Generation of adult hippocampal neural stem cells occurs in the early postnatal dentate gyrus and depends on cyclin D2.

Lifelong hippocampal neurogenesis is maintained by a pool of multipotent adult neural stem cells (aNSCs) residing in the subgranular zone of the dentate gyrus (DG). The mechanisms guiding transition of NSCs from the developmental to the adult state remain unclear. We show here, by using nestin-based reporter mice deficient for cyclin D2, that the aNSC pool is established through cyclin D2-dependent proliferation during the first two weeks of life. The absence of cyclin D2 does not affect normal development of the dentate gyrus until birth but prevents postnatal formation of radial glia-like aNSCs. Furthermore, retroviral fate mapping reveals that aNSCs are born on-site from precursors located in the dentate gyrus shortly after birth. Taken together, our data identify the critical time window and the spatial location of the precursor divisions that generate the persistent population of aNSCs and demonstrate the central role of cyclin D2 in this process.

BirthSeq, a new method to isolate and analyze dated cells in different vertebrates.

Embryonic development is a complex and dynamic process that unfolds over time and involves the production and diversification of increasing numbers of cells. The impact of developmental time on the formation of the central nervous system is well-documented, with evidence showing that time plays a critical role in establishing the identity of neuronal subtypes. However, the study of how time translates into genetic instructions driving cell fate is limited by the scarcity of suitable experimental tools. We introduce BirthSeq, a new method for isolating and analyzing cells based on their birth date. This innovative technique allows for in vivo labeling of cells, isolation via FACS, and analysis using high-throughput techniques. We tuned up BirthSeq in developmental organs across three vertebrate species (mouse, chick, and gecko), and fully made use of it for single-cell RNA sequencing and novel spatially resolved transcriptomic approaches in mouse and chick, respectively. Overall, BirthSeq provides a versatile tool for studying virtually any tissue in different vertebrate organism, helping to fill the necessity in developmental biology research by targeting cells and their temporal cues.

The Phylotypic Brain of Vertebrates, from Neural Tube Closure to Brain Diversification.

BACKGROUND: The phylotypic or intermediate stages are thought to be the most evolutionary conserved stages throughout embryonic development. The contrast with divergent early and later stages derived from the concept of the evo-devo hourglass model. Nonetheless, this developmental constraint has been studied as a whole embryo process, not at organ level. In this review, we explore brain development to assess the existence of an equivalent brain developmental hourglass. In the specific case of vertebrates, we propose to split the brain developmental stages into: (1) Early: Neurulation, when the neural tube arises after gastrulation. (2) Intermediate: Brain patterning and segmentation, when the neuromere identities are established. (3) Late: Neurogenesis and maturation, the stages when the neurons acquire their functionality. Moreover, we extend this analysis to other chordates brain development to unravel the evolutionary origin of this evo-devo constraint. SUMMARY: Based on the existing literature, we hypothesise that a major conservation of the phylotypic brain might be due to the pleiotropy of the inductive regulatory networks, which are predominantly expressed at this stage. In turn, earlier stages such as neurulation are rather mechanical processes, whose regulatory networks seem to adapt to environment or maternal geometries. The later stages are also controlled by inductive regulatory networks, but their effector genes are mostly tissue-specific and functional, allowing diverse developmental programs to generate current brain diversity. Nonetheless, all stages of the hourglass are highly interconnected: divergent neurulation must have a vertebrate shared end product to reproduce the vertebrate phylotypic brain, and the boundaries and transcription factor code established during the highly conserved patterning will set the bauplan for the specialised and diversified adult brain. KEY MESSAGES: The vertebrate brain is conserved at phylotypic stages, but the highly conserved mechanisms that occur during these brain mid-development stages (Inducing Regulatory Networks) are also present during other stages. Oppositely, other processes as cell interactions and functional neuronal genes are more diverse and majoritarian in early and late stages of development, respectively. These phenomena create an hourglass of transcriptomic diversity during embryonic development and evolution, with a really conserved bottleneck that set the bauplan for the adult brain around the phylotypic stage.

MEF2 transcription factors are key regulators of sprouting angiogenesis.

Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.

Time in Neurogenesis: Conservation of the Developmental Formation of the Cerebellar Circuitry.

The cerebellum is a conserved structure of vertebrate brains that develops at the most anterior region of the alar rhombencephalon. All vertebrates display a cerebellum, making it one of the most highly conserved structures of the brain. Although it greatly varies at the morphological level, several lines of research point to strong conservation of its internal neural circuitry. To test the conservation of the cerebellar circuit, we compared the developmental history of the neurons comprising this circuit in three amniote species: mouse, chick, and gecko. We specifically researched the developmental time of generation of the main neuronal types of the cerebellar cortex. This developmental trajectory is known for the mammalian cell types but barely understood for sauropsid species. We show that the neurogenesis of the GABAergic lineage proceeds following the same chronological sequence in the three species compared: Purkinje cells are the first ones generated in the cerebellar cortex, followed by Golgi interneurons of the granule cell layer, and lately by the interneurons of the molecular layer. In the cerebellar glutamatergic lineage, we observed the same conservation of neurogenesis throughout amniotes, and the same vastly prolonged neurogenesis of granule cells, extending much further than for any other brain region. Together these data show that the cerebellar circuitry develops following a tightly conserved chronological sequence of neurogenesis, which is responsible for the preservation of the cerebellum and its function. Our data reinforce the developmental perspective of homology, whereby similarities in neurons and circuits are likely due to similarities in developmental sequence.

Loss of Dmrt5 Affects the Formation of the Subplate and Early Corticogenesis.

Dmrt5 (Dmrta2) and Dmrt3 are key regulators of cortical patterning and progenitor proliferation and differentiation. In this study, we show an altered apical to intermediate progenitor transition, with a delay in SP neurogenesis and premature birth of Ctip2+ cortical neurons in Dmrt5-/- mice. In addition to the cortical progenitors, DMRT5 protein appears present in postmitotic subplate (SP) and marginal zone neurons together with some migrating cortical neurons. We observed the altered split of preplate and the reduced SP and disturbed radial migration of cortical neurons into cortical plate in Dmrt5-/- brains and demonstrated an increase in the proportion of multipolar cells in primary neuronal cultures from Dmrt5-/- embryonic brains. Dmrt5 affects cortical development with specific time sensitivity that we described in two conditional mice with slightly different deletion time. We only observed a transient SP phenotype at E15.5, but not by E18.5 after early (Dmrt5lox/lox;Emx1Cre), but not late (Dmrt5lox/lox;NestinCre) deletion of Dmrt5. SP was less disturbed in Dmrt5lox/lox;Emx1Cre and Dmrt3-/- brains than in Dmrt5-/- and affects dorsomedial cortex more than lateral and caudal cortex. Our study demonstrates a novel function of Dmrt5 in the regulation of early SP formation and radial cortical neuron migration. SUMMARY STATEMENT: Our study demonstrates a novel function of Dmrt5 in regulating marginal zone and subplate formation and migration of cortical neurons to cortical plate.

Update on forebrain evolution: From neurogenesis to thermogenesis.

Comparative developmental studies provide growing understanding of vertebrate forebrain evolution. This short review directs the spotlight to some newly emerging aspects, including the evolutionary origin of the proliferative region known as the subventricular zone (SVZ) and of intermediate progenitor cells (IPCs) that populate the SVZ, neural circuits that originated within homologous regions across all amniotes, and the role of thermogenesis in the acquisition of an increased brain size. These data were presented at the 8th European Conference on Comparative Neurobiology.

Mathematical Modeling of Cortical Neurogenesis Reveals that the Founder Population does not Necessarily Scale with Neurogenic Output.

The mammalian cerebral neocortex has a unique structure, composed of layers of different neuron types, interconnected in a stereotyped fashion. While the overall developmental program seems to be conserved, there are divergent developmental factors generating cortical diversity amongst species. In terms of cortical neuronal numbers, some of the determining factors are the size of the founder population, the duration of cortical neurogenesis, the proportion of different progenitor types, and the fine-tuned balance between self-renewing and differentiative divisions. We develop a mathematical model of neurogenesis that, accounting for these factors, aims at explaining the high diversity in neuronal numbers found across species. By framing our hypotheses in rigorous mathematical terms, we are able to identify paths of neurogenesis that match experimentally observed patterns in mouse, macaque and human. Additionally, we use our model to identify key parameters that would particularly benefit from accurate experimental investigation. We find that the timing of a switch in favor of symmetric neurogenic divisions produces the highest variation in cortical neuronal numbers. Surprisingly, assuming similar cell cycle lengths in primate progenitors, the increase in cortical neuronal numbers does not reflect a larger size of founder population, a prediction that has identified a specific need for experimental quantifications.

Subset of early radial glial progenitors that contribute to the development of callosal neurons is absent from avian brain.

The classical view of mammalian cortical development suggests that pyramidal neurons are generated in a temporal sequence, with all radial glial cells (RGCs) contributing to both lower and upper neocortical layers. A recent opposing proposal suggests there is a subgroup of fate-restricted RGCs in the early neocortex, which generates only upper-layer neurons. Little is known about the existence of fate restriction of homologous progenitors in other vertebrate species. We investigated the lineage of selected Emx2+ [vertebrate homeobox gene related to Drosophila empty spiracles (ems)] RGCs in mouse neocortex and chick forebrain and found evidence for both sequential and fate-restricted programs only in mouse, indicating that these complementary populations coexist in the developing mammalian but not avian brain. Among a large population of sequentially programmed RGCs in the mouse brain, a subset of self-renewing progenitors lack neurogenic potential during the earliest phase of corticogenesis. After a considerable delay, these progenitors generate callosal upper-layer neurons and glia. On the other hand, we found no homologous delayed population in any sectors of the chick forebrain. This finding suggests that neurogenic delay of selected RGCs may be unique to mammals and possibly associated with the evolution of the corpus callosum.

CLoNe is a new method to target single progenitors and study their progeny in mouse and chick.

Cell lineage analysis enables us to address pivotal questions relating to: the embryonic origin of cells and sibling cell relationships in the adult body; the contribution of progenitors activated after trauma or disease; and the comparison across species in evolutionary biology. To address such fundamental questions, several techniques for clonal labelling have been developed, each with its shortcomings. Here, we report a novel method, CLoNe that is designed to work in all vertebrate species and tissues. CLoNe uses a cocktail of labelling, targeting and transposition vectors that enables targeting of specific subpopulations of progenitor types with a combination of fluorophores resulting in multifluorescence that describes multiple clones per specimen. Furthermore, transposition into the genome ensures the longevity of cell labelling. We demonstrate the robustness of this technique in mouse and chick forebrain development, and show evidence that CLoNe will be broadly applicable to study clonal relationships in different tissues and species.

Cortical and Clonal Contribution of Tbr2 Expressing Progenitors in the Developing Mouse Brain.

The individual contribution of different progenitor subtypes towards the mature rodent cerebral cortex is not fully understood. Intermediate progenitor cells (IPCs) are key to understanding the regulation of neuronal number during cortical development and evolution, yet their exact contribution is much debated. Intermediate progenitors in the cortical subventricular zone are defined by expression of T-box brain-2 (Tbr2). In this study we demonstrate by using the Tbr2(Cre) mouse line and state-of-the-art cell lineage labeling techniques, that IPC derived cells contribute substantial proportions 67.5% of glutamatergic but not GABAergic or astrocytic cells to all cortical layers including the earliest generated subplate zone. We also describe the laminar dispersion of clonally derived cells from IPCs using a recently described clonal analysis tool (CLoNe) and show that pair-generated cells in different layers cluster closer (142.1 ± 76.8 µm) than unrelated cells (294.9 ± 105.4 µm). The clonal dispersion from individual Tbr2 positive intermediate progenitors contributes to increasing the cortical surface. Our study also describes extracortical contributions from Tbr2+ progenitors to the lateral olfactory tract and ventromedial hypothalamic nucleus.

Adult pallium transcriptomes surprise in not reflecting predicted homologies across diverse chicken and mouse pallial sectors.

The thorniest problem in comparative neurobiology is the identification of the particular brain region of birds and reptiles that corresponds to the mammalian neocortex [Butler AB, Reiner A, Karten HJ (2011) Ann N Y Acad Sci 1225:14-27; Wang Y, Brzozowska-Prechtl A, Karten HJ (2010) Proc Natl Acad Sci USA 107(28):12676-12681]. We explored which genes are actively transcribed in the regions of controversial ancestry in a representative bird (chicken) and mammal (mouse) at adult stages. We conducted four analyses comparing the expression patterns of their 5,130 most highly expressed one-to-one orthologous genes that considered global patterns of expression specificity, strong gene markers, and coexpression networks. Our study demonstrates transcriptomic divergence, plausible convergence, and, in two exceptional cases, conservation between specialized avian and mammalian telencephalic regions. This large-scale study potentially resolves the complex relationship between developmental homology and functional characteristics on the molecular level and settles long-standing evolutionary debates.

Compartmentalization of cerebral cortical germinal zones in a lissencephalic primate and gyrencephalic rodent.

Previous studies of macaque and human cortices identified cytoarchitectonically distinct germinal zones; the ventricular zone inner subventricular zone (ISVZ), and outer subventricular zone (OSVZ). To date, the OSVZ has only been described in gyrencephalic brains, separated from the ISVZ by an inner fiber layer and considered a milestone that triggered increased neocortical neurogenesis. However, this observation has only been assessed in a handful of species without the identification of the different progenitor populations. We examined the Amazonian rodent agouti (Dasyprocta agouti) and the marmoset monkey (Callithrix jacchus) to further understand relationships among progenitor compartmentalization, proportions of various cortical progenitors, and degree of cortical folding. We identified a similar cytoarchitectonic distinction between the OSVZ and ISVZ at midgestation in both species. In the marmoset, we quantified the ventricular and abventricular divisions and observed similar proportions as previously described for the human and ferret brains. The proportions of radial glia, intermediate progenitors, and outer radial glial cell (oRG) populations were similar in midgestation lissencephalic marmoset as in gyrencephalic human or ferret. Our findings suggest that cytoarchitectonic subdivisions of SVZ are an evolutionary trend and not a primate specific feature, and a large population of oRG can be seen regardless of cortical folding.

Gene expression analysis of the embryonic subplate.

The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of thalamocortical axons. At later developmental stages, they are predominantly involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. We aimed to further characterize the embryonic murine subplate population by establishing a gene expression profile at embryonic day (E) 15.5 using laser capture microdissection and microarrays. The microarray identified over 300 transcripts with higher expression in the subplate compared with the cortical plate at this stage. Using quantitative reverse transcription-polymerase chain reaction, in situ hybridization (ISH), and immunohistochemistry (IHC), we have confirmed specific expression in the E15.5 subplate for 13 selected genes, which have not been previously associated with this compartment (Abca8a, Cdh10, Cdh18, Csmd3, Gabra5, Kcnt2, Ogfrl1, Pls3, Rcan2, Sv2b, Slc8a2, Unc5c, and Zdhhc2). In the reeler mutant, the expression of the majority of these genes (9 of 13) was shifted in accordance with the altered position of subplate. These genes belong to several functional groups and likely contribute to synapse formation and axonal growth and guidance in subplate cells.

Functional evolutionary convergence of long noncoding RNAs involved in embryonic development.

Long noncoding RNAs have been identified in most vertebrates, but the functional characterization of these molecules is challenging, mainly due to the lack of linear sequence homology between species. In this work, we aimed to find functional evolutionary convergent lncRNAs involved in development by screening of k-mer content (nonlinear similarity) and secondary structure-based approaches combining in silico, in vitro and in vivo validation analysis. From the Madagascar gecko genes, we have found a non-orthologous lncRNA with a similar k-mer content and structurally concordant with the human lncRNA EVX1AS. Analysis of function-related characteristics together with locus-specific targeting of human EVX1AS and gecko EVX1AS-like (i.e., CRISPR Display) in human neuroepithelial cells and chicken mesencephalon have confirmed that gecko EVX1AS-like lncRNA mimics human EVX1AS function and induces EVX1 expression independently of the target species. Our data shows functional convergence of non-homologous lncRNAs and presents a useful approach for the definition and manipulation of lncRNA function within different model organisms.

Non-uniform temporal scaling of developmental processes in the mammalian cortex.

The time that it takes the brain to develop is highly variable across animals. Although staging systems equate major developmental milestones between mammalian species, it remains unclear how distinct processes of cortical development scale within these timeframes. Here, we compare the timing of cortical development in two mammals of similar size but different developmental pace: eutherian mice and marsupial fat-tailed dunnarts. Our results reveal that the temporal relationship between cell birth and laminar specification aligns to equivalent stages between these species, but that migration and axon extension do not scale uniformly according to the developmental stages, and are relatively more advanced in dunnarts. We identify a lack of basal intermediate progenitor cells in dunnarts that likely contributes in part to this timing difference. These findings demonstrate temporal limitations and differential plasticity of cortical developmental processes between similarly sized Therians and provide insight into subtle temporal changes that may have contributed to the early diversification of the mammalian brain.

Comparative aspects of subplate zone studied with gene expression in sauropsids and mammals.

There is currently a debate about the evolutionary origin of the earliest generated cortical preplate neurons and their derivatives (subplate and marginal zone). We examined the subplate with murine markers including nuclear receptor related 1 (Nurr1), monooxygenase Dbh-like 1 (Moxd1), transmembrane protein 163 (Tmem163), and connective tissue growth factor (Ctgf) in developing and adult turtle, chick, opossum, mouse, and rat. Whereas some of these are expressed in dorsal pallium in all species studied (Nurr1, Ctgf, and Tmem163), we observed that the closely related mouse and rat differed in the expression patterns of several others (Dopa decarboxylase, Moxd1, and thyrotropin-releasing hormone). The expression of Ctgf, Moxd1, and Nurr1 in the oppossum suggests a more dispersed subplate population in this marsupial compared with mice and rats. In embryonic and adult chick brains, our selected subplate markers are primarily expressed in the hyperpallium and in the turtle in the main cell dense layer of the dorsal cortex. These observations suggest that some neurons that express these selected markers were present in the common ancestor of sauropsids and mammals.

LIM-homeobox gene Lhx5 is required for normal development of Cajal-Retzius cells.

Cajal-Retzius (C-R) cells play important roles in the lamination of the mammalian cortex via reelin secretion. The genetic mechanisms underlying the development of these neurons have just begun to be unraveled. Here, we show that two closely related LIM-homeobox genes Lhx1 and Lhx5 are expressed in reelin+ cells in various regions in the mouse telencephalon at or adjacent to sites where the C-R cells are generated, including the cortical hem, the mantle region of the septal/retrobulbar area, and the ventral pallium. Whereas Lhx5 is expressed in all of these reelin-expressing domains, Lhx1 is preferentially expressed in the septal area and in a continuous domain spanning from lateral olfactory region to caudomedial territories. Genetic ablation of Lhx5 results in decreased reelin+ and p73+ cells in the neocortical anlage, in the cortical hem, and in the septal, olfactory, and caudomedial telencephalic regions. The overall reduction in number of C-R cells in Lhx5 mutants is accompanied by formation of ectopic reelin+ cell clusters at the caudal telencephalon. Based on differential expression of molecular markers and by fluorescent cell tracing in cultured embryos, we located the origin of reelin+ ectopic cell clusters at the caudomedial telencephalic region. We also confirmed the existence of a normal migration stream of reelin+ cells from the caudomedial area to telencephalic olfactory territories in wild-type embryos. These results reveal a complex role for Lhx5 in regulating the development and normal distribution of C-R cells in the developing forebrain.

Virtual meeting, real and sound science: report of the 17th Meeting of the Spanish Society for Developmental Biology (SEBD-2020).

The Spanish Society for Developmental Biology (SEBD) organized its 17th meeting in November 2020 (herein referred to as SEBD2020). This meeting, originally programmed to take place in the city of Bilbao, was forced onto an online format due to the SARS-CoV2, COVID-19 pandemic. Although, we missed the live personal interactions and missed out on the Bilbao social scene, we were able to meet online to present our work and discuss our latest results. An overview of the activities that took place around the meeting, the different scientific sessions and the speakers involved are presented here. The pros and cons of virtual meetings are discussed.