF2) controls diverse processes in cancer, development and regeneration, via Hippo pathway and cytoskeleton regulation. In humans, NF2 mutations can cause ocular abnormalities, including coloboma, however, its actual role in OF closure is unknown. Using conditional inactivation in the embryonic mouse eye, our data indicates that loss of Nf2 function results in a novel underlying cause for coloboma. In particular, mutant eyes show substantially increased RPE proliferation in the fissure region with concomitant acquisition of RPE cell fate. Cells lining the OF margin can maintain RPE fate ectopically and fail to transition from neuroepithelial to cuboidal shape. In the dorsal RPE of the optic cup, Nf2 inactivation leads to a robust increase in cell number, with local disorganization of the cytoskeleton components F-actin and pMLC2. We propose that RPE hyperproliferation is the primary cause for the observed defects causing insufficient alignment of the OF margins in Nf2 mutants and failure to fuse properly, resulting in persistent coloboma. Our findings indicate that limiting proliferation particularly in the RPE layer is a critical mechanism during optic fissure closure.

developmental biology↗