Eutherian Brain Research Articles

Imprinting as Basis for Complex Evolutionary Novelties in Eutherians.

The epigenetic phenomenon of genomic imprinting is puzzling. While epigenetic modifications in general are widely known in most species, genomic imprinting in the animal kingdom is restricted to autosomes of therian mammals, mainly eutherians, and to a lesser extent in marsupials. Imprinting causes monoallelic gene expression. It represents functional haploidy of certain alleles while bearing the evolutionary cost of diploidization, which is the need of a complex cellular architecture and the danger of producing aneuploid cells by mitotic and meiotic errors. The parent-of-origin gene expression has stressed many theories. Most prominent theories, such as the kinship (parental conflict) hypothesis for maternally versus paternally derived alleles, explain only partial aspects of imprinting. The implementation of single-cell transcriptome analyses and epigenetic research allowed detailed study of monoallelic expression in a spatial and temporal manner and demonstrated a broader but much more complex and differentiated picture of imprinting. In this review, we summarize all these aspects but argue that imprinting is a functional haploidy that not only allows a better gene dosage control of critical genes but also increased cellular diversity and plasticity. Furthermore, we propose that only the occurrence of allele-specific gene regulation mechanisms allows the appearance of evolutionary novelties such as the placenta and the evolutionary expansion of the eutherian brain.

Retrovirus-Derived RTL9 Plays an Important Role in Innate Antifungal Immunity in the Eutherian Brain.

Retrotransposon Gag-like (RTL) genes play a variety of essential and important roles in the eutherian placenta and brain. It has recently been demonstrated that RTL5 and RTL6 (also known as sushi-ichi retrotransposon homolog 8 (SIRH8) and SIRH3) are microglial genes that play important roles in the brain's innate immunity against viruses and bacteria through their removal of double-stranded RNA and lipopolysaccharide, respectively. In this work, we addressed the function of RTL9 (also known as SIRH10). Using knock-in mice that produce RTL9-mCherry fusion protein, we examined RTL9 expression in the brain and its reaction to fungal zymosan. Here, we demonstrate that RTL9 plays an important role, degrading zymosan in the brain. The RTL9 protein is localized in the microglial lysosomes where incorporated zymosan is digested. Furthermore, in Rtl9 knockout mice expressing RTL9ΔC protein lacking the C-terminus retroviral GAG-like region, the zymosan degrading activity was lost. Thus, RTL9 is essentially engaged in this reaction, presumably via its GAG-like region. Together with our previous study, this result highlights the importance of three retrovirus-derived microglial RTL genes as eutherian-specific constituents of the current brain innate immune system: RTL9, RTL5 and RTL6, responding to fungi, viruses and bacteria, respectively.

Development's Inaugural Outstanding Paper Prize.

Development's Inaugural Outstanding Paper Prize.

Retrovirus-derived RTL5 and RTL6 genes are novel constituents of the innate immune system in the eutherian brain.

Retrotransposon Gag-like 5 [RTL5, also known as sushi-ichi-related retrotransposon homolog 8 (SIRH8)] and RTL6 (also known as SIRH3) are eutherian-specific genes presumably derived from a retrovirus and phylogenetically related to each other. They, respectively, encode a strongly acidic and extremely basic protein, and are well conserved among the eutherians. Here, we report that RTL5 and RTL6 are microglial genes with roles in the front line of innate brain immune response. Venus and mCherry knock-in mice exhibited expression of RTL5-mCherry and RTL6-Venus fusion proteins in microglia and appeared as extracellular dots and granules in the central nervous system. These proteins display a rapid response to pathogens such as lipopolysaccharide (LPS), double-stranded (ds) RNA analog and non-methylated CpG DNA, acting both cooperatively and/or independently. Experiments using Rtl6 or Rtl5 knockout mice provided additional evidence that RTL6 and RTL5 act as factors against LPS and dsRNA, respectively, in the brain, providing the first demonstration that retrovirus-derived genes play a role in the eutherian innate immune system. Finally, we propose a model emphasizing the importance of extra-embryonic tissues as the origin site of retrovirus-derived genes. This article has an associated 'The people behind the papers' interview.

The role of eutherian-specific RTL1 in the nervous system and its implications for the Kagami-Ogata and Temple syndromes.

RTL1 (also termed paternal expressed 11 (PEG11)) is considered the major imprinted gene responsible for the placental and fetal/neonatal muscle defects that occur in the Kagami–Ogata and Temple syndromes (KOS14 and TS14, respectively). However, it remains elusive whether RTL1 is also involved in their neurological symptoms, such as behavioral and developmental delay/intellectual disability, feeding difficulties, motor delay, and delayed speech. Here, we demonstrate that the mouse RTL1 protein is widely expressed in the central nervous system (CNS), including the limbic system. Importantly, two disease model mice with over‐ and under‐expression of Rtl1 exhibited reduced locomotor activity, increased anxiety, and impaired amygdala‐dependent cued fear, demonstrating that Rtl1 also plays an important role in the CNS. These results indicate that the KOS14 and TS14 are neuromuscular as well as neuropsychiatric diseases caused by irregular CNS RTL1 expression, presumably leading to impaired innervation of motor neurons to skeletal muscles as well as malfunction of the hippocampus‐amygdala complex. It is of considerable interest that eutherian‐specific RTL1 is expressed in mammalian‐ and eutherian‐specific brain structures, that is, the corticospinal tract and corpus callosum, respectively, suggesting that RTL1 might have contributed to the acquisition of both these structures themselves and fine motor skill in eutherian brain evolution.

Numerical Analysis of the Cerebral Cortex in Diprotodontids (Marsupialia; Australidelphia) and Comparison with Eutherian Brains

Diprotodontids are a diverse group of Australian metatherians, which occupy a range of ecological niches from nectar and pollen-feeders to grazers and folivores. The group encompasses small-brained nectar-feeding species (Tarsipes) and large-brained grazing and browsing species (macropods). This group of Australian metatherians therefore represents an opportunity to examine how the cerebral cortex has expanded in an adaptive radiation quite independent of that occurring among eutherians. We have used the Nelson Brain Collection and online resources to perform a quantitative analysis of the isocortex, hippocampal formation and olfactory structures in diprotodontids. We found that the scaling relationship between iso- and periallocortical grey matter and brain size, and between subcortical white matter and iso- and periallocortex grey matter, are both almost identical among diprotodontids and eutherians. By contrast, the relationship between gyrification and brain size is strikingly different between diprotodontids and eutherians, with gyrification being much lower for a given brain size among the diprotodontids, although gyrification is much more varied among macropods than other diprotodontids. The scaling of iso- and periallocortical volume with dorsal striatal and dorsal thalamic volume is almost identical among the diprotodontids and eutherians, but the claustrum is smaller, and amygdala larger, for a given brain size among diprotodontids than eutherians. The hippocampal formation and central olfactory areas (anterior olfactory region and piriform cortex) both scale more steeply with brain size among diprotodontids compared to eutherians. Our findings suggest that, although white matter scaling is identical among all therians, there are significant differences between diprotodontids and eutherians in the way that cortical folding and expansion of allocortical structures occurs with brain enlargement.

Widespread cis-regulatory convergence between the extinct Tasmanian tiger and gray wolf.

The extinct marsupial Tasmanian tiger, or thylacine, and the eutherian gray wolf are among the most widely recognized examples of convergent evolution in mammals. Despite being distantly related, these large predators independently evolved extremely similar craniofacial morphologies, and evidence suggests that they filled similar ecological niches. Previous analyses revealed little evidence of adaptive convergence between their protein-coding genes. Thus, the genetic basis of their convergence is still unclear. Here, we identified candidate craniofacial cis-regulatory elements across vertebrates and compared their evolutionary rates in the thylacine and wolf, revealing abundant signatures of convergent positive selection. Craniofacial thylacine–wolf accelerated regions were enriched near genes involved in TGF beta (TGFB) and BMP signaling, both of which are key morphological signaling pathways with critical roles in establishing the identities and boundaries between craniofacial tissues. Similarly, enhancers of genes involved in craniofacial nerve development showed convergent selection and involvement in these pathways. Taken together, these results suggest that adaptation in cis-regulators of TGF beta and BMP signaling may provide a mechanism to explain the coevolution of developmentally and functionally integrated craniofacial structures in these species. We also found that despite major structural differences in marsupial and eutherian brains, accelerated regions in both species were common near genes with roles in brain development. Our findings support the hypothesis that, relative to protein-coding genes, positive selection on cis-regulatory elements is likely to be an essential driver of adaptive convergent evolution and may underpin thylacine–wolf phenotypic similarities.

Minimal variation in eutherian brain growth rates during fetal neurogenesis.

A central question in the evolution of brain development is whether species differ in rates of brain growth during fetal neurogenesis. Studies of neonatal data have found allometric evidence for brain growth rate differences according to physiological variables such as relative metabolism and placental invasiveness, but these findings have not been tested against fetal data directly. Here, we measure rates of exponential brain growth acceleration in 10 eutherian mammals, two marsupials, and two birds. Eutherian brain acceleration exhibits minimal variation relative to body and visceral organ growth, varies independently of correlated growth patterns in other organs, and is unrelated to proposed physiological constraints such as metabolic rate or placental invasiveness. Brain growth rates in two birds overlap with eutherian variation, while marsupial brain growth is exceptionally slow. Peak brain growth velocity is linked in time with forebrain myelination and eye opening, reliably separates altricial species born before it from precocial species born afterwards, and is an excellent predictor of adult brain size (r2 = 0.98). Species with faster body growth exhibit larger relative brain size in early ontogeny, while brain growth is unrelated to allometric measures. These findings indicate a surprising conservation of brain growth rates during fetal neurogenesis in eutherian mammals, clarify sources of variation in neonatal brain size, and suggest that slow body growth rates cause species to be more encephalized at birth.

An LTR Retrotransposon-Derived Gene Displays Lineage-Specific Structural and Putative Species-Specific Functional Variations in Eutherians.

Amongst the 11 eutherian-specific genes acquired from a sushi-ichi retrotransposon is the CCHC type zinc-finger protein-encoding gene SIRH11/ZCCHC16. Its contribution to eutherian brain evolution is implied because of its involvement in cognitive function in mice, possibly via the noradrenergic system. Although, the possibility that Sirh11/Zcchc16 functions as a non-coding RNA still remains, dN/dS ratios in pairwise comparisons between its orthologs have provided supportive evidence that it acts as a protein. It became a pseudogene in armadillos (Cingulata) and sloths (Pilosa), the only two extant orders of xenarthra, which prompted us to examine the lineage-specific variations of SIRH11/ZCCHC16 in eutherians. We examined the predicted SIRH11/ZCCHC16 open reading frame (ORF) in 95 eutherian species based on the genomic DNA information in GenBank. A large variation in the SIRH11/ZCCHC16 ORF was detected in several lineages. These include a lack of a CCHC RNA-binding domain in its C-terminus, observed in gibbons (Hylobatidae: Primates) and megabats (Megachiroptera: Chiroptera). A lack of the N-terminal half, on the other hand, was observed in New World monkeys (Platyrrhini: Primates) and species belonging to New World and African Hystricognaths (Caviomorpha and Bathyergidae: Rodents) along with Cetacea and Ruminantia (Cetartiodactyla). Among the hominoids, interestingly, three out of four genera of gibbons have lost normal SIRH11/ZCCHC16 function by deletion or the lack of the CCHC RNA-binding domain. Our extensive dN/dS analysis suggests that such truncated SIRH11/ZCCHC16 ORFs are functionally diversified even within lineages. Combined, our results show that SIRH11/ZCCHC16 may contribute to the diversification of eutherians by lineage-specific structural changes after its domestication in the common eutherian ancestor, followed by putative species-specific functional changes that enhanced fitness and occurred as a consequence of complex natural selection events.

Evolution of brain functions in mammals and LTR retrotransposon-derived genes.

In the human genome, there are approximately 30 LTR retrotransposon-derived genes, such as the sushi-ichi retrotransposon homologues (SIRH) and the paraneoplastic Ma antigen (PNMA) family genes. They are derivatives from the original LTR retrotransposons and each gene seems to have its own unique function. PEG10/SIRH1 as well as PEG11/RTL1/SIRH2 and SIRH7/LDOC1 play essential roles in placenta formation, maintenance of fetal capillaries and the differentiation/maturation of a variety of placental cells, respectively. All of this evidence provides strong support for their contribution to the evolution of viviparity in mammals via their eutherian-specific functions. SIRH11/ZCCHC16 is an X-linked gene that encodes a CCHC type of zinc-finger protein that exhibits high sequence identity to the LTR retrotransposon Gag protein and its deletion causes abnormal behavior related to cognition, including attention, impulsivity and working memory, possibly via the locus coeruleus noradrenaergic system in mice. Therefore, we have suggested that the acquisition of SIRH11/ZCCHC16 was involved in eutherian brain evolution. Interestingly, SIRH11/ZCCHC16 displays lineage-specific structural and putative species-specific functional variations in eutherians, suggesting that it contributed to the diversification of eutherians via increasing evolutionary fitness by these changes.

Topography and chemoarchitecture of the striatum and pallidum in a monotreme, the short-beaked echidna (Tachyglossus aculeatus)

The topography and chemoarchitecture of the striatum and pallidum in a monotreme, the short-beaked echidna (Tachyglossus aculeatus) have been studied using Nissl staining in conjunction with myelin staining, enzyme reactivity to acetylcholinesterase and NADPH diaphorase, and immunoreactivity to parvalbumin, calbindin, calretinin, tyrosine hydroxylase, neuropeptide Y, and neurofilament protein (SMI-32 antibody). All those components of the striatum and pallidum found in eutherian mammals could also be identified in the echidna's brain, with broad chemoarchitectural similarities to those regions in eutherian brains also apparent. There was a clear chemoarchitectural gradient visible with parvalbumin immunoreactivity of neurons and fibers, suggesting a subdivision of the echidna caudatoputamen into weakly reactive rostrodorsomedial and strongly reactive caudoventrolateral components. This may, in turn, relate to subdivision into associative versus sensorimotor CPu and reflect homology to the caudate and putamen of primates. Moreover, the chemoarchitecture of the echidna striatum suggested the presence of striosome-matrix architecture. The morphology of identified neuronal groups (i.e., parvalbumin, calbindin, and neuropeptide Y immunoreactive) in the echidna striatum and pallidum showed many similarities to those seen in eutherians, although the pattern of distribution of calbindin immunoreactive neurons was more uniform in the caudatoputamen of the echidna than in therians. These observations indicate that the same broad features of striatal and pallidal organization apply across all mammals and suggest that these common features may have arisen before the divergence of the monotreme and therian lineages.

The corpus callosum as an evolutionary innovation

The corpus callosum (CC) is the major interhemispheric fibre bundle in the eutherian brain and has been described as a true evolutionary innovation. This paper reviews the current literature with regard to functional, developmental and genetic concepts that may help elucidate the evolutionary origin of this structure. It has been suggested that the CC arose in the eutherian brain as a more direct and, therefore, more effective system for the interhemispheric integration of topographically organized sensory cortices than the anterior commissure (AC) and hippocampal commissure (HC) already present in nonplacental mammals. It can also be argued, however, that the ability of the CC to integrate the newly evolving motor cortices of placental mammals may have played a role in the evolutionary fixation of this structure. Investigations into the developmental mechanism involved in the formation of the CC and their underlying patterns of gene expression make it possible to formulate a tentative hypothesis about the evolutionary origin of this commissure. This paper suggests that changes in the developmental patterns of the expression of certain regulatory genes may have allowed a first group of callosal pioneering axons to cross the cortical midline. These pioneering fibres may have used the axons of the HC to find their way across the midline. Additional callosal fibres may then have fasciculated with these pioneers. Once the CC had formed in this way, more complex systems of axonal guidance may have evolved over time, thus enabling a gradual increase in the size and complexity of the CC.

CD15 immunoreactivity in the developing brain of a marsupial, the tammar wallaby (Macropus eugenii)

We have studied the distribution of the CD15 epitope in the developing brain of an Australian diprotodontid metatherian mammal, the tammar wallaby ( Macropus eugenii), using immunohistochemistry in conjunction with hematoxylin and eosin staining. At the time of birth (28 days after conception), CD15 immunoreactivity labeled somata in the primordial plexiform layer of the parietal cortex in a similar position to that seen in the early fetal eutherian brain. CD15 immunoreactivity in the brain of the developing pouch-young wallaby was found to be localized on the surface of radial glia at boundaries between developmentally significant forebrain compartments in a similar distribution to that seen in developing eutherian brain. These were best seen in the developing diencephalon, delineating epithalamus, ventral and dorsal thalamus and hypothalamic anlage, and in the striatum. Immunoreactivity for CD15 identified radial glia marking the lateral migratory stream at the striatopallial boundary, peaking in intensity at P19 to P25. From P37 to P54, CD15 immunoreactivity also demarcated patch compartments in the developing striatum. In contrast, CD15 immunoreactivity in hindbrain structures showed some differences from the temporospatial pattern seen in eutherian brain. These may reflect the relatively early brainstem maturation required for the newborn wallaby to be able to traverse the distance from the maternal genital tract to the pouch. The wallaby provides a convenient model for testing hypotheses concerning the role of CD15 in forebrain development because all events in which CD15 may play a critical role in forebrain morphogenesis occur during pouch life, when the young wallaby is accessible to experimental manipulation.

Visual abnormalities in albino wallabies: A brief note

Abnormally reduced uncrossed retinofugal pathways and abnormally low retinal cell densities have been described in albinos of a number of species of eutherian mammal, but have not been studied in marsupials. The optic chiasm of marsupials differs from that of the eutherian brains that have been studied, with uncrossed fibres segregating from crossed fibres prechiasmatically in the marsupials, but not in the eutherians. We have examined the optic chiasm and the retina in albino and normally pigmented wallabies (Macropus rufogriseus) to determine whether the abnormalities found in eutherian albinos are also present in this marsupial. The uncrossed pathway of the albino wallaby is smaller than that of the pigmented animals and the cell density in the retinal ganglion cell layer is reduced at the area centralis. We conclude that the characteristic albino abnormalities are present in both groups of mammals in spite of the other known differences in the visual pathways. The albino mutation acts at the chiasm in a similar manner in both groups even though the determinants of the chiasmatic pathway differ.

Visual abnormalities in albino wallabies: A brief note

Abnormally reduced uncrossed retinofugal pathways and abnormally low retinal cell densities have been described in albinos of a number of species of eutherian mammal, but have not been studied in marsupials. The optic chiasm of marsupials differs from that of the eutherian brains that have been studied, with uncrossed fibres segregating from crossed fibres prechiasmatically in the marsupials, but not in the eutherians. We have examined the optic chiasm and the retina in albino and normally pigmented wallabies (Macropus rufogriseus) to determine whether the abnormalities found in eutherian albinos are also present in this marsupial. The uncrossed pathway of the albino wallaby is smaller than that of the pigmented animals and the cell density in the retinal ganglion cell layer is reduced at the area centralis. We conclude that the characteristic albino abnormalities are present in both groups of mammals in spite of the other known differences in the visual pathways. The albino mutation acts at the chiasm in a similar manner in both groups even though the determinants of the chiasmatic pathway differ. J. Comp. Neurol. 403:33–38, 1999. © 1999 Wiley-Liss, Inc.

Angiotensin receptors in an Australian marsupial, the brushtail possum Trichosurus vulpecula

In this study, the binding properties of angiotensin receptors were examined in the liver, adrenal, brain, and vascular tissue of the brushtail possum, Trichosurus vulpecula. With 125I-Ile 5-angiotensin II as the radioligand, the binding affinity ( K a) and receptor number ( R 0) were estimated for the liver ( K a = 3.60 ± 0.31 liters/nmol; R 0 = 23.8 ± 1.30 pmol/g tissue; n = 8) and adrenal ( K a = 1.68 ± 0.29 liters/nmol; R 0 = 1.67 ± 0.23 pmol/g tissue; n = 8). Specific binding was not found in any of seven areas of the possum brain ( n = 6), whereas the expected binding was present in similar areas of the rat brain. Using angiotensin III or the antagonist Sar 1-Ala 8-angiotensin II as radioligands or changing the composition of the incubation buffer did not alter the outcome. Moreover, the intracerebroventricular injection of 1 and 5 nmol of angiotensin II did not elicit an increase in blood pressure which could be attributed to brain angiotensin II (AII) receptors. Ligand affinities of the adrenal and liver receptors were found to be in the following decreasing order: Val 5-AII > Ile 5-AII = Ile 5-AIII > Sar 1-Ala 8-AII > Sar 1-Gly 8-AII > Sar 1-Leu 8-AII > Ile5-AI > hexapeptide > Phe 3-Tyr 8-AII. The cardiovascular All receptor was investigated by generating dose-response curves of the pressor activity of Ile 5-AII and six AII analogs infused intravenously. It was concluded that liver, adrenal, and vascular AII receptors in the marsupial possum have characteristics similar to those in eutherian mammals. However, the failure to find brain AII receptors raises the possibility that those functions mediated by such receptors in the eutherian brain are absent in the possum and perhaps other marsupials.

Retinofugal projections in the opossum. an anterograde degeneration and radioautographic study

A study of anterograde degeneration and anterograde transport was undertaken in the opossum primary optic system in order to clarify several points regarding fiber organization and patterns of terminal fields. Through the radioautographic technique of axon tracing, it was demonstrated that the accessory optic system follows the generalized scheme of Hayhow, consisting of two fascicles and three terminal nuclei. The existence of retinal projections to the suprachiasmatic nucleus was confirmed in radioautographic material. New interpretations on the pattern of terminations of retinal fibers in the thalamus, pretectum and superior colliculus have also been reached. A quasi-laminar arrangement of retinal endings in the dorsal lateral geniculate nucleus was observed in radioautographic but not in degenerating material. Both techniques indicated a pattern of projections to the ventral lateral geniculate nucleus and to the pretectal complex (nucleus pretectalis olivaris and nucleus of the optic tract) basically similar to that described in eutherian mammals. In addition, radioautographic data suggested a projection to the pretectal posterior nucleus. A segregation of retinal endings in the rostral half of the superior colliculus was detected in radioautographic but not in degenerating material. It was concluded that the general organization of the retinofugal projections in the opossum is essentially similar to that found in eutherian brains.