LINE-1 retrotransposons contribute to mouse PV interneuron development.

Retrotransposons are mobile DNA sequences duplicated via transcription and reverse transcription of an RNA intermediate. Cis-regulatory elements encoded by retrotransposons can also promote the transcription of adjacent genes. Somatic LINE-1 (L1) retrotransposon insertions have been detected in mammalian neurons. It is, however, unclear whether L1 sequences are mobile in only some neuronal lineages or therein promote neurodevelopmental gene expression. Here we report programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV) interneuron development. Mouse PV interneurons permit L1 mobilization in vitro and in vivo, harbor unmethylated L1 promoters and express full-length L1 mRNAs and proteins. Using nanopore long-read sequencing, we identify unmethylated L1s proximal to PV interneuron genes, including a novel L1 promoter-driven Caps2 transcript isoform that enhances neuron morphological complexity in vitro. These data highlight the contribution made by L1 cis-regulatory elements to PV interneuron development and transcriptome diversity, uncovered due to L1 mobility in this milieu.

Brain plasticity following corpus callosum agenesis or loss: a review of the Probst bundles.

The corpus callosum is the largest axonal tract in the human brain, connecting the left and right cortical hemipheres. This structure is affected in myriad human neurodevelopmental disorders, and can be entirely absent as a result of congenital or surgical causes. The age when callosal loss occurs, for example via surgical section in cases of refractory epilepsy, correlates with resulting brain morphology and neuropsychological outcomes, whereby an earlier loss generally produces relatively improved interhemispheric connectivity compared to a loss in adulthood (known as the "Sperry's paradox"). However, the mechanisms behind these age-dependent differences remain unclear. Perhaps the best documented and most striking of the plastic changes that occur due to developmental, but not adult, callosal loss is the formation of large, bilateral, longitudinal ectopic tracts termed Probst bundles. Despite over 100 years of research into these ectopic tracts, which are the largest and best described stereotypical ectopic brain tracts in humans, much remains unclear about them. Here, we review the anatomy of the Probst bundles, along with evidence for their faciliatory or detrimental function, the required conditions for their formation, patterns of etiology, and mechanisms of development. We provide hypotheses for many of the remaining mysteries of the Probst bundles, including their possible relationship to preserved interhemispheric communication following corpus callosum absence. Future research into naturally occurring plastic tracts such as Probst bundles will help to inform the general rules governing axon plasticity and disorders of brain miswiring.

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.

Clade-specific forebrain cytoarchitectures of the extinct Tasmanian tiger.

The thylacine, or Tasmanian tiger, is the largest of modern-day carnivorous marsupials and was hunted to extinction by European settlers in Australia. Its physical resemblance to eutherian wolves is a striking example of evolutionary convergence to similar ecological niches. However, whether the neuroanatomical organization of the thylacine brain resembles that of canids and how it compares with other mammals remain unknown due to the scarcity of available samples. Here, we gained access to a century-old hematoxylin-stained histological series of a thylacine brain, digitalized it at high resolution, and compared its forebrain cellular architecture with 34 extant species of monotremes, marsupials, and eutherians. Phylogenetically informed comparisons of cortical folding, regional volumes, and cell sizes and densities across cortical areas and layers provide evidence against brain convergences with canids, instead demonstrating features typical of marsupials, and more specifically Dasyuridae, along with traits that scale similarly with brain size across mammals. Enlarged olfactory, limbic, and neocortical areas suggest a small-prey predator and/or scavenging lifestyle, similar to extant quolls and Tasmanian devils. These findings are consistent with a nonuniformity of trait convergences, with brain traits clustering more with phylogeny and head/body traits with lifestyle. By making this resource publicly available as rapid web-accessible, hierarchically organized, multiresolution images for perpetuity, we anticipate that additional comparative insights might arise from detailed studies of the thylacine brain and encourage researchers and curators to share, annotate, and preserve understudied material of outstanding biological relevance.

Cortical activity emerges in region-specific patterns during early brain development.

The development of precise neural circuits in the brain requires spontaneous patterns of neural activity prior to functional maturation. In the rodent cerebral cortex, patchwork and wave patterns of activity develop in somatosensory and visual regions, respectively, and are present at birth. However, whether such activity patterns occur in noneutherian mammals, as well as when and how they arise during development, remain open questions relevant for understanding brain formation in health and disease. Since the onset of patterned cortical activity is challenging to study prenatally in eutherians, here we offer an approach in a minimally invasive manner using marsupial dunnarts, whose cortex forms postnatally. We discovered similar patchwork and travelling waves in the dunnart somatosensory and visual cortices at stage 27 (equivalent to newborn mice) and examined earlier stages of development to determine the onset of these patterns and how they first emerge. We observed that these patterns of activity emerge in a region-specific and sequential manner, becoming evident as early as stage 24 in somatosensory and stage 25 in visual cortices (equivalent to embryonic day 16 and 17, respectively, in mice), as cortical layers establish and thalamic axons innervate the cortex. In addition to sculpting synaptic connections of existing circuits, evolutionarily conserved patterns of neural activity could therefore help regulate other early events in cortical development.

Cortical activity emerges in region-specific patterns during early brain development.

The development of precise neural circuits in the brain requires spontaneous patterns of neural activity prior to functional maturation. In the rodent cerebral cortex patchwork and wave patterns of activity develop in somatosensory and visual regions, respectively, and are present at birth. However, whether such activity patterns occur in non-eutherian mammals, as well as when and how they arise during development remain open questions relevant to understand brain formation in health and disease. Since the onset of patterned cortical activity is challenging to study prenatally in eutherians, here we offer a new approach in a minimally invasive manner using marsupial dunnarts, whose cortex forms postnatally. We discovered similar patchwork and travelling waves in the dunnart somatosensory and visual cortices at stage 27 (equivalent to newborn mice), and examined progressively earlier stages of development to determine their onset and how they first emerge. We observed that these patterns of activity emerge in a region-specific and sequential manner, becoming evident as early as stage 24 in somatosensory and stage 25 in visual cortices (equivalent to embryonic day 16 and 17, respectively, in mice), as cortical layers establish and thalamic axons innervate the cortex. In addition to sculpting synaptic connections of existing circuits, evolutionarily conserved patterns of neural activity could therefore help regulate early events in cortical development. Significance Statement: Region-specific patterns of neural activity are present at birth in rodents and are thought to refine synaptic connections during critical periods of cerebral cortex development. Marsupials are born much more immature than rodents, allowing the investigation of how these patterns arise in vivo. We discovered that cortical activity patterns are remarkably similar in marsupial dunnarts and rodents, and that they emerge very early, before cortical neurogenesis is complete. Moreover, they arise from the outset in different patterns specific to somatosensory and visual areas (i.e., patchworks and waves) indicating they may also play evolutionarily conserved roles in cortical regionalization during development.

Divergent evolution of developmental timing in the neocortex revealed by marsupial and eutherian transcriptomes.

Only mammals evolved a neocortex, which integrates sensory-motor and cognitive functions. Significant diversifications in the cellular composition and connectivity of the neocortex occurred between the two main therian groups: marsupials and eutherians. However, the developmental mechanisms underlying these diversifications are largely unknown. Here, we compared the neocortical transcriptomes of Sminthopsis crassicaudata, a mouse-sized marsupial, with those of eutherian mice at two developmentally equivalent time points corresponding to deeper and upper layer neuron generation. Enrichment analyses revealed more mature gene networks in marsupials at the early stage, which reverted at the later stage, suggesting a more precocious but protracted neuronal maturation program relative to birth timing of cortical layers. We ranked genes expressed in different species and identified important differences in gene expression rankings between species. For example, genes known to be enriched in upper-layer cortical projection neuron subtypes, such as Cux1, Lhx2 and Satb2, likely relate to corpus callosum emergence in eutherians. These results show molecular heterochronies of neocortical development in Theria, and highlight changes in gene expression and cell type composition that may underlie neocortical evolution and diversification. This article has an associated 'The people behind the papers' interview.

Timing as a Mechanism of Development and Evolution in the Cerebral Cortex.

One of the biggest mysteries in neurobiology concerns the mechanisms responsible for the diversification of the brain over different time scales, i.e. during development and evolution. Subtle differences in the timing of biological processes during development, e.g. onset, offset, duration, speed and sequence, can trigger large changes in phenotypic outcomes. At the level of a single organism, altered timing of developmental events can lead to individual variability, as well as malformation and disease. At the level of phylogeny, there are known interspecies differences in the timing of developmental events, and this is thought to be an important factor that drives phenotypic variation across evolution, known as heterochrony. A particularly striking example of phenotypic variation is the evolution of human cognitive abilities, which has largely been attributed to the development of the mammalian-specific neocortex and its subsequent expansion in higher primates. Here, I review how the timing of different aspects of cortical development specifies developmental outcomes within species, including processes of cell proliferation and differentiation, neuronal migration and lamination, and axonal targeting and circuit maturation. Some examples of the ways that different processes might "keep time" in the cortex are explored, reviewing potential cell-intrinsic and -extrinsic mechanisms. Further, by combining this knowledge with known differences in timing across species, timing changes that may have occurred during evolution are identified, which perhaps drove the phylogenetic diversification of neocortical structure and function.

The evolution, formation and connectivity of the anterior commissure.

The anterior commissure is the most ancient of the forebrain interhemispheric connections among all vertebrates. Indeed, it is the predominant pallial commissure in all non-eutherian vertebrates, universally subserving basic functions related to olfaction and survival. A key feature of the anterior commissure is its ability to convey connections from diverse brain areas, such as most of the neocortex in non-eutherian mammals, thereby mediating the bilateral integration of diverse functions. Shared developmental mechanisms between the anterior commissure and more evolutionarily recent commissures, such as the corpus callosum in eutherians, have led to the hypothesis that the former may have been a precursor for additional expansion of commissural circuits. However, differences between the formation of the anterior commissure and other telencephalic commissures suggest that independent developmental mechanisms underlie the emergence of these connections in extant species. Here, we review the developmental mechanisms and connectivity of the anterior commissure across evolutionarily distant species, and highlight its potential functional importance in humans, both in the course of normal neurodevelopment, and as a site of plastic axonal rerouting in the absence or damage of other connections.

DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation.

The forebrain hemispheres are predominantly separated during embryogenesis by the interhemispheric fissure (IHF). Radial astroglia remodel the IHF to form a continuous substrate between the hemispheres for midline crossing of the corpus callosum (CC) and hippocampal commissure (HC). Deleted in colorectal carcinoma (DCC) and netrin 1 (NTN1) are molecules that have an evolutionarily conserved function in commissural axon guidance. The CC and HC are absent in Dcc and Ntn1 knockout mice, while other commissures are only partially affected, suggesting an additional aetiology in forebrain commissure formation. Here, we find that these molecules play a critical role in regulating astroglial development and IHF remodelling during CC and HC formation. Human subjects with DCC mutations display disrupted IHF remodelling associated with CC and HC malformations. Thus, axon guidance molecules such as DCC and NTN1 first regulate the formation of a midline substrate for dorsal commissures prior to their role in regulating axonal growth and guidance across it.

Altered structural connectivity networks in a mouse model of complete and partial dysgenesis of the corpus callosum.

Corpus callosum dysgenesis (CCD) describes a collection of brain malformations in which the main fiber tract connecting the two hemispheres is either absent (complete CCD, or 'agenesis of the corpus callosum') or reduced in size (partial CCD). Humans with these neurodevelopmental disorders have a wide range of cognitive outcomes, including seemingly preserved features of interhemispheric communication in some cases. However, the structural substrates that could underlie this variability in outcome remain to be fully elucidated. Here, for the first time, we characterize the global brain connectivity of a mouse model of complete and partial CCD. We demonstrate features of structural brain connectivity that model those predicted in humans with CCD, including Probst bundles in complete CCD and heterotopic sigmoidal connections in partial CCD. Crucially, we also histologically validate the recently predicted ectopic sigmoid bundle present in humans with partial CCD, validating the utility of this mouse model for fine anatomical studies of this disorder. Taken together, this work describes a mouse model of altered structural connectivity in variable severity CCD and forms a foundation for future studies investigating the function and mechanisms of development of plastic tracts in developmental disorders of brain connectivity.

Differential timing of a conserved transcriptional network underlies divergent cortical projection routes across mammalian brain evolution.

A unique combination of transcription factor expression and projection neuron identity demarcates each layer of the cerebral cortex. During mouse and human cortical development, the transcription factor CTIP2 specifies neurons that project subcerebrally, while SATB2 specifies neuronal projections via the corpus callosum, a large axon tract connecting the two neocortical hemispheres that emerged exclusively in eutherian mammals. Marsupials comprise the sister taxon of eutherians but do not have a corpus callosum; their intercortical commissural neurons instead project via the anterior commissure, similar to egg-laying monotreme mammals. It remains unknown whether divergent transcriptional networks underlie these cortical wiring differences. Here, we combine birth-dating analysis, retrograde tracing, gene overexpression and knockdown, and axonal quantification to compare the functions of CTIP2 and SATB2 in neocortical development, between the eutherian mouse and the marsupial fat-tailed dunnart. We demonstrate a striking degree of structural and functional homology, whereby CTIP2 or SATB2 of either species is sufficient to promote a subcerebral or commissural fate, respectively. Remarkably, we reveal a substantial delay in the onset of developmental SATB2 expression in mice as compared to the equivalent stage in dunnarts, with premature SATB2 overexpression in mice to match that of dunnarts resulting in a marsupial-like projection fate via the anterior commissure. Our results suggest that small alterations in the timing of regulatory gene expression may underlie interspecies differences in neuronal projection fate specification.

A pan-mammalian map of interhemispheric brain connections predates the evolution of the corpus callosum.

The brain of mammals differs from that of all other vertebrates, in having a six-layered neocortex that is extensively interconnected within and between hemispheres. Interhemispheric connections are conveyed through the anterior commissure in egg-laying monotremes and marsupials, whereas eutherians evolved a separate commissural tract, the corpus callosum. Although the pattern of interhemispheric connectivity via the corpus callosum is broadly shared across eutherian species, it is not known whether this pattern arose as a consequence of callosal evolution or instead corresponds to a more ancient feature of mammalian brain organization. Here we show that, despite cortical axons using an ancestral commissural route, monotremes and marsupials share features of interhemispheric connectivity with eutherians that likely predate the origin of the corpus callosum. Based on ex vivo magnetic resonance imaging and tractography, we found that connections through the anterior commissure in both fat-tailed dunnarts (Marsupialia) and duck-billed platypus (Monotremata) are spatially segregated according to cortical area topography. Moreover, cell-resolution retrograde and anterograde interhemispheric circuit mapping in dunnarts revealed several features shared with callosal circuits of eutherians. These include the layered organization of commissural neurons and terminals, a broad map of connections between similar (homotopic) regions of each hemisphere, and regions connected to different areas (heterotopic), including hyperconnected hubs along the medial and lateral borders of the cortex, such as the cingulate/motor cortex and claustrum/insula. We therefore propose that an interhemispheric connectome originated in early mammalian ancestors, predating the evolution of the corpus callosum. Because these features have been conserved throughout mammalian evolution, they likely represent key aspects of neocortical organization.

Transcriptional control of long-range cortical projections.

Long-range projection neurons of the neocortex form the major tracts of the mammalian brain and are crucial for sensory-motor, associative and executive functions. Development of such circuits involves neuronal proliferation, specification and migration, as well as axonal elongation, navigation and targeting, where growing axons encounter multiple guidance cues and integrate these signals to execute guidance decisions. The complexity of axon guidance mechanisms in the formation of long-range neuronal projections has suggested that they might be under control of transcription factors, which are DNA-binding proteins that regulate the expression of downstream genes. Here we discuss recent advances in our understanding of the control of axon guidance by transcriptional regulation, as well as future directions for the elucidation of the mechanisms and pathological relevance of this process.

Pharmacogenetic stimulation of neuronal activity increases myelination in an axon-specific manner.

Mounting evidence suggests that neuronal activity influences myelination, potentially allowing for experience-driven modulation of neural circuitry. The degree to which neuronal activity is capable of regulating myelination at the individual axon level is unclear. Here we demonstrate that stimulation of somatosensory axons in the mouse brain increases proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) within the underlying white matter. Stimulated axons display an increased probability of being myelinated compared to neighboring non-stimulated axons, in addition to being ensheathed with thicker myelin. Conversely, attenuating neuronal firing reduces axonal myelination in a selective activity-dependent manner. Our findings reveal that the process of selecting axons for myelination is strongly influenced by the relative activity of individual axons within a population. These observed cellular changes are consistent with the emerging concept that adaptive myelination is a key mechanism for the fine-tuning of neuronal circuitry in the mammalian CNS.

Multiple events of gene manipulation via in pouch electroporation in a marsupial model of mammalian forebrain development.

BACKGROUND: The technique of in utero electroporation has been widely used in eutherians, such as mice and rats, to investigate brain development by selectively manipulating gene expression in specific neuronal populations. A major challenge, however, is that surgery is required to access the embryos, affecting animal survival and limiting the number of times it can be performed within the same litter. NEW METHOD: Marsupials are born at an early stage of brain development as compared to eutherians. Forebrain neurogenesis occurs mostly postnatally, allowing electroporation to be performed while joeys develop attached to the teat. Here we describe the method of in pouch electroporation using the Australian marsupial fat-tailed dunnart (Sminthopsis crassicaudata, Dasyuridae). RESULTS: In pouch electroporation is minimally invasive, quick, successful and anatomically precise. Moreover, as no surgery is required, it can be performed several times in the same individual, and littermates can undergo independent treatments. COMPARISON WITH EXISTING METHOD: As compared to in utero electroporation in rodents, in pouch electroporation in marsupials offers unprecedented opportunities to study brain development in a minimally invasive manner. Continuous access to developing joeys during a protracted period of cortical development allows multiple and independent genetic manipulations to study the interaction of different systems during brain development. CONCLUSIONS: In pouch electroporation in marsupials offers an excellent in vivo assay to study forebrain development and evolution. By combining developmental, functional and comparative approaches, this system offers new avenues to investigate questions of biological and medical relevance, such as the precise mechanisms of brain wiring and the organismic and environmental influences on neural circuit formation.

Development of body, head and brain features in the Australian fat-tailed dunnart (Sminthopsis crassicaudata; Marsupialia: Dasyuridae); A postnatal model of forebrain formation.

Most of our understanding of forebrain development comes from research of eutherian mammals, such as rodents, primates, and carnivores. However, as the cerebral cortex forms largely prenatally, observation and manipulation of its development has required invasive and/or ex vivo procedures. Marsupials, on the other hand, are born at comparatively earlier stages of development and most events of forebrain formation occur once attached to the teat, thereby permitting continuous and non-invasive experimental access. Here, we take advantage of this aspect of marsupial biology to establish and characterise a resourceful laboratory model of forebrain development: the fat-tailed dunnart (Sminthopsis crassicaudata), a mouse-sized carnivorous Australian marsupial. We present an anatomical description of the postnatal development of the body, head and brain in dunnarts, and provide a staging system compatible with human and mouse developmental stages. As compared to eutherians, the orofacial region develops earlier in dunnarts, while forebrain development is largely protracted, extending for more than 40 days versus ca. 15 days in mice. We discuss the benefits of fat-tailed dunnarts as laboratory animals in studies of developmental biology, with an emphasis on how their accessibility in the pouch can help address new experimental questions, especially regarding mechanisms of brain development and evolution.

The anatomy, organisation and development of contralateral callosal projections of the mouse somatosensory cortex.

BACKGROUND: Alterations in the development of neuronal connectivity can result in dramatic outcomes for brain function. In the cerebral cortex, most sensorimotor and higher-order functions require coordination between precise regions of both hemispheres through the axons that form the corpus callosum. However, little is known about how callosal axons locate and innervate their contralateral targets. METHODS: Here, we use a combination of in utero electroporation, retrograde tracing, sensory deprivation and high-resolution axonal quantification to investigate the development, organisation and activity dependence of callosal axons arising from the primary somatosensory cortex of mice. RESULTS: We show that distinct contralateral projections arise from different neuronal populations and form homotopic and heterotopic circuits. Callosal axons innervate the contralateral hemisphere following a dorsomedial to ventrolateral and region-specific order. Furthermore, we identify two periods of region- and layer-specific developmental exuberance that correspond to initial callosal axon innervation and subsequent arborisation. Early sensory deprivation affects only the latter of these events. CONCLUSION: Taken together, these results reveal the main developmental events of contralateral callosal targeting and may aid future understanding of the formation and pathologies of brain connectivity.

EMX1 regulates NRP1-mediated wiring of the mouse anterior cingulate cortex.

Transcription factors act during cortical development as master regulatory genes that specify cortical arealization and cellular identities. Although numerous transcription factors have been identified as being crucial for cortical development, little is known about their downstream targets and how they mediate the emergence of specific neuronal connections via selective axon guidance. The EMX transcription factors are essential for early patterning of the cerebral cortex, but whether EMX1 mediates interhemispheric connectivity by controlling corpus callosum formation remains unclear. Here, we demonstrate that in mice on the C57Bl/6 background EMX1 plays an essential role in the midline crossing of an axonal subpopulation of the corpus callosum derived from the anterior cingulate cortex. In the absence of EMX1, cingulate axons display reduced expression of the axon guidance receptor NRP1 and form aberrant axonal bundles within the rostral corpus callosum. EMX1 also functions as a transcriptional activator of Nrp1 expression in vitro, and overexpression of this protein in Emx1 knockout mice rescues the midline-crossing phenotype. These findings reveal a novel role for the EMX1 transcription factor in establishing cortical connectivity by regulating the interhemispheric wiring of a subpopulation of neurons within the mouse anterior cingulate cortex.

Formation of functional areas in the cerebral cortex is disrupted in a mouse model of autism spectrum disorder.

BACKGROUND: Autism spectrum disorders (ASD) are a group of poorly understood behavioural disorders, which have increased in prevalence in the past two decades. Animal models offer the opportunity to understand the biological basis of these disorders. Studies comparing different mouse strains have identified the inbred BTBR T + tf/J (BTBR) strain as a mouse model of ASD based on its anti-social and repetitive behaviours. Adult BTBR mice have complete agenesis of the corpus callosum, reduced cortical thickness and changes in early neurogenesis. However, little is known about the development or ultimate organisation of cortical areas devoted to specific sensory and motor functions in these mice that may also contribute to their behavioural phenotype. RESULTS: In this study, we performed diffusion tensor imaging and tractography, together with histological analyses to investigate the emergence of functional areas in the cerebral cortex and their connections in BTBR mice and age-matched C57Bl/6 control mice. We found evidence that neither the anterior commissure nor the hippocampal commissure compensate for the loss of callosal connections, indicating that no interhemispheric neocortical connectivity is present in BTBR mice. We also found that both the primary visual and somatosensory cortical areas are shifted medially in BTBR mice compared to controls and that cortical thickness is differentially altered in BTBR mice between cortical areas and throughout development. CONCLUSIONS: We demonstrate that interhemispheric connectivity and cortical area formation are altered in an age- and region-specific manner in BTBR mice, which may contribute to the behavioural deficits previously observed in this strain. Some of these developmental patterns of change are also present in human ASD patients, and elucidating the aetiology driving cortical changes in BTBR mice may therefore help to increase our understanding of this disorder.