Molecular mechanisms controlling vertebrate retinal patterning, neurogenesis, and cell fate specification.

This review covers recent advances in understanding the molecular mechanisms controlling neurogenesis and specification of the developing retina, with a focus on insights obtained from comparative single cell multiomic analysis. We discuss recent advances in understanding the mechanisms by which extrinsic factors trigger transcriptional changes that spatially pattern the optic cup (OC) and control the initiation and progression of retinal neurogenesis. We also discuss progress in unraveling the core evolutionarily conserved gene regulatory networks (GRNs) that specify early- and late-state retinal progenitor cells (RPCs) and neurogenic progenitors and that control the final steps in determining cell identity. Finally, we discuss findings that provide insight into regulation of species-specific aspects of retinal patterning and neurogenesis, including consideration of key outstanding questions in the field.

Chondroitin sulfate enhances the barrier function of basement membrane assembled by heparan sulfate.

Glycosaminoglycans are ubiquitously expressed polysaccharides that are attached to proteoglycans. Here, we showed that ablation of the heparan sulfate (HS) polymerase Ext1 in retinal progenitor cells did not affect initial progression of retinal angiogenesis, but it disrupted the pruning of blood vessels and establishment of arterioles and venules. In the absence of retinal HS, blood vessels were also vulnerable to high oxygen tension in early postnatal stages, which could be rescued by exogenous vascular endothelial growth factor (VEGF), consistent with the role of retinal HS in the fine-tuning of VEGF signaling. Furthermore, we observed that the retinal inner limiting membrane (ILM) was disrupted by deletion of Ext1 in a timing-specific manner, suggesting that retinal HS is required for the assembly but not the maintenance of the basement membrane. Lastly, we showed that further deletion of C4st1, a chondroitin sulfate (CS) sulfation enzyme, did not affect the assembly of the ILM but, when combined with Ext1 deletion, it aggravated the retinal permeability by disrupting the retinal glycocalyx. These results demonstrate an important role of CS and HS in establishing the barrier function of the extracellular matrix.

Jack of all trades, master of each: the diversity of fibroblast growth factor signalling in eye development.

A central question in development biology is how a limited set of signalling pathways can instruct unlimited diversity of multicellular organisms. In this review, we use three ocular tissues as models of increasing complexity to present the astounding versatility of fibroblast growth factor (FGF) signalling. In the lacrimal gland, we highlight the specificity of FGF signalling in a one-dimensional model of budding morphogenesis. In the lens, we showcase the dynamics of FGF signalling in altering functional outcomes in a two-dimensional space. In the retina, we present the prolific utilization of FGF signalling from three-dimensional development to homeostasis. These examples not only shed light on the cellular basis for the perfection and complexity of ocular development, but also serve as paradigms for the diversity of FGF signalling.

Phase transition specified by a binary code patterns the vertebrate eye cup.

The developing vertebrate eye cup is partitioned into the neural retina (NR), the retinal pigmented epithelium (RPE), and the ciliary margin (CM). By single-cell analysis, we showed that fibroblast growth factor (FGF) signaling regulates the CM in its stem cell­like property of self-renewal, differentiation, and survival, which is balanced by an evolutionarily conserved Wnt signaling gradient. FGF promotes Wnt signaling in the CM by stabilizing ß-catenin in a GSK3ß-independent manner. While Wnt signaling converts the NR to either the CM or the RPE depending on FGF signaling, FGF transforms the RPE to the NR or CM dependent on Wnt activity. The default fate of the eye cup is the NR, but synergistic FGF and Wnt signaling promotes CM formation both in vivo and in human retinal organoid. Our study reveals that the vertebrate eye develops through phase transition determined by a combinatorial code of FGF and Wnt signaling.

A glycine substitution in the collagenous domain of Col4a3 in mice recapitulates late onset Alport syndrome.

Alport syndrome (AS) is a severe inherited glomerulopathy caused by mutations in the genes encoding the α-chains of type-IV collagen, the most abundant component of the extracellular glomerular basement membrane (GBM). Currently most AS mouse models are knockout models for one of the collagen-IV genes. In contrast, about half of AS patients have missense mutations, with single aminoacid substitutions of glycine being the most common. The only mouse model for AS with a homozygous knockin missense mutation, Col4a3-p.Gly1332Glu, was partly described before by our group. Here, a detailed in-depth description of the same mouse is presented, along with another compound heterozygous mouse that carries the glycine substitution in trans with a knockout allele. Both mice recapitulate essential features of AS, including shorten lifespan by 30-35%, increased proteinuria, increased serum urea and creatinine, pathognomonic alternate GBM thinning and thickening, and podocyte foot process effacement. Notably, glomeruli and tubuli respond differently to mutant collagen-IV protomers, with reduced expression in tubules but apparently normal in glomeruli. However, equally important is the fact that in the glomeruli the mutant α3-chain as well as the normal α4/α5 chains seem to undergo a cleavage at, or near the point of the mutation, possibly by the metalloproteinase MMP-9, producing a 35 kDa C-terminal fragment. These mouse models represent a good tool for better understanding the spectrum of molecular mechanisms governing collagen-IV nephropathies and could be used for pre-clinical studies aimed at better treatments for AS.

Etv transcription factors functionally diverge from their upstream FGF signaling in lens development.

The signal regulated transcription factors (SRTFs) control the ultimate transcriptional output of signaling pathways. Here, we examined a family of FGF-induced SRTFs - Etv1, Etv 4, and Etv 5 - in murine lens development. Contrary to FGF receptor mutants that displayed loss of ERK signaling and defective cell differentiation, Etv deficiency augmented ERK phosphorylation without disrupting the normal lens fiber gene expression. Instead, the transitional zone for lens differentiation was shifted anteriorly as a result of reduced Jag1-Notch signaling. We also showed that Etv proteins suppresses mTOR activity by promoting Tsc2 expression, which is necessary for the nuclei clearance in mature lens. These results revealed the functional divergence between Etv and FGF in lens development, demonstrating that these SRTFs can operate outside the confine of their upstream signaling.

Many cells contain proteins known as signal-induced transcription factors, which are poised to receive messages from the environment and then react by activating genes required for the cell to respond appropriately. It is commonly thought that these transcription factors faithfully follow the instructions they receive from the external signal: for instance, if the message was to encourage the cell to grow, the transcription factors would switch on growth-related genes. As the eyes of mice and other mammals develop, a signal known as FGF is required for certain cells to specialize into lens fiber cells: these long, thin, transparent cells form the bulk of the lens, the structure that allows focused vision. Previous studies suggest that FGF activates three transcription factors known as Etv1, Etv4 and Etv5, but their precise roles in the development of the lens has remained unclear. Here, Garg, Hannan, Wang et al. confirm that FGF signaling does indeed activate all three proteins. However, mutant mice that lacked Etv1, Etv4 and Etv5 still created lens fiber cells, suggesting that the transcription factors are largely unnecessary for lens fiber cells formation. Instead, the Etv proteins participated in a cascade of molecular events involving a protein called Notch; as a result, if the transcription factors were absent, the lens fiber cells formed prematurely. In addition, deactivating Etv1, Etv4 and Etv5 also promoted the activity of a protein which interfered with the removal of internal cell compartments, a process required for lens fiber cells to mature properly. These findings reveal that the roles of Etv1, Etv4 and Etv5 deviate from and even oppose FGF signaling in the lenses of mice. Transcription factors control the ultimate fate of a cell, and there is therefore increased interest in targeting them for therapy. The work by Garg, Hannan, Wang et al. reveals an unexpected complexity in how these proteins respond to upstream signals, highlighting the importance of further dissecting these relationships.

Evidence for miR-548c-5p regulation of FOXC2 transcription through a distal genomic target site in human podocytes.

Podocytes are highly differentiated epithelial cells outlining the glomerular vessels. FOXC2 is a transcription factor essential for inducing podocyte differentiation, development and maturation, and is considered to be the earliest podocyte marker. miRNA prediction analysis revealed a full-length target site for the primate-specific miR-548c-5p at a genomic region > 8 kb upstream of FOXC2. We hypothesised that the transcription rates of FOXC2 during podocyte differentiation might be tuned by miR-548c-5p through this target site. Experiments were performed with cultured human podocytes, transfected with luciferase reporter constructs bearing this target site region within an enhancer element of the native plasmid. The results confirmed a seed region-driven targeting potential by the miRNA, with mimics downregulating and inhibitors enhancing luciferase activity. Introducing mutations into the miRNA target seed region abolished the expected response. In cultured podocytes, FOXC2 mRNA and protein levels responded to miR-548c-5p abundance in a coordinated manner before and after induction of differentiation, with high statistical significance. Ago-ChIP experiments revealed occupancy of the miRNA target site by miRNA/RISC in undifferentiated cells and its release when differentiation is initiated, allowing its interaction with the gene's promoter region to amplify FOXC2 expression, as shown by chromosome conformation capture and qRT-PCR. Moreover, the expression pattern of FOXC2 during podocyte differentiation seems to be affected by miR-548c-5p, as removal of either endogenous or mimic miR-548c-5p results in increased FOXC2 protein levels and cells resembling those undergoing differentiation. Collectively, results indicate a well-orchestrated regulatory model of FOXC2 expression by a remote upstream target site for miR-548c-5p.

Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain.

Studies proposed a model for embryonic neurogenesis where the expression levels of the SOXB2 and SOXB1 factors regulate the differentiation status of the neural stem cells. However, the precise role of the SOXB2 genes remains controversial. Therefore, this study aims to investigate the effects of individual deletions of the SOX21 and SOX14 genes during the development of the dorsal midbrain. We show that SOX21 and SOX14 function distinctly during the commitment of the GABAergic lineage. More explicitly, deletion of SOX21 reduced the expression of the GABAergic precursor marker GATA3 and BHLHB5 while the expression of GAD6, which marks GABAergic terminal differentiation, was not affected. In contrast deletion of SOX14 alone was sufficient to inhibit terminal differentiation of the dorsal midbrain GABAergic neurons. Furthermore, we demonstrate through gain-of-function experiments, that despite the homology of SOX21 and SOX14, they have unique gene targets and cannot compensate for the loss of each other. Taken together, these data do not support a pan-neurogenic function for SOXB2 genes in the dorsal midbrain, but instead they influence, sequentially, the specification of GABAergic neurons.

Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers.

Oligonucleotide gene therapy has shown great promise for the treatment of muscular dystrophies. Nevertheless, the selective delivery to affected muscles has shown to be challenging because of their high representation in the body and the high complexity of their cell membranes. Current trials show loss of therapeutic molecules to non-target tissues leading to lower target efficacy. Therefore, strategies that increase uptake efficiency would be particularly compelling. To address this need, we applied a cell-internalization SELEX (Systematic Evolution of Ligands by Exponential Enrichment) approach and identified a skeletal muscle-specific RNA aptamer. A01B RNA aptamer preferentially internalizes in skeletal muscle cells and exhibits decreased affinity for off-target cells. Moreover, this in vitro selected aptamer retained its functionality in vivo, suggesting a potential new approach for targeting skeletal muscles. Ultimately, this will aid in the development of targeted oligonucleotide therapies against muscular dystrophies.

Vsx1 Transiently Defines an Early Intermediate V2 Interneuron Precursor Compartment in the Mouse Developing Spinal Cord.

Spinal ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities. Interneurons arise during embryonic development from distinct progenitor domains distributed orderly along the dorso-ventral axis of the neural tube. A single ventral progenitor population named p2 generates at least five V2 interneuron subsets. Whether the diversification of V2 precursors into multiple subsets occurs within the p2 progenitor domain or involves a later compartment of early-born V2 interneurons remains unsolved. Here, we provide evidence that the p2 domain produces an intermediate V2 precursor compartment characterized by the transient expression of the transcriptional repressor Vsx1. These cells display an original repertoire of cellular markers distinct from that of any V2 interneuron population. They have exited the cell cycle but have not initiated neuronal differentiation. They coexpress Vsx1 and Foxn4, suggesting that they can generate the known V2 interneuron populations as well as possible additional V2 subsets. Unlike V2 interneurons, the generation of Vsx1-positive precursors does not depend on the Notch signaling pathway but expression of Vsx1 in these cells requires Pax6. Hence, the p2 progenitor domain generates an intermediate V2 precursor compartment, characterized by the presence of the transcriptional repressor Vsx1, that contributes to V2 interneuron development.