Disruption of the Extracellular Matrix Progressively Impairs Central Nervous System Vascular Maturation Downstream of ß-Catenin Signaling.

Objective- The Wnt/ß-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/ß-catenin signaling by induced overexpression of Axin1, an inhibitor of ß-catenin signaling, specifically in endothelial cells ( Axin1 iEC- OE). AOE (Axin1 overexpression) in Axin1 iEC- OE mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/ß-catenin driven CNS vascular development in zebrafish also suggested that Axin1 iEC- OE led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, ß-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific ß-catenin-responsive ECM signature was also repressed in Axin1 iEC- OE and endothelial cell-specific ß-catenin-knockout mice ( Ctnnb1-KOiEC) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/ß-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-ß-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article.

Apolipoprotein C-I mediates Wnt/Ctnnb1 signaling during neural border formation and is required for neural crest development.

In vertebrates, the neural crest and placodes originate in the neural border, which is located between the neural plate and epidermal ectoderm. The neural crest and placodes give rise to a vast array of cell types. Formation of neural crest is a multi-step process, in which Wnt signals are used reiteratively, but it is currently not clear if a Wnt signal is required for neural border formation. Here, we have identified apolipoprotein C-I (apoc1) in a screen for genes regulated by Wnt/Ctnnb1 signaling in late blastula stage Xenopus tropicalis embryos. We show that Xenopus laevis apoc1 encodes a small, secreted protein, and is induced by Wnt/Ctnnb1 signaling. Depletion of Apoc1 protein results in a neural border formation defect and loss of border fates, including neural crest cells. However, unlike another Wnt/Ctnnb1 target, gbx2.2, apoc1 is not required for patterning of the neural border. We further show that gbx2.2 and apoc1 are independently regulated by Wnt signaling. Our results thus suggest that Wnt regulates border formation and patterning by distinct genetic mechanisms.

Gene expression profiles of brain endothelial cells during embryonic development at bulk and single-cell levels.

The blood-brain barrier is a dynamic interface that separates the brain from the circulatory system, and it is formed by highly specialized endothelial cells. To explore the molecular mechanisms defining the unique nature of vascular development and differentiation in the brain, we generated high-resolution gene expression profiles of mouse embryonic brain endothelial cells using translating ribosome affinity purification and single-cell RNA sequencing. We compared the brain vascular translatome with the vascular translatomes of other organs and analyzed the vascular translatomes of the brain at different time points during embryonic development. Because canonical Wnt signaling is implicated in the formation of the blood-brain barrier, we also compared the brain endothelial translatome of wild-type mice with that of mice lacking the transcriptional cofactor ß-catenin (Ctnnb1). Our analysis revealed extensive molecular changes during the embryonic development of the brain endothelium. We identified genes encoding brain endothelium-specific transcription factors (Foxf2, Foxl2, Foxq1, Lef1, Ppard, Zfp551, and Zic3) that are associated with maturation of the blood-brain barrier and act downstream of the Wnt-ß-catenin signaling pathway. Profiling of individual brain endothelial cells revealed substantial heterogeneity in the population. Nevertheless, the high abundance of Foxf2, Foxq1, Ppard, or Zic3 transcripts correlated with the increased expression of genes encoding markers of brain endothelial cell differentiation. Expression of Foxf2 and Zic3 in human umbilical vein endothelial cells induced the production of blood-brain barrier differentiation markers. This comprehensive data set may help to improve the engineering of in vitro blood-brain barrier models.

Evaluation of TRAP-sequencing technology with a versatile conditional mouse model.

Gene expression profiling of various cell lineages has provided invaluable insights into the molecular mechanisms regulating cellular development and differentiation. However, in vivo molecular profiling of rare and interspersed cell populations, such as endothelial cells, has remained challenging. We have generated a versatile floxed translating ribosome affinity purification (TRAP) mouse model, mCherryTRAP, for Cre-dependent translational profiling of distinct cell lineages from intact tissues. To identify cell type-specific transcripts using TRAP, the data have to be filtered to remove both background transcripts not expressed in the profiled cell type and transcripts expressed in all cell populations of the tissue/organ. Filtering has previously been achieved using transcribed RNA from the tissue/organ. Using the mCherryTRAP model, we demonstrate extensive differential expression of RNAs between the translatome and transcriptome of embryonic brains and kidneys. We evaluate the implications of these data for TRAP studies of abundant and rare cell populations. Finally, we demonstrate the applicability of the technology to study organ-specific endothelial cell differentiation.

Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature.

Every organ depends on blood vessels for oxygen and nutrients, but the vasculature associated with individual organs can be structurally and molecularly diverse. The central nervous system (CNS) vasculature consists of a tightly sealed endothelium that forms the blood-brain barrier, whereas blood vessels of other organs are more porous. Wnt7a and Wnt7b encode two Wnt ligands produced by the neuroepithelium of the developing CNS coincident with vascular invasion. Using genetic mouse models, we found that these ligands directly target the vascular endothelium and that the CNS uses the canonical Wnt signaling pathway to promote formation and CNS-specific differentiation of the organ's vasculature.

Mouse brain organization revealed through direct genome-scale TF expression analysis.

In the developing brain, transcription factors (TFs) direct the formation of a diverse array of neurons and glia. We identifed 1445 putative TFs in the mouse genome. We used in situ hybridization to map the expression of over 1000 of these TFs and TF-coregulator genes in the brains of developing mice. We found that 349 of these genes showed restricted expression patterns that were adequate to describe the anatomical organization of the brain. We provide a comprehensive inventory of murine TFs and their expression patterns in a searchable brain atlas database.

Sequential phases of cortical specification involve Neurogenin-dependent and -independent pathways.

Neocortical projection neurons, which segregate into six cortical layers according to their birthdate, have diverse morphologies, axonal projections and molecular profiles, yet they share a common cortical regional identity and glutamatergic neurotransmission phenotype. Here we demonstrate that distinct genetic programs operate at different stages of corticogenesis to specify the properties shared by all neocortical neurons. Ngn1 and Ngn2 are required to specify the cortical (regional), glutamatergic (neurotransmitter) and laminar (temporal) characters of early-born (lower-layer) neurons, while simultaneously repressing an alternative subcortical, GABAergic neuronal phenotype. Subsequently, later-born (upper-layer) cortical neurons are specified in an Ngn-independent manner, requiring instead the synergistic activities of Pax6 and Tlx, which also control a binary choice between cortical/glutamatergic and subcortical/GABAergic fates. Our study thus reveals an unanticipated heterogeneity in the genetic mechanisms specifying the identity of neocortical projection neurons.

Tlx controls proliferation and patterning of lateral telencephalic progenitor domains.

We showed previously that the orphan nuclear receptor Tlx is required for the correct establishment of the pallio-subpallial boundary. Loss of Tlx results in a dorsal expansion of ventral markers (e.g., the homeodomain protein GSH2) into the ventralmost pallial region, i.e., the ventral pallium. We also observed a disproportionate reduction in the size of the Tlx mutant lateral ganglionic eminence (LGE) from embryonic day 14.5 onward. Here we show that this reduction is caused, at least in large part, by a proliferation defect. Interestingly, in Tlx mutants, the LGE derivatives are differentially affected. Although the development of the Tlx mutant striatum is compromised, an apparently normal number of olfactory bulb interneurons are observed. Consistent with this observation, we found that Tlx is required for the normal establishment of the ventral LGE that gives rise to striatal projection neurons. This domain is reduced by the dorsal and ventral expansion of molecular markers normally confined to progenitor domains flanking the ventral LGE. Finally, we investigated possible genetic interactions between Gsh2 and Tlx in lateral telencephalic development. Our results show that, although Gsh2 and Tlx have additive effects on striatal development, they differentially regulate the establishment of ventral pallial identity.