Maternal corticosterone but not testosterone level is associated with the ratio of second-to-fourth digit length (2D:4D) in field vole offspring (Microtus agrestis)

The steroid environment encountered by a foetus can strongly affect its post-natal physiology and behaviour. It has been proposed that steroid concentrations experienced in utero could be estimated from adults by measuring their second-to-fourth digit length ratio (2D:4D). However, there is still little direct evidence that intra-uterine steroid levels affect individual 2D:4D. We examined whether maternal pre-pregnancy testosterone and corticosterone levels (as estimates of intra-uterine testosterone and corticosterone exposure) affected the 2D:4D of pups in non-domesticated field voles (Microtus agrestis), measured by X-rays at the age of weaning (21 days). Furthermore, for the first time in a non-human species, we studied whether testosterone and corticosterone levels correlated with 2D:4D in adult females. We found that the maternal pre-pregnancy level of testosterone was not associated with offspring 2D:4D in either the left or the right paw. Instead, maternal pre-pregnancy corticosterone level was positively correlated with offspring 2D:4D in the right paw, but unrelated to 2D:4D in the left paw. In addition, the 2D:4D of adult females was not associated with either their circulating testosterone or corticosterone levels. Our results suggest that in field voles maternally administered testosterone is not a major determinant of offspring 2D:4D, whereas maternal stress appears to account for some of the variation in the 2D:4D of their offspring.     

Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age

Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.     

Synaptic formation in subsets of glutamatergic terminals in the mouse hippocampal formation is affected by a deficiency in the neural cell recognition molecule NB-3

The neural cell recognition molecule NB-3, which is also referred to as contactin-6, is a member of the contactin subgroup molecules that are expressed prominently in the developing nervous system after birth. In mice, an NB-3 deficiency impairs motor coordination and reduces the synaptic density between parallel fibers and Purkinje cells in the cerebellum. Here, we studied the role of NB-3 in the formation of glutamatergic synapses in the hippocampal formation. At postnatal day 5, NB-3 immunoreactivity was detected in the subiculum, the stratum lacunosum–moleculare of the CA1 region and the hilus of the dentate gyrus. NB-3 expression in the strata radiatum and oriens was weak, and it was very weak in the granule cell layer of the dentate gyrus, the pyramidal cell layer of regions CA3 to CA1 and the stratum lucidum. NB-3-positive puncta partially overlapped with vesicular glutamate transporter 1 (VGLUT1) and 2 (VGLUT2), excitatory presynaptic markers, but not with vesicular GABA transporter (VGAT), an inhibitory presynaptic marker. The density of VGLUT1 and VGLUT2 puncta in the regions where NB-3 was strongly expressed in wild-type mice was reduced by ∼20–30% in NB-3 knockout mice relative to wild-type mice, whereas that of VGAT puncta was not affected by NB-3 deficiency. Thus, NB-3 has key roles in the formation of glutamatergic, but not GABAergic, synapses during postnatal development of the hippocampal formation as well as the cerebellum.     

T lymphocyte development in p56lck deficient mice: allelic exclusion of the TcR β locus is incomplete but thymocyte development is not restored by TcR β or TcR αβ transgenes

The protein tyrosine kinase, p56^lck, is involved in signal transduction in mature T cells and in the molecular events controlling early thymocyte differentiation. Thymuses of mice deficient for p56^lck expression (p56^lck-/-) consist of immature CD4-CD8- double-negative (DN) and CD4⁺CD8⁺ double-positive (DP) thymocytes and are severely reduced in total cell number. In this report we have studied DN thymocytes from p56^lck-/- mice and found an increase in the proportion of the CD44^?CD25⁺ subset, suggesting that transit through this stage, which is known to require T cell receptor (TcR) β expression, may be delayed in the absence of p56^lck expression. In addition, the expression of a transgenic TcR β chain or TcR αβ pair did not restore thymic development in p56^lck-/- mice. However, in contrast to mice expressing a dominant negative isoform of p56^lck in which DP thymocytes do not develop, DP thymocytes still develop in nontransgenic and TcR transgenic p56^lck-/- mice. These results demonstrate that expansion of the DP subset is impaired in p56^lck-/- mice. In contrast, allelic exclusion is not severely compromised. Although there was an increase in the number of peripheral T cells expressing more than one Vβ chain in TcR transgenic p56^lck-/- mice, we found that inhibition of endogenous TcR β gene rearrangement was almost complete in thymocytes of Vβ transgenic p56^lck-/- mice and we could not detect any peripheral T cells that expressed more than one Vβ chain in non-transgenic p56^lck-/- mice.     

The generation of human innate lymphoid cells is influenced by the source of hematopoietic stem cells and by the use of G‐CSF

NK cells play a central role in the haploidentical HSC transplantation (HSCT) to cure high‐risk leukemias. Other innate lymphoid cells (ILCs) have been proposed to exert a protective role in graft‐versus‐host disease and could also contribute to anti‐microbial defence and to lymphoid tissue remodeling. Thus, we investigated the ILC differentiation potential of HSCs isolated from BM, mobilized peripheral blood (PB), and umbilical cord blood (UCB). BM CD34⁺ cells are enriched in lymphoid‐committed precursors, while PB CD34⁺ cells preferentially contain myeloid precursors. In vitro differentiation experiments revealed that the highest and the lowest CD56⁺CD161⁺ ILC recovery was detected in UCB and PB HSC cultures, respectively. Among CD56⁺CD161⁺ ILCs, the ratio between NK cells and ILC3s was similar for all HSC analyzed. ILC recovery in PB CD34⁺ cultures was lower for G‐CSF‐mobilized HSCs (good mobilizers) than for G‐CSF+plerixafor‐mobilized HSC (poor mobilizers). Moreover, G‐CSF inhibited in vitro ILC recovery and the degree of inhibition was proportional to the time of exposure to the cytokine. Thus, although all common sources of HSC for transplant differentiate towards ILCs, substantial differences exist among different sources and G‐CSF may influence ILC recovery. These data offer new clues for a better understanding of the immune reconstitution after HSCT.     

The homogeneity of the TCRδ repertoire expressed by the Thy-1dull γ δ T cell population is due to cellular selection

Thy-1^dull γ δ T cells are an unusual subset of mature TCRγ δ T cells characterized by their highly restricted TCR repertoire. In DBA/2 mice, they predominantly express the product of the Vγ1 gene together with that of a member of the Vδ6 subfamily (the Vδ6.4 gene) and their junctional sequences show very little diversity. To address the mechanisms underlying the expression of the restricted TCRγ δ repertoire, we have cloned all Vδ6 subfamily members present in DBA/2 mice and studied their frequency of expression in Thy-1^dull and Thy-1^bright γ δ thymocyte populations. Furthermore, we have also cloned non-functional Vδ6DδJδ1 rearrangements present in the Thy-1^dull γ δ T cell population and compared their Vδ6 gene utilization and their junctional sequences with those expressed by this population. Our results indicate that the restricted TCRδ repertoire expressed by the Thy-1^dull γ δ thymocytes results from cellular selection, rather than molecular constraints suggesting the existence of a limited set of self-ligands. Finally, phenotypic, functional and TCRγ δ repertoire analysis of Thy-1^dull γ δ T cells in β2 -microglobulin (β₂m)-deficient mice indicated that these putative ligands are not β₂m-dependent major histocompatibility complex class I or class I-like molecules.     

The intrinsic innervation of the lung is derived from neural crest cells as shown by optical projection tomography in Wnt1-Cre;YFP reporter mice

Within the embryonic lung, intrinsic nerve ganglia, which innervate airway smooth muscle, are required for normal lung development and function. We studied the development of neural crest-derived intrinsic neurons within the embryonic mouse lung by crossing Wnt1-Cre mice with R26R-EYFP reporter mice to generate double transgenic mice that express yellow fluorescent protein (YFP) in all neural crest cells (NCCs) and their derivatives. In addition to utilizing conventional immunohistochemistry on frozen lung sections, the complex organization of lung innervation was visualized in three dimensions by combining the genetic labelling of NCCs with optical projection tomography, a novel imaging technique that is particularly useful for the 3D examination of developing organs within embryos. YFP-positive NCCs migrated into the mouse lung from the oesophagus region at embryonic day 10.5. These cells subsequently accumulated around the bronchi and epithelial tubules of the lung and, as shown by 3D lung reconstructions with optical projection tomography imaging, formed an extensive, branching network in association with the developing airways. YFP-positive cells also colonized lung maintained in organotypic culture, and responded in a chemoattractive manner to the proto-oncogene, rearranged during transfection (RET) ligand, glial-cell-line-derived neurotrophic factor (GDNF), suggesting that the RET signalling pathway is involved in neuronal development within the lung. However, when the lungs of Ret^−/− and Gfrα1^−/− embryos, deficient in the RET receptor and GDNF family receptor α 1 (GFRα1) co-receptor respectively, were examined, no major differences in the extent of lung innervation were observed. Our findings demonstrate that intrinsic neurons of the mouse lung are derived from NCCs and that, although implicated in the development of these cells, the role of the RET signalling pathway requires further investigation.