Ret signaling in ureteric bud epithelial cells controls cell movements, cell clustering and bud formation.

Ret signaling promotes branching morphogenesis during kidney development, but the underlying cellular mechanisms remain unclear. While Ret-expressing progenitor cells proliferate at the ureteric bud tips, some of these cells exit the tips to generate the elongating collecting ducts, and in the process turn off Ret. Genetic ablation of Ret in tip cells promotes their exit, suggesting that Ret is required for cell rearrangements that maintain the tip compartments. Here, we examine the behaviors of ureteric bud cells that are genetically forced to maintain Ret expression. These cells move to the nascent tips, and remain there during many cycles of branching; this tip-seeking behavior may require positional signals from the mesenchyme, as it occurs in whole kidneys but not in epithelial ureteric bud organoids. In organoids, cells forced to express Ret display a striking self-organizing behavior, attracting each other to form dense clusters within the epithelium, which then evaginate to form new buds. The ability of forced Ret expression to promote these events suggests that similar Ret-dependent cell behaviors play an important role in normal branching morphogenesis.

Brn3a/Pou4f1 regulates dorsal root ganglion sensory neuron specification and axonal projection into the spinal cord.

The sensory neurons of the dorsal root ganglia (DRG) must project accurately to their central targets to convey proprioceptive, nociceptive and mechanoreceptive information to the spinal cord. How these different sensory modalities and central connectivities are specified and coordinated still remains unclear. Given the expression of the POU homeodomain transcription factors Brn3a/Pou4f1 and Brn3b/Pou4f2 in DRG and spinal cord sensory neurons, we determined the subtype specification of DRG and spinal cord sensory neurons as well as DRG central projections in Brn3a and Brn3b single and double mutant mice. Inactivation of either or both genes causes no gross abnormalities in early spinal cord neurogenesis; however, in Brn3a single and Brn3a;Brn3b double mutant mice, sensory afferent axons from the DRG fail to form normal trajectories in the spinal cord. The TrkA(+) afferents remain outside the dorsal horn and fail to extend into the spinal cord, while the projections of TrkC(+) proprioceptive afferents into the ventral horn are also impaired. Moreover, Brn3a mutant DRGs are defective in sensory neuron specification, as marked by the excessive generation of TrkB(+) and TrkC(+) neurons as well as TrkA(+)/TrkB(+) and TrkA(+)/TrkC(+) double positive cells at early embryonic stages. At later stages in the mutant, TrkB(+), TrkC(+) and parvalbumin(+) neurons diminish while there is a significant increase of CGRP(+) and c-ret(+) neurons. In addition, Brn3a mutant DRGs display a dramatic down-regulation of Runx1 expression, suggesting that the regulation of DRG sensory neuron specification by Brn3a is mediated in part by Runx1. Our results together demonstrate a critical role for Brn3a in generating DRG sensory neuron diversity and regulating sensory afferent projections to the central targets.

Respecification of ectoderm and altered Nodal expression in sea urchin embryos after cobalt and nickel treatment.

In the sea urchin embryo, Nodal is the earliest known signal to play a role in the specification of the oral ectodermal territory. Nodal, a TGF-beta ligand, is first expressed in the presumptive oral ectoderm at approximately 7 H of development. Nodal overexpression produces a distinctive bell-shaped phenotype with expanded oral ectoderm, which resembles the oralized phenotype obtained as a result of nickel (Ni) treatment. To date, a detailed analysis of gene expression in Ni-treated embryos has not been undertaken. Because treatment with cobalt (Co) produces similar results to those seen with Ni treatment in other systems, we were interested in determining how Co influences sea urchin embryonic development. Here we report that Co also induces oralization of the ectoderm, and the effects of Ni and Co depend on functional Nodal signaling. Although both metals upregulate nodal gene expression, they do not initiate nodal transcription precociously. Analysis of the perturbation of Nodal receptor function suggests that Ni and Co contribute to nodal upregulation in the absence of nodal autoregulation, but cannot fully oralize the ectoderm in the absence of Nodal signaling.

Retinal ganglion cell death and optic nerve degeneration by genetic ablation in adult mice.

Despite the magnitude of the problem, no effective treatments exist to prevent retinal ganglion cell (RGC) death and optic nerve degeneration from occurring in diseases affecting the human eye. Animal models currently available for developing treatment strategies suffer from cumbersome procedures required to induce RGC death or rely on mutations that induce defects in developing retinas rather than in mature retinas of adults. Our objective was to develop a robust genetically engineered adult mouse model for RGC loss and optic nerve degeneration based on genetic ablation. To achieve this, we took advantage of Pou4f2 (Brn3b), a gene activated immediately as RGCs begin to differentiate and expressed throughout life. We generated adult mice whose genomes harbored a conditional Pou4f2 allele containing a floxed-lacZ-stop-diphtheria toxin A cassette and a CAGG-Cre-ER transgene. In this bigenic model, Cre recombinase is fused to a modified estrogen nuclear receptor in which the estrogen-binding domain binds preferentially to the estrogen agonist tamoxifen rather than to endogenous estradiol. Upon binding to the estrogen-binding domain, tamoxifen derepresses Cre recombinase, leading to the efficient genomic deletion of the floxed-lacZ-stop DNA sequence and expression of diphtheria toxin A. Tamoxifen administered to adult mice at different ages by intraperitoneal injection led to rapid RGC loss, reactive gliosis, progressive degradation of the optic nerve over a period of several months, and visual impairment. Perhaps more reflective of human disease, partial loss of RGCs was achieved by modulating the tamoxifen treatment. Especially relevant for RGC death and optic nerve degeneration in human retinal pathologies, RGC-ablated retinas maintained their structural integrity, and other retinal neurons and their connections in the inner and outer plexiform layers appeared unaffected by RGC ablation. These events are hallmarks of progressive optic nerve degeneration observed in human retinal pathologies and demonstrate the validity of this model for use in developing stem cell therapies for replacing dead RGCs with healthy ones.

The succinate receptor GPR91 in neurons has a major role in retinal angiogenesis.

Vascularization is essential for tissue development and in restoration of tissue integrity after an ischemic injury. In studies of vascularization, the focus has largely been placed on vascular endothelial growth factor (VEGF), yet other factors may also orchestrate this process. Here we show that succinate accumulates in the hypoxic retina of rodents and, via its cognate receptor G protein-coupled receptor-91 (GPR91), is a potent mediator of vessel growth in the settings of both normal retinal development and proliferative ischemic retinopathy. The effects of GPR91 are mediated by retinal ganglion neurons (RGCs), which, in response to increased succinate levels, regulate the production of numerous angiogenic factors including VEGF. Accordingly, succinate did not have proangiogenic effects in RGC-deficient rats. Our observations show a pathway of metabolite signaling where succinate, acting through GPR91, governs retinal angiogenesis and show the propensity of RGCs to act as sensors of ischemic stress. These findings provide a new therapeutic target for modulating revascularization.