FGFR4 does not contribute to progression of chronic kidney disease.

In chronic kidney disease (CKD), elevated serum levels of the phosphate regulating hormone fibroblast growth factor (FGF) 23 have emerged as powerful risk factors for cardiovascular disease and death. Mechanistically, FGF23 can bind and activate fibroblast growth factor receptor (FGFR) 4 independently of α-klotho, the canonical co-receptor for FGF23 in the kidney, which stimulates left ventricular hypertrophy and hepatic production of inflammatory cytokines. FGF23 has also been shown to independently predict progression of renal disease, however, whether FGF23 and FGFR4 also contribute to CKD remains unknown. Here, we generated a mouse model with dual deletions of FGFR4 and α-klotho, and we induced CKD in mice with either global deletion or constitutive activation of FGFR4. We demonstrate that FGF23 is not capable of inducing phosphaturia via FGFR4 and that FGFR4 does not promote or mitigate renal injury in animal models of CKD. Taken together our results suggest FGFR4 inhibition as a safe alternative strategy to target cardiovascular disease and chronic inflammation in patients with CKD without interrupting the necessary phosphaturic effects of FGF23.

Dre - Cre sequential recombination provides new tools for retinal ganglion cell labeling and manipulation in mice.

BACKGROUND: Genetic targeting methods have greatly advanced our understanding of many of the 20 Retinal Ganglion Cell (RGC) types conveying visual information from the eyes to the brain. However, the complexity and partial overlap of gene expression patterns in RGCs call for genetic intersectional or sparse labeling strategies. Loci carrying the Cre recombinase in conjunction with conditional knock-out, reporter or other genetic tools can be used for targeted cell type ablation and functional manipulation of specific cell populations. The three members of the Pou4f family of transcription factors, Brn3a, Brn3b and Brn3c, expressed early during RGC development and in combinatorial pattern amongst RGC types are excellent candidates for such gene manipulations. METHODS AND FINDINGS: We generated conditional Cre knock-in alleles at the Brn3a and Brn3b loci, Brn3a(CKOCre) and Brn3b(CKOCre). When crossed to mice expressing the Dre recombinase, the endogenous Brn3 gene expressed by Brn3a(CKOCre) or Brn3b(CKOCre) is removed and replaced with a Cre recombinase, generating Brn3a(Cre) and Brn3b(Cre) knock-in alleles. Surprisingly both Brn3a(Cre) and Brn3b(Cre) knock-in alleles induce early ubiquitous recombination, consistent with germline expression. However in later stages of development, their expression is limited to the expected endogenous pattern of the Brn3a and Brn3b genes. We use the Brn3a(Cre) and Brn3b(Cre) alleles to target a Cre dependent Adeno Associated Virus (AAV) reporter to RGCs and demonstrate its use in morphological characterization, early postnatal gene delivery and tracing the expression of Brn3 genes in RGCs. CONCLUSIONS: Dre recombinase effectively recombines the Brn3a(CKOCre) and Brn3b(CKOCre) alleles containing its roxP target sites. Sequential Dre to Cre recombination reveals Brn3a and Brn3b expression in early mouse development. The generated Brn3a(Cre) and Brn3b(Cre) alleles are useful tools that can target exogenously delivered Cre dependent reagents to RGCs in early postnatal development, opening up a large range of potential applications.

Genetic interactions between Brn3 transcription factors in retinal ganglion cell type specification.

BACKGROUND: Visual information is conveyed from the retina to the brain via 15-20 Retinal Ganglion Cell (RGC) types. The developmental mechanisms by which RGC types acquire their distinct molecular, morphological, physiological and circuit properties are essentially unknown, but may involve combinatorial transcriptional regulation. Brn3 transcription factors are expressed in RGCs from early developmental stages, and are restricted in adults to distinct, partially overlapping populations of RGC types. Previously, we described cell autonomous effects of Brn3b (Pou4f2) and Brn3a (Pou4f1) on RGC axon and dendrites development. METHODS AND FINDINGS: We now have investigated genetic interactions between Brn3 transcription factors with respect to RGC development, by crossing conventional knock-out alleles of each Brn3 gene with conditional knock-in reporter alleles of a second Brn3 gene, and analyzing the effects of single or double Brn3 knockouts on RGC survival and morphology. We find that Brn3b loss results in axon defects and dendritic arbor area and lamination defects in Brn3a positive RGCs, and selectively affects survival and morphology of specific Brn3c (Pou4f3) positive RGC types. Brn3a and Brn3b interact synergistically to control RGC numbers. Melanopsin positive ipRGCs are resistant to combined Brn3 loss but are under the transcriptional control of Isl1, expanding the combinatorial code of RGC specification. CONCLUSIONS: Taken together these results complete our knowledge on the mechanisms of transcriptional control of RGC type specification. They demonstrate that Brn3b is required for the correct development of more RGC cell types than suggested by its expression pattern in the adult, but that several cell types, including some Brn3a, Brn3c or Melanopsin positive RGCs are Brn3b independent.

Combinatorial expression of Brn3 transcription factors in somatosensory neurons: genetic and morphologic analysis.

The three members of the Brn3 family of POU-domain transcription factors (Brn3a/Pou4f1, Brn3b/Pou4f2, and Brn3c/Pou4f3) are expressed in overlapping subsets of visual, auditory/vestibular, and somatosensory neurons. Using unmarked Brn3-null alleles and Brn3 conditional alleles in which gene loss is coupled to expression of an alkaline phosphatase reporter, together with sparse Cre-mediated recombination, we describe the following: (1) the overlapping patterns of Brn3 gene expression in somatosensory neurons; (2) the manner in which these patterns correlate with molecular markers, peripheral afferent arbor morphologies, and dorsal horn projections; and (3) the consequences for these neurons of deleting individual Brn3 genes in the mouse. We observe broad expression of Brn3a among DRG neurons, but subtype-restricted expression of Brn3b and Brn3c. We also observe a nearly complete loss of hair follicle-associated sensory endings among Brn3a(-/-) neurons. Together with earlier analyses of Brn3 gene expression patterns in the retina and inner ear, these experiments suggest a deep functional similarity among primary somatosensory neurons, spiral and vestibular ganglion neurons, and retinal ganglion cells. This work also demonstrates the utility of sparse genetically directed labeling for visualizing individual somatosensory afferent arbors and for defining cell-autonomous mutant phenotypes.