Multifaceted actions of Zeb2 in postnatal neurogenesis from the ventricular-subventricular zone to the olfactory bulb.

The transcription factor Zeb2 controls fate specification and subsequent differentiation and maturation of multiple cell types in various embryonic tissues. It binds many protein partners, including activated Smad proteins and the NuRD co-repressor complex. How Zeb2 subdomains support cell differentiation in various contexts has remained elusive. Here, we studied the role of Zeb2 and its domains in neurogenesis and neural differentiation in the young postnatal ventricular-subventricular zone (V-SVZ), in which neural stem cells generate olfactory bulb-destined interneurons. Conditional Zeb2 knockouts and separate acute loss- and gain-of-function approaches indicated that Zeb2 is essential for controlling apoptosis and neuronal differentiation of V-SVZ progenitors before and after birth, and we identified Sox6 as a potential downstream target gene of Zeb2. Zeb2 genetic inactivation impaired the differentiation potential of the V-SVZ niche in a cell-autonomous fashion. We also provide evidence that its normal function in the V-SVZ also involves non-autonomous mechanisms. Additionally, we demonstrate distinct roles for Zeb2 protein-binding domains, suggesting that Zeb2 partners co-determine neuronal output from the mouse V-SVZ in both quantitative and qualitative ways in early postnatal life.

Defective DNA Polymerase α-Primase Leads to X-Linked Intellectual Disability Associated with Severe Growth Retardation, Microcephaly, and Hypogonadism.

Replicating the human genome efficiently and accurately is a daunting challenge involving the duplication of upward of three billion base pairs. At the core of the complex machinery that achieves this task are three members of the B family of DNA polymerases: DNA polymerases α, δ, and ε. Collectively these multimeric polymerases ensure DNA replication proceeds at optimal rates approaching 2 × 103 nucleotides/min with an error rate of less than one per million nucleotides polymerized. The majority of DNA replication of undamaged DNA is conducted by DNA polymerases δ and ε. The DNA polymerase α-primase complex performs limited synthesis to initiate the replication process, along with Okazaki-fragment synthesis on the discontinuous lagging strand. An increasing number of human disorders caused by defects in different components of the DNA-replication apparatus have been described to date. These are clinically diverse and involve a wide range of features, including variable combinations of growth delay, immunodeficiency, endocrine insufficiencies, lipodystrophy, and cancer predisposition. Here, by using various complementary approaches, including classical linkage analysis, targeted next-generation sequencing, and whole-exome sequencing, we describe distinct missense and splice-impacting mutations in POLA1 in five unrelated families presenting with an X-linked syndrome involving intellectual disability, proportionate short stature, microcephaly, and hypogonadism. POLA1 encodes the p180 catalytic subunit of DNA polymerase α-primase. A range of replicative impairments could be demonstrated in lymphoblastoid cell lines derived from affected individuals. Our findings describe the presentation of pathogenic mutations in a catalytic component of a B family DNA polymerase member, DNA polymerase α.

A practical staging atlas to study embryonic development of Octopus vulgaris under controlled laboratory conditions.

BACKGROUND: Octopus vulgaris has been an iconic cephalopod species for neurobiology research as well as for cephalopod aquaculture. It is one of the most intelligent and well-studied invertebrates, possessing both long- and short-term memory and the striking ability to perform complex cognitive tasks. Nevertheless, how the common octopus developed these uncommon features remains enigmatic. O. vulgaris females spawn thousands of small eggs and remain with their clutch during their entire development, cleaning, venting and protecting the eggs. In fact, eggs incubated without females usually do not develop normally, mainly due to biological contamination (fungi, bacteria, etc.). This high level of parental care might have hampered laboratory research on the embryonic development of this intriguing cephalopod. RESULTS: Here, we present a completely parameter-controlled artificial seawater standalone egg incubation system that replaces maternal care and allows successful embryonic development of a small-egged octopus species until hatching in a laboratory environment. We also provide a practical and detailed staging atlas based on bright-field and light sheet fluorescence microscopy imaging for precise monitoring of embryonic development. The atlas has a comparative section to benchmark stages to the different scales published by Naef (1928), Arnold (1965) and Boletzky (2016). Finally, we provide methods to monitor health and wellbeing of embryos during organogenesis. CONCLUSION: Besides introducing the study of O. vulgaris embryonic development to a wider community, this work can be a high-quality reference for comparative evolutionary developmental biology.

N-cadherin specifies first asymmetry in developing neurons.

The precise polarization and orientation of developing neurons is essential for the correct wiring of the brain. In pyramidal excitatory neurons, polarization begins with the sprouting of opposite neurites, which later define directed migration and axo-dendritic domains. We here show that endogenous N-cadherin concentrates at one pole of the newborn neuron, from where the first neurite subsequently emerges. Ectopic N-cadherin is sufficient to favour the place of appearance of the first neurite. The Golgi and centrosome move towards this newly formed morphological pole in a second step, which is regulated by PI3K and the actin/microtubule cytoskeleton. Moreover, loss of function experiments in vivo showed that developing neurons with a non-functional N-cadherin misorient their cell axis. These results show that polarization of N-cadherin in the immediate post-mitotic stage is an early and crucial mechanism in neuronal polarity.

Onecut transcription factors act upstream of Isl1 to regulate spinal motoneuron diversification.

During development, spinal motoneurons (MNs) diversify into a variety of subtypes that are specifically dedicated to the motor control of particular sets of skeletal muscles or visceral organs. MN diversification depends on the coordinated action of several transcriptional regulators including the LIM-HD factor Isl1, which is crucial for MN survival and fate determination. However, how these regulators cooperate to establish each MN subtype remains poorly understood. Here, using phenotypic analyses of single or compound mutant mouse embryos combined with gain-of-function experiments in chick embryonic spinal cord, we demonstrate that the transcriptional activators of the Onecut family critically regulate MN subtype diversification during spinal cord development. We provide evidence that Onecut factors directly stimulate Isl1 expression in specific MN subtypes and are therefore required to maintain Isl1 production at the time of MN diversification. In the absence of Onecut factors, we observed major alterations in MN fate decision characterized by the conversion of somatic to visceral MNs at the thoracic levels of the spinal cord and of medial to lateral MNs in the motor columns that innervate the limbs. Furthermore, we identify Sip1 (Zeb2) as a novel developmental regulator of visceral MN differentiation. Taken together, these data elucidate a comprehensive model wherein Onecut factors control multiple aspects of MN subtype diversification. They also shed light on the late roles of Isl1 in MN fate decision.

A complex Xp11.22 deletion in a patient with syndromic autism: exploration of FAM120C as a positional candidate gene for autism.

We present a male patient with sporadic Aarskog syndrome, cleft palate, mild intellectual disability, and autism spectrum disorder (ASD). A submicroscopic discontiguous deletion was detected on chromosome Xp11.2 encompassing FGD1, FAM120C, and PHF8. That the deletion encompassed FGD1 (exons 2-8) explains the Aarskog features while the deletion of PHF8 most likely explains the cleft palate and mild intellectual disability. We identify FAM120C as a novel X-linked candidate gene for autism for two reasons: first, a larger deletion encompassing FAM120C segregates with autism in a previously reported family and second, there is recent evidence that FAM120C interacts with CYFIP1, part of the FMRP (Fragile X Mental Retardation Protein) network. In the current study, resequencing of FAM120C in 87 Belgian male patients with autism spectrum disorder identified no novel mutations. Expression of Fam120c in mouse tissues showed enriched expression in pituitary, cerebellum, cortex, and pancreatic islets of Langerhans. Additionally, we found a cortical expression pattern of Fam120c similar to that of Fmr1. In conclusion, FAM120C is a novel candidate gene for autism spectrum disorder based on genetic evidence and the brain expression pattern. Thereby we highlight a role for FMRP network genes in ASD.

Embryonic development of a centralised brain in coleoid cephalopods.

The last common ancestor of cephalopods and vertebrates lived about 580 million years ago, yet coleoid cephalopods, comprising squid, cuttlefish and octopus, have evolved an extraordinary behavioural repertoire that includes learned behaviour and tool utilization. These animals also developed innovative advanced defence mechanisms such as camouflage and ink release. They have evolved unique life cycles and possess the largest invertebrate nervous systems. Thus, studying coleoid cephalopods provides a unique opportunity to gain insights into the evolution and development of large centralised nervous systems. As non-model species, molecular and genetic tools are still limited. However, significant insights have already been gained to deconvolve embryonic brain development. Even though coleoid cephalopods possess a typical molluscan circumesophageal bauplan for their central nervous system, aspects of its development are reminiscent of processes observed in vertebrates as well, such as long-distance neuronal migration. This review provides an overview of embryonic coleoid cephalopod research focusing on the cellular and molecular aspects of neurogenesis, migration and patterning. Additionally, we summarize recent work on neural cell type diversity in embryonic and hatchling cephalopod brains. We conclude by highlighting gaps in our knowledge and routes for future research.

Single-cell sequencing unveils the impact of aging on the progenitor cell diversity in the telencephalon of the female killifish N. furzeri.

The African turquoise killifish (Nothobranchius furzeri) combines a short lifespan with spontaneous age-associated loss of neuro-regenerative capacity, an intriguing trait atypical for a teleost. The impact of aging on the cellular composition of the adult stem cell niches, leading to this dramatic decline in the postnatal neuro- and gliogenesis, remains elusive. Single-cell RNA sequencing of the telencephalon of young adult female killifish of the short-lived GRZ-AD strain unveiled progenitors of glial and non-glial nature, different excitatory and inhibitory neuron subtypes, as well as non-neural cell types. Sub-clustering of the progenitors identified four radial glia (RG) cell types, two non-glial progenitor (NGP) and four intermediate (intercell) cell states. Two astroglia-like, one ependymal, and one neuroepithelial-like (NE) RG subtype were found at different locations in the forebrain in line with their role, while proliferative, active NGPs were spread throughout. Lineage inference pointed to NE-RG and NGPs as start and intercessor populations for glio- and neurogenesis. Upon aging, single-cell RNA sequencing revealed major perturbations in the proportions of the astroglia and intercell states, and in the molecular signatures of specific subtypes, including altered MAPK, mTOR, Notch, and Wnt pathways. This cell catalog of the young regeneration-competent killifish telencephalon, combined with the evidence for aging-related transcriptomic changes, presents a useful resource to understand the molecular basis of age-dependent neuroplasticity. This data is also available through an online database (killifishbrain_scseq).

Altered socio-affective communication and amygdala development in mice with protocadherin10-deficient interneurons.

Autism spectrum disorder (ASD) is a group of neurodevelopmental conditions associated with deficits in social interaction and communication, together with repetitive behaviours. The cell adhesion molecule protocadherin10 (PCDH10) is linked to ASD in humans. Pcdh10 is expressed in the nervous system during embryonic and early postnatal development and is important for neural circuit formation. In mice, strong expression of Pcdh10 in the ganglionic eminences and in the basolateral complex (BLC) of the amygdala was observed at mid and late embryonic stages, respectively. Both inhibitory and excitatory neurons expressed Pcdh10 in the BLC at perinatal stages and vocalization-related genes were enriched in Pcdh10-expressing neurons in adult mice. An epitope-tagged Pcdh10-HAV5 mouse line revealed endogenous interactions of PCDH10 with synaptic proteins in the young postnatal telencephalon. Nuanced socio-affective communication changes in call emission rates, acoustic features and call subtype clustering were primarily observed in heterozygous pups of a conditional knockout (cKO) with selective deletion of Pcdh10 in Gsh2-lineage interneurons. These changes were less prominent in heterozygous ubiquitous Pcdh10 KO pups, suggesting that altered anxiety levels associated with Gsh2-lineage interneuron functioning might drive the behavioural effects. Together, loss of Pcdh10 specifically in interneurons contributes to behavioural alterations in socio-affective communication with relevance to ASD.

The EMT regulator Zeb2/Sip1 is essential for murine embryonic hematopoietic stem/progenitor cell differentiation and mobilization.

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs), and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo, but is essential for normal HSC/HPC differentiation. In addition, Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased ß1 integrin and Cxcr4 expression. Moreover, deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and, subsequently, improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult.

Molecular and Biochemical Analyses of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish.

As modern society is graying, aging research and biogerontology models, in which the aging process can be studied, are becoming increasingly important. A proper aging model can be defined as one that displays many of the aging hallmarks. Here, we provide two different practical approaches-namely, real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting-that can be used to investigate cellular senescence (RT-qPCR for p21 and p27), altered intercellular communication/inflammaging (RT-qPCR for il-10, sirt-1, il-6, il-1b, il-8, and tnf), and oxidative stress (western blotting for 4-HNE) in the killifish central nervous system, and, more specifically, in the retina, optic tectum, and telencephalon. These molecular and biochemical analyses are a first step in confirming the aging characteristics but should preferably be combined with morphological analyses.

A short dasatinib and quercetin treatment is sufficient to reinstate potent adult neuroregenesis in the aged killifish.

The young African turquoise killifish has a high regenerative capacity, but loses it with advancing age, adopting several aspects of the limited form of mammalian regeneration. We deployed a proteomic strategy to identify pathways that underpin the loss of regenerative power caused by aging. Cellular senescence stood out as a potential brake on successful neurorepair. We applied the senolytic cocktail Dasatinib and Quercetin (D + Q) to test clearance of chronic senescent cells from the aged killifish central nervous system (CNS) as well as rebooting the neurogenic output. Our results show that the entire aged killifish telencephalon holds a very high senescent cell burden, including the parenchyma and the neurogenic niches, which could be diminished by a short-term, late-onset D + Q treatment. Reactive proliferation of non-glial progenitors increased substantially and lead to restorative neurogenesis after traumatic brain injury. Our results provide a cellular mechanism for age-related regeneration resilience and a proof-of-concept of a potential therapy to revive the neurogenic potential in an already aged or diseased CNS.

Neuroanatomical characterization of the Nmu-Cre knock-in mice reveals an interconnected network of unique neuropeptidergic cells.

Neuromedin U (NMU) is an evolutionary conserved neuropeptide that has been implicated in multiple processes, such as circadian regulation, energy homeostasis, reward processing and stress coping. Although the central expression of NMU has been addressed previously, the lack of specific and sensitive tools has prevented a comprehensive characterization of NMU-expressing neurons in the brain. We have generated a knock-in mouse model constitutively expressing Cre recombinase under the Nmu promoter. We have validated the model using a multi-level approach based on quantitative reverse-transcription polymerase chain reactions, in situ hybridization, a reporter mouse line and an adenoviral vector driving Cre-dependent expression of a fluorescent protein. Using the Nmu-Cre mouse, we performed a complete characterization of NMU expression in adult mouse brain, unveiling a potential midline NMU modulatory circuit with the ventromedial hypothalamic nucleus (VMH) as a key node. Moreover, immunohistochemical analysis suggested that NMU neurons in the VMH mainly constitute a unique population of hypothalamic cells. Taken together, our results suggest that Cre expression in the Nmu-Cre mouse model largely reflects NMU expression in the adult mouse brain, without altering endogenous NMU expression. Thus, the Nmu-Cre mouse model is a powerful and sensitive tool to explore the role of NMU neurons in mice.

A chromosome-level reference genome for the common octopus, Octopus vulgaris (Cuvier, 1797).

Cephalopods are emerging animal models and include iconic species for studying the link between genomic innovations and physiological and behavioral complexities. Coleoid cephalopods possess the largest nervous system among invertebrates, both for cell counts and brain-to-body ratio. Octopus vulgaris has been at the center of a long-standing tradition of research into diverse aspects of cephalopod biology, including behavioral and neural plasticity, learning and memory recall, regeneration, and sophisticated cognition. However, no chromosome-scale genome assembly was available for O. vulgaris to aid in functional studies. To fill this gap, we sequenced and assembled a chromosome-scale genome of the common octopus, O. vulgaris. The final assembly spans 2.8 billion basepairs, 99.34% of which are in 30 chromosome-scale scaffolds. Hi-C heatmaps support a karyotype of 1n = 30 chromosomes. Comparisons with other octopus species' genomes show a conserved octopus karyotype and a pattern of local genome rearrangements between species. This new chromosome-scale genome of O. vulgaris will further facilitate research in all aspects of cephalopod biology, including various forms of plasticity and the neural machinery underlying sophisticated cognition, as well as an understanding of cephalopod evolution.

Cephalopod-omics: Emerging Fields and Technologies in Cephalopod Biology.

Few animal groups can claim the level of wonder that cephalopods instill in the minds of researchers and the general public. Much of cephalopod biology, however, remains unexplored: the largest invertebrate brain, difficult husbandry conditions, and complex (meta-)genomes, among many other things, have hindered progress in addressing key questions. However, recent technological advancements in sequencing, imaging, and genetic manipulation have opened new avenues for exploring the biology of these extraordinary animals. The cephalopod molecular biology community is thus experiencing a large influx of researchers, emerging from different fields, accelerating the pace of research in this clade. In the first post-pandemic event at the Cephalopod International Advisory Council (CIAC) conference in April 2022, over 40 participants from all over the world met and discussed key challenges and perspectives for current cephalopod molecular biology and evolution. Our particular focus was on the fields of comparative and regulatory genomics, gene manipulation, single-cell transcriptomics, metagenomics, and microbial interactions. This article is a result of this joint effort, summarizing the latest insights from these emerging fields, their bottlenecks, and potential solutions. The article highlights the interdisciplinary nature of the cephalopod-omics community and provides an emphasis on continuous consolidation of efforts and collaboration in this rapidly evolving field.

Optimization of Whole Mount RNA Multiplexed in situ Hybridization Chain Reaction With Immunohistochemistry, Clearing and Imaging to Visualize Octopus Embryonic Neurogenesis.

Gene expression analysis has been instrumental to understand the function of key factors during embryonic development of many species. Marker analysis is also used as a tool to investigate organ functioning and disease progression. As these processes happen in three dimensions, the development of technologies that enable detection of gene expression in the whole organ or embryo is essential. Here, we describe an optimized protocol of whole mount multiplexed RNA in situ hybridization chain reaction version 3.0 (HCR v3.0) in combination with immunohistochemistry (IHC), followed by fructose-glycerol clearing and light sheet fluorescence microscopy (LSFM) imaging on Octopus vulgaris embryos. We developed a code to automate probe design which can be applied for designing HCR v3.0 type probe pairs for fluorescent in situ mRNA visualization. As proof of concept, neuronal (Ov-elav) and glial (Ov-apolpp) markers were used for multiplexed HCR v3.0. Neural progenitor (Ov-ascl1) and precursor (Ov-neuroD) markers were combined with immunostaining for phosphorylated-histone H3, a marker for mitosis. After comparing several tissue clearing methods, fructose-glycerol clearing was found optimal in preserving the fluorescent signal of HCR v3.0. The expression that was observed in whole mount octopus embryos matched with the previous expression data gathered from paraffin-embedded transverse sections. Three-dimensional reconstruction revealed additional spatial organization that had not been discovered using two-dimensional methods.

Modifying PCDH19 levels affects cortical interneuron migration.

PCDH19 is a transmembrane protein and member of the protocadherin family. It is encoded by the X-chromosome and more than 200 mutations have been linked to the neurodevelopmental PCDH-clustering epilepsy (PCDH19-CE) syndrome. A disturbed cell-cell contact that arises when random X-inactivation creates mosaic absence of PCDH19 has been proposed to cause the syndrome. Several studies have shown roles for PCDH19 in neuronal proliferation, migration, and synapse function, yet most of them have focused on cortical and hippocampal neurons. As epilepsy can also be caused by impaired interneuron migration, we studied the role of PCDH19 in cortical interneurons during embryogenesis. We show that cortical interneuron migration is affected by altering PCDH19 dosage by means of overexpression in brain slices and medial ganglionic eminence (MGE) explants. We also detect subtle defects when PCDH19 expression was reduced in MGE explants, suggesting that the dosage of PCDH19 is important for proper interneuron migration. We confirm this finding in vivo by showing a mild reduction in interneuron migration in heterozygote, but not in homozygote PCDH19 knockout animals. In addition, we provide evidence that subdomains of PCDH19 have a different impact on cell survival and interneuron migration. Intriguingly, we also observed domain-dependent differences in migration of the non-targeted cell population in explants, demonstrating a non-cell-autonomous effect of PCDH19 dosage changes. Overall, our findings suggest new roles for the extracellular and cytoplasmic domains of PCDH19 and support that cortical interneuron migration is dependent on balanced PCDH19 dosage.

MicroRNAs are deeply linked to the emergence of the complex octopus brain.

Soft-bodied cephalopods such as octopuses are exceptionally intelligent invertebrates with a highly complex nervous system that evolved independently from vertebrates. Because of elevated RNA editing in their nervous tissues, we hypothesized that RNA regulation may play a major role in the cognitive success of this group. We thus profiled messenger RNAs and small RNAs in three cephalopod species including 18 tissues of the Octopus vulgaris. We show that the major RNA innovation of soft-bodied cephalopods is an expansion of the microRNA (miRNA) gene repertoire. These evolutionarily novel miRNAs were primarily expressed in adult neuronal tissues and during the development and had conserved and thus likely functional target sites. The only comparable miRNA expansions happened, notably, in vertebrates. Thus, we propose that miRNAs are intimately linked to the evolution of complex animal brains.

Cell type diversity in a developing octopus brain.

Octopuses are mollusks that have evolved intricate neural systems comparable with vertebrates in terms of cell number, complexity and size. The brain cell types that control their sophisticated behavioral repertoire are still unknown. Here, we profile the cell diversity of the paralarval Octopus vulgaris brain to build a cell type atlas that comprises mostly neural cells, but also multiple glial subtypes, endothelial cells and fibroblasts. We spatially map cell types to the vertical, subesophageal and optic lobes. Investigation of cell type conservation reveals a shared gene signature between glial cells of mouse, fly and octopus. Genes related to learning and memory are enriched in vertical lobe cells, which show molecular similarities with Kenyon cells in Drosophila. We construct a cell type taxonomy revealing transcriptionally related cell types, which tend to appear in the same brain region. Together, our data sheds light on cell type diversity and evolution in the octopus brain.

Novel Perspectives on the Development of the Amygdala in Rodents.

The amygdala is a hyperspecialized brain region composed of strongly inter- and intraconnected nuclei involved in emotional learning and behavior. The cellular heterogeneity of the amygdalar nuclei has complicated straightforward conclusions on their developmental origin, and even resulted in contradictory data. Recently, the concentric ring theory of the pallium and the radial histogenetic model of the pallial amygdala have cleared up several uncertainties that plagued previous models of amygdalar development. Here, we provide an extensive overview on the developmental origin of the nuclei of the amygdaloid complex. Starting from older gene expression data, transplantation and lineage tracing studies, we systematically summarize and reinterpret previous findings in light of the novel perspectives on amygdalar development. In addition, migratory routes that these cells take on their way to the amygdala are explored, and known transcription factors and guidance cues that seemingly drive these cells toward the amygdala are emphasized. We propose some future directions for research on amygdalar development and highlight that a better understanding of its development could prove critical for the treatment of several neurodevelopmental and neuropsychiatric disorders.