Joaquim Egea

Cortical gyrification, characterized by the formation of sulcal fissures and gyral peaks, is a distinctive feature of large mammalian brains and is associated with the emergence of higher cognitive functions. This contrasts with small mammals, like mice, which generally display smooth (lissencephalic) cortices. Gyrification results from the integration of several complex processes such as neuron progenitor amplification and migration. However, the underlying molecular mechanisms remain poorly understood. Here, we show that genetic ablation of Rnd3 , encoding the atypical RHO-GTPase RND3/RHOE, induces spontaneous cortical sulci in a subset of mouse embryos without increasing neural progenitor amplification. Instead, RND3 regulates cortical neuron migration, as RND3 overexpression delays neuronal migration, whereas its loss accelerates this process. Similar phenotypes reported in the double Flrt1 / 3 mutants, suggested a functional interaction between the transmembrane FLRTs and RND3 during cortex development. We demonstrate that Rnd3 and Flrt3 are co-expressed in migrating cortical neurons and interact through a β-strand-mediated binding mechanism, involving a conserved motif in RND3. Disruption of this motif abolishes FLRT3 binding, impairs RND3-mediated regulation of neuronal migration and promotes sulcus formation, indicating that FLRT3-RND3 suppresses cortical folding by regulating neuronal migration in mice. Consistently, Rnd3 expression is reduced in the outer subventricular zone of the prospective sulcal regions in the gyrencephalic ferret brain, supporting the notion that RND3 downregulation contributes to sulci development. Together, these findings identify FLRT3-RND3 signalling as a conserved negative regulator of cortical neuron migration and cortical folding.

During nervous system development, the interplay between cell cycle regulation and neurogenesis is fundamental to achieving the correct timing for neuronal differentiation. However, the molecules regulating this transition are poorly understood. Among these, the cell-cycle regulatory cyclins and their cyclin-dependent kinases (CDKs) play a pivotal role. In the present work we uncover an unknown function of cyclin D1 (CCND1) during cortex development which is independent of cell cycle regulation and that relies on its cytoplasmic localization and membrane association. We show that CCND1 is localized in the cytoplasm of the radial glial process (RGP) of neuron progenitors in different regions of the developing brain, including the pallium. Cytoplasmic CCND1 is enriched at the distal tip of the RGP, adjacent to the meningeal basement membrane, and overlaps with β1-integrin at the plasma membrane. Ccnd1 knock-out embryos show an abnormal cortical layering in which the distribution of TBR2 + and CTIP2 + cells are affected without displaying proliferation defects. This is consistent with a cytoplasmic function of CCND1 as overexpression by in utero electroporation of a dominant negative CCND1, unable to activate CDKs, and targeted to the cytoplasmic membranes, reproduces some of these TBR2 and CTIP2 defects. Finally, we provide evidence that cytoplasmic CCND1 affects neuron morphology and that it is required for the proper detachment of the RGP from the meningeal basement membrane by a mechanism involving the phosphorylation of the integrin effector protein paxillin. Hence, we propose that CCND1 has an important cytoplasmic function for cortical development independently of cell cycle regulation.

During nervous system development, the interplay between cell cycle regulation and neurogenesis is fundamental to achieve the correct timing for neuronal differentiation. However, the molecular players regulating this transition are poorly understood. Among these, the cell-cycle regulatory cyclins and their cyclin-dependent kinases (Cdks) play a pivotal role. In the present work we uncover an unknown function of cyclin D1 (Ccnd1) during cortex development which is independent of cell cycle regulation and that relies on its cytoplasmic localization and membrane association. We show that Ccnd1 is localized in the cytoplasm of the radial glial process (RGP) of neuron progenitors in different regions of the developing brain, including the cortex. Cytoplasmic Ccnd1 is enriched at the distal tip of the RGP, adjacent to the meningeal basement membrane, and overlaps with β1-integrin at the plasma membrane. CCND1 knock-out animals show an abnormal cortical layering in which the distribution of Tbr2+ and Ctip2+ cells are affected without displaying proliferation defects. This is consistent with a cytoplasmic function of Ccnd1 as overexpression by in utero electroporation of a dominant negative Ccnd1, unable to activate Cdks, and targeted to the cytoplasmic membranes, reproduces some of these Tbr2 and Ctip2 defects. Finally, we provide evidence that cytoplasmic Ccnd1 affects neuron morphology and that it is required for the proper detachment of the RGP from the meningeal basement membrane by a mechanism involving the phosphorylation of the integrin effector protein paxillin. Hence, we propose that Ccnd1 has an important cytoplasmic function for cortical development independently of cell cycle regulation. Significant Statement: A key developmental step during nervous system formation is the transition from proliferating progenitors to postmitotic neurons. However, the molecular mechanisms regulating this process are not fully understood. Cyclin D1 (Ccnd1) is a canonical regulator of cell cycle in the cell nucleus. Surprisingly, we show that Ccnd1 is also located in the radial glial process of neuron progenitors and associated to the plasma membrane in different regions of the developing mouse brain. We uncover a novel function for this cytoplasmic Ccnd1 and show that it is required for proper cortical layering, independent of cell cycle regulation. Mechanistically, we provide evidence that this function is mediated by the integrin effector paxillin. We propose therefore that cytoplasmic Ccnd1 is important for cortex development independent of cell cycle regulation.

Genes of the Sprouty family (Spry1-4) are feedback inhibitors of receptor tyrosine kinases, especially of Ret and the FGF receptors. As such, they play distinct and overlapping roles in embryo morphogenesis and are considered to be tumor suppressors in adult life. Genetic experiments in mice have defined in great detail the role of these genes during embryonic development, however their function in adult mice is less clearly established. Here we generate adult-onset, whole body Spry1/2/4 triple knockout mice. Tumor incidence in triple mutant mice is comparable to that of wild type littermates of up to one year of age, indicating that Sprouty loss per se is not sufficient to initiate tumorigenesis. On the other hand, triple knockout mice do not gain weight as they age, show less visceral fat, and have lower plasma glucose levels than wild type littermates, despite showing similar food intake and slightly reduced motor function. They also show alopecia, eyelid inflammation, and mild hyperthyroidism. Finally, triple knockout mice present phosphaturia and hypophosphatemia, suggesting exacerbated signaling downstream of FGF23. In conclusion, triple knockout mice develop a series of endocrine abnormalities but do not show increased tumor incidence.

Phosphatase and TENsin homolog (Pten) and p53 are two of the most frequently mutated tumor suppressor genes in endometrial cancer. However, the functional consequences and histopathological manifestation of concomitant p53 and Pten loss of function alterations in the development of endometrial cancer is still controversial. Here, it is demonstrated that simultaneous Pten and p53 deletion is sufficient to cause epithelial to mesenchymal transition phenotype in endometrial organoids. By a novel intravaginal delivery method using HIV1 trans‐activator of transcription cell penetrating peptide fused with a Cre recombinase protein (TAT‐Cre), local ablation of both p53 and Pten is achieved specifically in the uterus. These mice developed high‐grade endometrial carcinomas and a high percentage of uterine carcinosarcomas resembling those found in humans. To further demonstrate that carcinosarcomas arise from epithelium, double Pten/p53 deficient epithelial cells are mixed with wild type stromal and myometrial cells and subcutaneously transplanted to Scid mice. All xenotransplants resulted in the development of uterine carcinosarcomas displaying high nuclear pleomorphism and metastatic potential. Accordingly, in vivo CRISPR/Cas9 disruption of Pten and p53 also triggered the development of metastatic carcinosarcomas. The results unfadingly demonstrate that simultaneous deletion of p53 and Pten in endometrial epithelial cells is enough to trigger epithelial to mesenchymal transition that is consistently translated to the formation of uterine carcinosarcomas in vivo.

Editorial: Molecular and Cellular Crosstalk on Neuronal Functionality and Regulation, From Development to Pathology

The Wolffian Ducts (WD) are paired epithelial tubules central to the development of the mammalian genitourinary tract. Outgrowths from the WD known as the ureteric buds (UB) generate the collecting ducts of the kidney. Later during development, the caudal portion of the WD will form the vas deferens, epididymis and seminal vesicle in males, and will degenerate in females. While the genetic pathways controlling the development of the UB are firmly established, less is known about those governing development of WD portions caudal to the UB. Sprouty proteins are inhibitors of receptor tyrosine kinase (RTK) signaling in vivo. We have recently shown that homozygous mutation of a conserved tyrosine (Tyr53) of Spry1 results in UB defects indistinguishable from that of Spry1 null mice. Here we show that heterozygosity for the Spry1 Y53A allele causes caudal WD developmental defects consisting on ectopically branched seminal vesicles in males and persistent WD in females, without affecting kidney development. Detailed analysis reveals that this phenotype also occurs in Spry1 ^+/- mice but with a much lower penetrance, indicating that removal of tyrosine 53 generates a dominant negative mutation in vivo. Supporting this notion, concomitant deletion of one allele of Spry1 and Spry2 also recapitulates the genital phenotype of Spry1 ^Y53A/+ mice with high penetrance. Mechanistically, we show that unlike the effects of Spry1 in kidney development, these caudal WD defects are independent of Ret signaling, but can be completely rescued by lowering the genetic dosage of Fgf10. In conclusion, mutation of tyrosine 53 of Spry1 generates a dominant-negative allele that uncovers fine-tuning of caudal WD development by Sprouty genes.

TGF-β has a dichotomous function, acting as tumor suppressor in premalignant cells but as a tumor promoter for cancerous cells. These contradictory functions of TGF-β are caused by different cellular contexts, including both intracellular and environmental determinants. The TGF-β/SMAD and the PI3K/PTEN/AKT signal transduction pathways have an important role in the regulation of epithelial cell homeostasis and perturbations in either of these two pathways’ contributions to endometrial carcinogenesis. We have previously demonstrated that both PTEN and SMAD2/3 display tumor-suppressive functions in the endometrium, and genetic ablation of either gene results in sustained activation of PI3K/AKT signaling that suppresses TGF-β-induced apoptosis and enhances cell proliferation of mouse endometrial cells. However, the molecular and cellular effects of PTEN deficiency on TGF-β/SMAD2/3 signaling remain controversial. Here, using an in vitro and in vivo model of endometrial carcinogenesis, we have demonstrated that loss of PTEN leads to a constitutive SMAD2/3 nuclear translocation. To ascertain the function of nuclear SMAD2/3 downstream of PTEN deficiency, we analyzed the effects of double deletion PTEN and SMAD2/3 in mouse endometrial organoids. Double PTEN/SMAD2/3 ablation results in a further increase of cell proliferation and enlarged endometrial organoids compared to those harboring single PTEN, suggesting that nuclear translocation of SMAD2/3 constrains tumorigenesis induced by PTEN deficiency.

In recent decades, neurogenesis in the adult brain has been well demonstrated in a number of animal species, including humans. Interestingly, work with rodents has shown that adult neurogenesis in the dentate gyrus (DG) of the hippocampus is vital for some cognitive aspects, as increasing neurogenesis improves memory, while its disruption triggers the opposite effect. Adult neurogenesis declines with age and has been suggested to play a role in impaired progressive learning and memory loss seen in Alzheimer’s disease (AD). Therefore, therapeutic strategies designed to boost adult hippocampal neurogenesis may be beneficial for the treatment of AD. The precursor forms of neurotrophins, such as pro-NGF, display remarkable increase during AD in the hippocampus and entorhinal cortex. In contrast to mature NGF, pro-NGF exerts adverse functions in survival, proliferation, and differentiation. Hence, we hypothesized that pro-NGF and its p75 neurotrophin receptor (p75NTR) contribute to disrupting adult hippocampal neurogenesis during AD. To test this hypothesis, in this study, we took advantage of the availability of mouse models of AD (APP/PS1), which display memory impairment, and AD human samples to address the role of pro-NGF/p75NTR signaling in different aspects of adult neurogenesis. First, we observed that DG doublecortin (DCX) + progenitors express p75NTR both, in healthy humans and control animals, although the percentage of DCX+ cells are significantly reduced in AD. Interestingly, the expression of p75NTR in these progenitors is significantly decreased in AD conditions compared to controls. In order to assess the contribution of the pro-NGF/p75NTR pathway to the memory deficits of APP/PS1 mice, we injected pro-NGF neutralizing antibodies (anti-proNGF) into the DG of control and APP/PS1 mice and animals are subjected to a Morris water maze test. Intriguingly, we observed that anti-pro-NGF significantly restored memory performance of APP/PS1 animals and significantly increase the percentage of DCX+ progenitors in the DG region of these animals. In summary, our results suggest that pro-NGF is involved in disrupting spatial memory in AD, at least in part by blocking adult neurogenesis. Moreover, we propose that adult neurogenesis alteration should be taken into consideration for better understanding of AD pathology. Additionally, we provide a new molecular entry point (pro-NGF/p75NTR signaling) as a promising therapeutic target in AD.

Neuron migration is a hallmark of nervous system development that allows gathering of neurons from different origins for assembling of functional neuronal circuits. Cortical inhibitory interneurons arise in the ventral telencephalon and migrate tangentially forming three transient migratory streams in the cortex before reaching the final laminar destination. Although migration defects lead to the disruption of inhibitory circuits and are linked to aspects of psychiatric disorders such as autism and schizophrenia, the molecular mechanisms controlling cortical interneuron development and final layer positioning are incompletely understood. Here, we show that mouse embryos with a double deletion of FLRT2 and FLRT3 genes encoding cell adhesion molecules exhibit an abnormal distribution of interneurons within the streams during development, which in turn, affect the layering of somatostatin+ interneurons postnatally. Mechanistically, FLRT2 and FLRT3 proteins act in a noncell-autonomous manner, possibly through a repulsive mechanism. In support of such a conclusion, double knockouts deficient in the repulsive receptors for FLRTs, Unc5B and Unc5D, also display interneuron defects during development, similar to the FLRT2/FLRT3 mutants. Moreover, FLRT proteins are chemorepellent ligands for developing interneurons in vitro, an effect that is in part dependent on FLRT-Unc5 interaction. Together, we propose that FLRTs act through Unc5 receptors to control cortical interneuron distribution in a mechanism that involves cell repulsion. SIGNIFICANCE STATEMENT Disruption of inhibitory cortical circuits is responsible for some aspects of psychiatric disorders such as schizophrenia or autism. These defects include interneuron migration during development. A crucial step during this process is the formation of three transient migratory streams within the developing cortex that determine the timing of interneuron final positioning and the formation of functional cortical circuits in the adult. We report that FLRT proteins are required for the proper distribution of interneurons within the cortical migratory streams and for the final laminar allocation in the postnatal cortex. These results expand the multifunctional role of FLRTs during nervous system development in addition to the role of FLRTs in axon guidance and the migration of excitatory cortical neurons.