Antibody validation for Western blot: By the user, for the user.

Well-characterized antibody reagents play a key role in the reproducibility of research findings, and inconsistent antibody performance leads to variability in Western blotting and other immunoassays. The current lack of clear, accepted standards for antibody validation and reporting of experimental details contributes to this problem. Because the performance of primary antibodies is strongly influenced by assay context, recommendations for validation and usage are unique to each type of immunoassay. Practical strategies are proposed for the validation of primary antibody specificity, selectivity, and reproducibility using Western blot analysis. The antibody should produce reproducible results within and between Western blotting experiments and the observed effect confirmed with a complementary or orthogonal method. Routine implementation of standardized antibody validation and reporting in immunoassays such as Western blotting may promote improved reproducibility across the global life sciences community.

A systematic approach to quantitative Western blot analysis.

Attaining true quantitative data from WB requires that all the players involved in the procedure are quality controlled including the user. Appropriate protein extraction method, electrophoresis, and transfer of proteins, immunodetection of blotted protein by antibodies, and the ultimate step of imaging and analyzing the data is nothing short of a symphony. Like with any other technology in life-sciences research, Western blotting can produce erroneous and irreproducible data. We provide a systematic approach to generate quantitative data from Western blot experiments that incorporates critical validation steps to identify and minimize sources of error and variability throughout the Western blot process.

Sox11 is required to maintain proper levels of Hedgehog signaling during vertebrate ocular morphogenesis.

Ocular coloboma is a sight-threatening malformation caused by failure of the choroid fissure to close during morphogenesis of the eye, and is frequently associated with additional anomalies, including microphthalmia and cataracts. Although Hedgehog signaling is known to play a critical role in choroid fissure closure, genetic regulation of this pathway remains poorly understood. Here, we show that the transcription factor Sox11 is required to maintain specific levels of Hedgehog signaling during ocular development. Sox11-deficient zebrafish embryos displayed delayed and abnormal lens formation, coloboma, and a specific reduction in rod photoreceptors, all of which could be rescued by treatment with the Hedgehog pathway inhibitor cyclopamine. We further demonstrate that the elevated Hedgehog signaling in Sox11-deficient zebrafish was caused by a large increase in shha transcription; indeed, suppressing Shha expression rescued the ocular phenotypes of sox11 morphants. Conversely, over-expression of sox11 induced cyclopia, a phenotype consistent with reduced levels of Sonic hedgehog. We screened DNA samples from 79 patients with microphthalmia, anophthalmia, or coloboma (MAC) and identified two novel heterozygous SOX11 variants in individuals with coloboma. In contrast to wild type human SOX11 mRNA, mRNA containing either variant failed to rescue the lens and coloboma phenotypes of Sox11-deficient zebrafish, and both exhibited significantly reduced transactivation ability in a luciferase reporter assay. Moreover, decreased gene dosage from a segmental deletion encompassing the SOX11 locus resulted in microphthalmia and related ocular phenotypes. Therefore, our study reveals a novel role for Sox11 in controlling Hedgehog signaling, and suggests that SOX11 variants contribute to pediatric eye disorders.

Sox4 regulates choroid fissure closure by limiting Hedgehog signaling during ocular morphogenesis.

SoxC transcription factors play critical roles in many developmental processes, including neurogenesis, cardiac formation, and skeletal differentiation. In vitro and in vivo loss-of-function studies have suggested that SoxC genes are required for oculogenesis; however the mechanism was poorly understood. Here, we have explored the function of the SoxC factor Sox4 during zebrafish eye development. We show that sox4a and sox4b are expressed in the forebrain and periocular mesenchyme adjacent to the optic stalk during early eye development. Knockdown of sox4 in zebrafish resulted in coloboma, a structural malformation of the eye that is a significant cause of pediatric visual impairment in humans, in which the choroid fissure fails to close. Sox4 morphants displayed altered proximo-distal patterning of the optic vesicle, including expanded pax2 expression in the optic stalk, as well as ectopic cell proliferation in the retina. We show that the abnormal ocular morphogenesis observed in Sox4-deficient zebrafish is caused by elevated Hedgehog (Hh) signaling, and this is due to increased expression of the Hh pathway ligand Indian Hedgehog b (ihhb). Consistent with these results, coloboma in sox4 morphants could be rescued by pharmacological treatment with the Hh inhibitor cyclopamine, or by co-knockdown of ihhb. Conversely, overexpression of sox4 reduced Hh signaling and ihhb expression, resulting in cyclopia. Finally, we demonstrate that sox4 and sox11 have overlapping, but not completely redundant, functions in regulating ocular morphogenesis. Taken together, our data demonstrate that Sox4 is required to limit the extent of Hh signaling during eye development, and suggest that mutations in SoxC factors could contribute to the development of coloboma.

Tracking the fate of her4 expressing cells in the regenerating retina using her4:Kaede zebrafish.

The Basic-Helix-Loop-Helix-Orange (bHLH-O) transcription factor Hairy-related 4 (her4) is a downstream effector of Notch-Delta signaling that represses expression of typically pro-neural genes in proliferative domains of the central nervous system. Notch-Delta signaling in the retina has been shown to increase in response to injury and influences neuroprotective properties of Müller glia. In contrast to mammals, teleost fish are able to regenerate retinal neurons in response to injury. In zebrafish, her4 is upregulated in the regenerating neural retina in response to both acute and chronic photoreceptor damage, but the contribution of her4 expressing cells to neurogenesis following acute or chronic retinal damage has remained unexplored. Here we investigate the role of her4 in the regenerating retina in a background of chronic, rod-specific degeneration as well as following acute light damage. We demonstrate that her4 is expressed in the persistently neurogenic ciliary marginal zone (CMZ), as well as in small subsets of slowly proliferating Müller glia in the inner nuclear layer (INL) of the central retina. We generated a transgenic line of zebrafish that expresses the photoconvertible Kaede reporter driven by a her4 promoter and validated that expression of the transgene faithfully recapitulates endogenous her4 expression. Lineage tracing analysis revealed that her4-expressing cells in the INL contribute to the rod lineage, and her4 expressing cells in the CMZ are capable of generating any retinal cell type except rod photoreceptors. Our results indicate that her4 is involved in a replenishing pathway that maintains populations of stem cells in the central retina, and that the magnitude of the her4-associated proliferative response mirrors the extent of retinal damage.

Keeping an eye on SOXC proteins.

The formation of a mature, functional eye requires a complex series of cell proliferation, migration, induction among different germinal layers, and cell differentiation. These processes are regulated by extracellular cues such as the Wnt/BMP/Hh/Fgf signaling pathways, as well as cell intrinsic transcription factors that specify cell fate. In this review article, we provide an overview of stages of embryonic eye morphogenesis, extrinsic and intrinsic factors that are required for each stage, and pediatric ocular diseases that are associated with defective eye development. In addition, we focus on recent findings about the roles of the SOXC proteins in regulating vertebrate ocular development and implicating SOXC mutations in human ocular malformations.