Impaired Recall of Positional Memory following Chemogenetic Disruption of Place Field Stability.

The neural network of the temporal lobe is thought to provide a cognitive map of our surroundings. Functional analysis of this network has been hampered by coarse tools that often result in collateral damage to other circuits. We developed a chemogenetic system to temporally control electrical input into the hippocampus. When entorhinal input to the perforant path was acutely silenced, hippocampal firing patterns became destabilized and underwent extensive remapping. We also found that spatial memory acquired prior to neural silencing was impaired by loss of input through the perforant path. Together, our experiments show that manipulation of entorhinal activity destabilizes spatial coding and disrupts spatial memory. Moreover, we introduce a chemogenetic model for non-invasive neuronal silencing that offers multiple advantages over existing strategies in this setting.

Specificity and efficiency of reporter expression in adult neural progenitors vary substantially among nestin-CreER(T2) lines.

Transgenic lines expressing a controllable form of Cre recombinase have become valuable tools for manipulating gene expression in adult neural progenitors and their progeny. Neural progenitors express several proteins that distinguish them from mature neurons, and the promoters for these genes have been co-opted to produce selective transgene expression within this population. To date, nine CreER(T2) transgenic lines have been designed using the nestin promoter; however, only a subset are capable of eliciting expression within both neurogenic zones of the adult brain. Here we compare three such nestin-CreER(T2) lines to evaluate specificity of expression and efficiency of recombination. Each line was examined by using three different Cre reporter strains that varied in sensitivity. We found that all three nestin-CreER(T2) strains induced reporter expression within the main neurogenic areas, albeit to varying degrees depending on the reporter. Unexpectedly, we found that two of the three lines induced substantial reporter expression outside of neurogenic areas. These lines produced strong labeling in cerebellar granule neurons, with additional expression in the cortex, hippocampus, striatum, and thalamus. Reporter expression in the third nestin-CreER(T2) line was considerably more specific, but was also less efficient, labeling a smaller percentage of the target population than the other two drivers. Our findings suggest that each nestin-CreER(T2) line may best serve different experimental needs, depending on whether specificity or efficiency is of greatest concern. Our study further demonstrates that each new pair of driver and responder lines should be evaluated independently, as both components can significantly influence the resulting expression pattern.

EGFR signaling promotes TGFß-dependent renal fibrosis.

The mechanisms by which angiotensin II (Ang II) promotes renal fibrosis remain incompletely understood. Ang II both stimulates TGFß signaling and activates the EGF receptor (EGFR), but the relative contribution of these pathways to renal fibrogenesis is unknown. Using a murine model with EGFR-deficient proximal tubules, we demonstrate that upstream activation of EGFR-dependent ERK signaling is critical for mediating sustained TGFß expression in renal fibrosis. Persistent activation of the Ang II receptor stimulated ROS-dependent phosphorylation of Src, leading to sustained EGFR-dependent signaling for TGFß expression. Either genetic or pharmacologic inhibition of EGFR significantly decreased TGFß-mediated fibrogenesis. We conclude that TGFß-mediated tissue fibrosis relies on a persistent feed-forward mechanism of EGFR/ERK activation through an unexpected signaling pathway, highlighting EGFR as a potential therapeutic target for modulating tissue fibrogenesis.

Epidermal growth factor receptor plays an anabolic role in bone metabolism in vivo.

While the epidermal growth factor receptor (EGFR)-mediated signaling pathway has been shown to have vital roles in many developmental and pathologic processes, its functions in the development and homeostasis of the skeletal system has been poorly defined. To address its in vivo role, we constructed transgenic and pharmacologic mouse models and used peripheral quantitative computed tomography (pQCT), micro-computed tomography (µCT) and histomorphometry to analyze their trabecular and cortical bone phenotypes. We initially deleted the EGFR in preosteoblasts/osteoblasts using a Cre/loxP system (Col-Cre Egfr(f/f)), but no bone phenotype was observed because of incomplete deletion of the Egfr genomic locus. To further reduce the remaining osteoblastic EGFR activity, we introduced an EGFR dominant-negative allele, Wa5, and generated Col-Cre Egfr(Wa5/f) mice. At 3 and 7 months of age, both male and female mice exhibited a remarkable decrease in tibial trabecular bone mass with abnormalities in trabecular number and thickness. Histologic analyses revealed decreases in osteoblast number and mineralization activity and an increase in osteoclast number. Significant increases in trabecular pattern factor and structural model index indicate that trabecular microarchitecture was altered. The femurs of these mice were shorter and smaller with reduced cortical area and periosteal perimeter. Moreover, colony-forming unit-fibroblast (CFU-F) assay indicates that these mice had fewer bone marrow mesenchymal stem cells and committed progenitors. Similarly, administration of an EGFR inhibitor into wild-type mice caused a significant reduction in trabecular bone volume. In contrast, Egfr(Dsk5/+) mice with a constitutively active EGFR allele displayed increases in trabecular and cortical bone content. Taken together, these data demonstrate that the EGFR signaling pathway is an important bone regulator and that it primarily plays an anabolic role in bone metabolism.

Targeted inactivation of EGF receptor inhibits renal collecting duct development and function.

The ureteric bud (UB) expresses high levels of the EGF receptor (EGFR) during kidney development, but its function in this setting is unclear. Here, Egfr mRNA was abundant in medullary portions of the UB trunk but absent from the branching UB tips during embryogenesis. Homozygous Egfr knockout did not affect the pattern of UB arborization, but renal papillae were hypoplastic and exhibited widespread apoptosis of tubular cells. Because these EGFR-deficient mice die within 1 week of life, we targeted Egfr inactivation to the renal collecting ducts using Cre-lox technology with a Hoxb7-Cre transgene. This targeted inactivation of Egfr led to a thin renal medulla, and at 7 weeks of age, the mice had moderate polyuria and reduced urine-concentrating ability. At 30 to 33 weeks, water deprivation demonstrated a continued urine-concentrating defect despite similar levels of vasopressin between knockout mice and littermate controls. Taken together, these results suggest that unlike other tyrosine kinases expressed at the UB tip, EGFR functions primarily to drive elongation of the emerging collecting ducts and to optimize urine-concentrating ability.

EGFR regulates the expression of keratinocyte-derived granulocyte/macrophage colony-stimulating factor in vitro and in vivo.

Recent advances in the knowledge of the EGFR pathway have revealed its contribution to distinct immune/inflammatory functions of the epidermis. The purpose of our study was to evaluate the role of EGFR in the regulation of keratinocyte GM-CSF expression. In cultured human keratinocytes, proinflammatory cytokines synergized with TGF-alpha to induce GM-CSF expression. Accordingly, high epidermal levels of EGFR activation are associated with enhanced expression of GM-CSF in lesional skin of patients with psoriasis or allergic contact dermatitis. In cultured keratinocytes, pharmacological inhibition of EGFR activity reduced GM-CSF promoter transactivation, whereas genetic inhibition of AP-1 reduced expression of GM-CSF. Furthermore, EGFR activation enhanced TNF-alpha-induced c-Jun phosphorylation and DNA binding, whereas c-Jun silencing reduced GM-CSF expression. Using two different mouse models, we showed that the lack of a functional EGFR pathway was associated with reduced cytokine-induced phosphorylation of ERK1/2, JNK1/2, c-Jun and reduced keratinocyte-derived GM-CSF expression both in vitro and in vivo. Finally, the analysis of GM-CSF expression in the skin of cancer patients treated with anti EGFR drugs showed an association between ERK activity, c-Jun phosphorylation, and epidermal GM-CSF expression. These data demonstrate that the EGFR pathway is critical for the upregulation of keratinocyte GM-CSF expression under conditions of cytokine stimulation.

Generation and validation of mice carrying a conditional allele of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is important for normal homeostasis in a variety of tissues and, when abnormally expressed or mutated, contributes to the development of many diseases. However, in vivo functional studies are hindered by the lack of adult mice lacking EGFR because of the pre- and postnatal lethality of EGFR deficient mice. We generated a conditional allele of Egfr (Egfr(tm1Dwt)) by flanking exon 3 with loxP sites in order to investigate tissue-specific functions of this widely expressed receptor tyrosine kinase. The activity of the Egfr(tm1Dwt) allele is indistinguishable from wildtype Egfr. Conversely, the Egfr(Delta) allele, generated by Cre-mediated deletion of exon 3 using the germline EIIa-Cre transgenic line, functions as a null allele. Egfr(Delta/Delta) embryos that have complete ablation of EGFR activity and die at mid-gestation with placental defects identical to those reported for mice homozygous for the Egfr(tm1Mag) null allele. We also inactivated the Egfr(tm1Dwt) allele tissue-specifically in the skin epithelium using the K14-Cre transgenic line. These mice were viable but exhibited wavy coat hair remarkably similar to mice homozygous for the Egfr(wa2) hypomorphic allele or heterozygous for the Egfr(Wa5) antimorphic allele. These results suggest that the hairless phenotype of Egfr nullizygous mice is not solely due to absence of EGFR in the epithelium, but that EGFR activity is required also in skin stromal cells for normal hair morphogenesis. This new mouse model should have wide utility to inactivate Egfr conditionally for functional analysis of EGFR in adult tissues and disease states.

The EGFR is required for proper innervation to the skin.

EGFR family members are essential for proper peripheral nervous system development. A role for EGFR itself in peripheral nervous system development in vivo, however, has not been reported. We investigated whether EGFR is required for cutaneous innervation using Egfr null and skin-targeted Egfr mutant mice. Neuronal markers; including PGP9.5, GAP-43, acetylated tubulin, and neurofilaments; revealed that Egfr null dorsal skin was hyperinnervated with a disorganized pattern of innervation. In addition, receptor subtypes such as lanceolate endings were disorganized and immature. To determine whether the hyperinnervation phenotype resulted from a target-derived effect of loss of EGFR, mice lacking EGFR expression in the cutaneous epithelium were examined. These mice retained other aspects of the cutaneous Egfr null phenotype but exhibited normal innervation. The sensory deficits in Egfr null dorsal skin were not associated with any abnormality in the morphology or density of dorsal root ganglion (DRG) neurons or Schwann cells. However, explant and dissociated cell cultures of DRG revealed more extensive branching in Egfr null cultures. These data demonstrate that EGFR is required for proper cutaneous innervation during development and suggest that it limits axonal outgrowth and branching in a DRG-autonomous manner.