Identification of natural killer cell receptor genes in the genome of the marsupial Tasmanian devil (Sarcophilus harrisii)

Within the mammalian immune system, natural killer (NK) cells contribute to the first line of defence against infectious agents and tumours. Their activity is regulated, in part, by cell surface NK cell receptors. NK receptors can be divided into two unrelated, but functionally analogous superfamilies based on the structure of their extracellular ligand-binding domains. Receptors belonging to the C-type lectin superfamily are predominantly encoded in the natural killer complex (NKC), while receptors belonging to the immunoglobulin superfamily are predominantly encoded in the leukocyte receptor complex (LRC). Natural killer cell receptors are emerging as a rapidly evolving gene family which can display significant intra- and interspecific variation. To date, most studies have focused on eutherian mammals, with significantly less known about the evolution of these receptors in marsupials. Here, we describe the identification of 43 immunoglobulin domain-containing LRC genes in the genome of the Tasmanian devil (Sarcophilus harrisii), the largest remaining marsupial carnivore and only the second marsupial species to be studied. We also identify orthologs of NKC genes KLRK1, CD69, CLEC4E, CLEC1B, CLEC1A and an ortholog of an opossum NKC receptor. Characterisation of these regions in a second, distantly related marsupial provides new insights into the dynamic evolutionary histories of these receptors in mammals. Understanding the functional role of these genes is also important for the development of therapeutic agents against Devil Facial Tumour Disease, a contagious cancer that threatens the Tasmanian devil with extinction.     

The fungal pattern recognition receptor, Dectin-1, and the associated cluster of C-type lectin-like receptors

The mammalian natural killer gene complex (NKC) contains several families of type II transmembrane C-type lectin-like receptors (CLRs) that are best known for their involvement in the detection of virally infected or transformed cells, through the recognition of endogenous (or self) proteinacious ligands. However, certain CLR families within the NKC, particularly those expressed by myeloid cells, recognize structurally diverse ligands and perform a variety of other immune and homoeostatic functions. One such family is the 'Dectin-1 cluster' of CLRs, which includes MICL, CLEC-2, CLEC12B, CLEC9A, CLEC-1, Dectin-1 and LOX-1. Here, we review each of these CLRs, exploring our current understanding of their ligands and functions and highlighting where they have provided new insights into the underlying mechanisms of immunity and homeostasis.     

Genomic structure, alternative splicing, and physical mapping of the killer cell lectin-like receptor G1 gene (KLRG1), the mouse homologue of MAFA

The mouse killer cell lectin-like receptor G1 (KLRG1), the mouse homologue of the mast cell function-associated antigen (MAFA), is an inhibitory C-type lectin expressed on natural killer (NK) cells and activated CD8 T cells. Here we report the complete nucleotide sequence, alternatively spliced variants, and the physical mapping of the KLRG1 gene in the mouse. The gene spans about 13 kb and consists of five exons. Short interspersed repeats of the B1 and B2 family, a LINE-1-like element, and a (CTT)170 triplet repeat were found in intron sequences. In contrast to human KLRG1 and to the murine KLR family members, mouse KLRG1 locates outside the NK complex on Chromosome 6 between the genes encoding CD9 and CD4.     

Molecular Basis for E-cadherin Recognition by Killer Cell Lectin-like Receptor G1 (KLRG1)

The killer cell lectin-like receptor G1, KLRG1, is a cell surface receptor expressed on subsets of natural killer (NK) cells and T cells. KLRG1 was recently found to recognize E-cadherin and thus inhibit immune responses by regulating the effector function and the developmental processes of NK and T cells. E-cadherin is expressed on epithelial cells and exhibits Ca²⁺-dependent homophilic interactions that contribute to cell-cell junctions. However, the mechanism underlying the molecular recognition of KLRG1 by E-cadherin remains unclear. Here, we report structural, binding, and functional analyses of this interaction using multiple methods. Surface plasmon resonance demonstrated that KLRG1 binds the E-cadherin N-terminal domains 1 and 2 with low affinity (K_d ∼7–12 μm), typical of cell-cell recognition receptors. NMR binding studies showed that only a limited N-terminal region of E-cadherin, comprising the homodimer interface, exhibited spectrum perturbation upon KLRG1 complex formation. It was confirmed by binding studies using a series of E-cadherin mutants. Furthermore, killing assays using KLRG1⁺NK cells and reporter cell assays demonstrated the functional significance of the N-terminal region of E-cadherin. These results suggest that KLRG1 recognizes the N-terminal homodimeric interface of domain 1 of E-cadherin and binds only the monomeric form of E-cadherin to inhibit the immune response. This raises the possibility that KLRG1 detects monomeric E-cadherin at exposed cell surfaces to control the activation threshold of NK and T cells.Natural killer (NK)³ cells play a critical role in the innate immune system because of their ability to kill other cells. For example, NK cells can kill virus-infected cells and tumor cells without presensitization to a specific antigen, and they produce various cytokines, including interferon-γ and tumor necrosis factor-α (1). NK cells are controlled by both inhibitory and activating receptors that are expressed on their surfaces (2). The killer cell Ig-like receptor, Ly49, CD94/NKG2, and paired Ig-like type 2 receptor families include both inhibitory and activating members and thus are designated as paired receptor families. On the other hand, some inhibitory receptors, including KLRG1 (killer cell lectin-like receptor G1), and activating receptors, such as NKG2D, also exist. The integration of the signals from these receptors determines the final functional outcome of NK cells.These inhibitory and activating receptors can also be divided into two structurally different groups, the Ig-like receptors and the C-type lectin-like receptors, based on the structural aspects of their extracellular regions. The Ig-like receptors include killer cell Ig-like receptors and the leukocyte Ig-like receptors, and the C-type lectin-like receptors include CD94/NKG2(KLRD/KLRC), Ly49(KLRA), NKG2D(KLRK), NKR-P1(KLRB), and KLRG1. Many of these immune receptors recognize major histocompatibility complex class I molecules or their relatives (2–4), but there are still many orphan receptors expressed on NK cells. KLRG1 was one such orphan receptor; however, E-cadherin was recently found to be a ligand of KLRG1 (5, 6). Although major histocompatibility complex-receptor interactions have been extensively examined, the molecular basis of non-major histocompatibility complex ligand-receptor recognition is poorly understood.KLRG1 is a type II membrane protein, with one C-type lectin domain in the extracellular region, one transmembrane region, and one immunoreceptor tyrosine-based inhibitory motif. KLRG1 is expressed on a subset of mature NK cells in spleen, lungs, and peripheral blood during normal development. KLRG1 expression is induced on the surface of NK cells during viral responses (7, 8). NK cells expressing KLRG1 produce low levels of interferon-γ and cytokines and have a slow in vivo turnover rate and low proliferative responsiveness to interleukin-15 (9). Furthermore, KLRG1 is recognized as a marker of some T cell subsets, as follows. KLRG1 defines a subset of T cells, short lived effector CD8 T cells (SLECs), which are mature effector cells that express high levels of KLRG1 and cannot be differentiated into long lived memory CD8 T cells. In addition, memory precursor effector cells express low levels of KLRG1 and harbor the potential to become long lived memory CD8 T cells (10). Since SLECs exhibit stronger effector function than memory precursor effector cells, it is potentially beneficial, in terms of preventing harmful excess cytotoxicity, that SLECs express KLRG1 at a higher level to inhibit the immune response. Taken together, the expression of KLRG1 during the viral response and normal development might confer the inhibition of effector function and the regulation of NK and T cell proliferation (9).E-cadherin plays a pivotal role in Ca²⁺-dependent cell-cell adhesion and also contributes to tissue organization and development (11–14). E-cadherin is primarily expressed on epithelial cells, and its extracellular region consists of several domains that include cadherin motifs (15, 16). These domains mediate Ca²⁺-dependent homophilic interactions to facilitate cell adhesion. When E-cadherins form cis- or trans-homodimers, they utilize their N-terminal regions as an interface, which can dock with domain 1 of another E-cadherin to form strand exchange (17). Therefore, the N-terminal region plays important roles in homophilic binding and cell adhesion.KLRG1 recognizes E-cadherins (and other class I cadherins), which are widely expressed in tissues and form tight adhesive cell-cell junctions, and Ito et al. (5) demonstrated that E-cadherin binding by KLRG1 inhibits NK cytotoxicity. Further, Gründermann et al. (6) showed that the E-cadherin-KLRG1 interaction inhibits the antigen-induced proliferation and induction of the cytolytic activity of CD8 T cells. Therefore, it is plausible that E-cadherin recognition by KLRG1, expressed on the surfaces of NK cells and T cells, may raise their activation thresholds by transducing inhibitory signals. Such an inhibition would prevent the excess injury of normal cells, which might result in inflammatory autoimmune diseases. KLRG1 may also have an important role in monitoring and removing cancer cells that lose E-cadherin expression. A recent report demonstrated that N-terminal domains 1 and 2 of E-cadherin are critical for KLRG1 recognition (18); however, despite accumulating evidence supporting the functional importance of the E-cadherin-KLRG1 interaction, the molecular basis of this interaction is poorly understood. Here, we report that the N-terminal region of E-cadherin, comprising the dimer interface, is the binding site for KLRG1. This suggests that KLRG1 does not recognize the dimeric form of E-cadherin but rather recognizes the monomeric form, which is exposed on the cell surfaces of disrupted or infected cells. This may suppress excess immune responses.     

CLEC4F Is an Inducible C-Type Lectin in F4/80-Positive Cells and Is Involved in Alpha-Galactosylceramide Presentation in Liver

CLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal), N-acetylgalactosamine (GalNAc), and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f−/−) mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5) but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes) infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3∼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer) in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT) cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.     

Human CLEC18 Gene Cluster Contains C-type Lectins with Differential Glycan-binding Specificity

The human C-type lectin 18 (clec18) gene cluster, which contains three clec18a, clec18b, and clec18c loci, is located in human chromosome 16q22. Although the amino acid sequences of CLEC18A, CLEC18B, and CLEC18C are almost identical, several amino acid residues located in the C-type lectin-like domain (CTLD) and the sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain, also known as the cysteine-rich secretory proteins/antigen 5/pathogenesis-related 1 proteins (CAP) domain, are distinct from each other. Genotyping by real-time PCR and sequencing further shows the presence of multiple alleles in clec18a/b/c loci. Flow cytometry analysis demonstrates that CLEC18 (CLEC18A, -B, and -C) are expressed abundantly in human peripheral blood cells. Moreover, CLEC18 expression is further up-regulated when monocytes differentiate into macrophages and dendritic cells. Immunofluorescence staining reveals that CLEC18 are localized in the endoplasmic reticulum, Golgi apparatus, and endosome. Interestingly, CLEC18 are also detectable in human sera and culture supernatants from primary cells and 293T cells overexpressing CLEC18. Moreover, CLEC18 bind polysaccharide in Ca²⁺-independent manner, and amino acid residues Ser/Arg³³⁹ and Asp/Asn⁴²¹ in CTLD domain contribute to their differential binding abilities to polysaccharides isolated from Ganoderma lucidum (GLPS-F3). The Ser³³⁹ (CLEC18A) → Arg³³⁹ (CLEC18A-1) mutation completely abolishes CLEC18A-1 binding to GLPS-F3, and a sugar competition assay shows that CLEC18 preferentially binds to fucoidan, β-glucans, and galactans. Because proteins with the SCP/TAPS/CAP domain are able to bind sterol and acidic glycolipid, and are involved in sterol transport and β-amyloid aggregation, it would be interesting to investigate whether CLEC18 modulates host immunity via binding to glycolipids, and are also involved in glycolipid transportation and protein aggregation in the future.     

Cloning of Clr, a new family of lectin-like genes localized between mouse Nkrp1a and Cd69

We report the identification of a novel family of genes, named Clr, encoding C-type lectin-like molecules, which maps in the natural killer (NK) gene complex (NKC) on mouse Chromosome 6. Genomic sequence analysis indicates the presence of at least seven members between Nkrpla and Cd69. By RT-PCR, at least three members of the family are expressed on interleukin-2-activated NK cells. Sequence analysis revealed complete open reading frames of 203-205 amino acids, with a carboxyl-terminal C-type lectin-like carbohydrate recognition domain (CRD). The CRDs of the Clr proteins exhibit a significant degree of homology with the known NKC-encoded NK-cell receptors. However, a key cysteine usually present in the CRD is missing in the Clr proteins, suggesting that their ligands and functions are distinct from other molecules encoded in the NKC.     

Identification of natural killer cell receptor clusters in the platypus genome reveals an expansion of C-type lectin genes

Natural killer (NK) cell receptors belong to two unrelated, but functionally analogous gene families: the immunoglobulin superfamily, situated in the leukocyte receptor complex (LRC) and the C-type lectin superfamily, located in the natural killer complex (NKC). Here, we describe the largest NK receptor gene expansion seen to date. We identified 213 putative C-type lectin NK receptor homologs in the genome of the platypus. Many have arisen as the result of a lineage-specific expansion. Orthologs of OLR1, CD69, KLRE, CLEC12B, and CLEC16p genes were also identified. The NKC is split into at least two regions of the genome: 34 genes map to chromosome 7, two map to a small autosome, and the remainder are unanchored in the current genome assembly. No NK receptor genes from the LRC were identified. The massive C-type lectin expansion and lack of Ig-domain-containing NK receptors represents the most extreme polarization of NK receptors found to date. We have used this new data from platypus to trace the possible evolutionary history of the NK receptor clusters. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

KLRG1 Negatively Regulates Natural Killer Cell Functions through the Akt Pathway in Individuals with Chronic Hepatitis C Virus Infection

In this study, we demonstrate that killer cell lectin-like receptor subfamily G member 1 (KLRG1), a transmembrane protein preferentially expressed on T cells, is highly expressed on CD56⁺ NK cells, which are significantly reduced in their numbers and functions in the peripheral blood of patients with chronic hepatitis C virus (HCV) infection compared to subjects without infection. KLRG1 expression is also upregulated on healthy NK cells exposed to Huh-7 hepatocytes infected with HCV in vitro. Importantly, the expression levels of KLRG1 are inversely associated with the capacity of NK cells to proliferate and to produce gamma interferon (IFN-γ) but positively associated with apoptosis of NK cells in response to inflammatory cytokine stimulation. KLRG1⁺ NK cells, including CD56^bright and CD56^dim subsets, exhibit impaired cell activation and IFN-γ production but increased apoptosis compared to KLRG1⁻ NK cells, particularly in HCV-infected individuals. Importantly, blockade of KLRG1 signaling significantly recovered the impaired IFN-γ production by NK cells from HCV-infected subjects. Blockade of KLRG1 also enhanced the impaired phosphorylation of Akt (Ser473) in NK cells from HCV-infected subjects. Taken together, these results indicate that KLRG1 negatively regulates NK cell numbers and functions via the Akt pathway, thus providing a novel marker and therapeutic target for HCV infection.     

Kupffer cell receptor CLEC4F is important for the destruction of desialylated platelets in mice

The liver has recently been identified as a major organ for destruction of desialylated platelets. However, the underlying mechanism remains unclear. Kupffer cells, which are professional phagocytic cells in the liver, comprise the largest population of resident tissue macrophages in the body. Kupffer cells express a C-type lectin receptor, CLEC4F, that recognizes desialylated glycans with an unclear in vivo role in mediating platelet destruction. In this study, we generated a CLEC4F-deficient mouse model (Clec4f^−/−) and found that CLEC4F was specifically expressed by Kupffer cells. Using the Clec4f^−/− mice and a newly generated platelet-specific reporter mouse line, we revealed a critical role for CLEC4F on Kupffer cells in mediating destruction of desialylated platelets in the liver in vivo. Platelet clearance experiments and ultrastructural analysis revealed that desialylated platelets were phagocytized predominantly by Kupffer cells in a CLEC4F-dependent manner in mice. Collectively, these findings identify CLEC4F as a Kupffer cell receptor important for the destruction of desialylated platelets induced by bacteria-derived neuraminidases, which provide new insights into the pathogenesis of thrombocytopenia in disease conditions such as sepsis.Subject terms: Glycobiology, Cell death and immune response, Haematological diseases     

CLEC5A Regulates Japanese Encephalitis Virus-Induced Neuroinflammation and Lethality

CLEC5A/MDL-1, a member of the myeloid C-type lectin family expressed on macrophages and neutrophils, is critical for dengue virus (DV)-induced hemorrhagic fever and shock syndrome in Stat1 ^−/− mice and ConA-treated wild type mice. However, whether CLEC5A is involved in the pathogenesis of viral encephalitis has not yet been investigated. To investigate the role of CLEC5A to regulate JEV-induced neuroinflammation, antagonistic anti-CLEC5A mAb and CLEC5A-deficient mice were generated. We find that Japanese encephalitis virus (JEV) directly interacts with CLEC5A and induces DAP12 phosphorylation in macrophages. In addition, JEV activates macrophages to secrete proinflammatory cytokines and chemokines, which are dramatically reduced in JEV-infected Clec5a^−/− macrophages. Although blockade of CLEC5A cannot inhibit JEV infection of neurons and astrocytes, anti-CLEC5A mAb inhibits JEV-induced proinflammatory cytokine release from microglia and prevents bystander damage to neuronal cells. Moreover, JEV causes blood-brain barrier (BBB) disintegrity and lethality in STAT1-deficient (Stat1 ^−/−) mice, whereas peripheral administration of anti-CLEC5A mAb reduces infiltration of virus-harboring leukocytes into the central nervous system (CNS), restores BBB integrity, attenuates neuroinflammation, and protects mice from JEV-induced lethality. Moreover, all surviving mice develop protective humoral and cellular immunity against JEV infection. These observations demonstrate the critical role of CLEC5A in the pathogenesis of Japanese encephalitis, and identify CLEC5A as a target for the development of new treatments to reduce virus-induced brain damage.     

Mapping the CLEC12A expression on myeloid progenitors in normal bone marrow; implications for understanding CLEC12A‐related cancer stem cell biology

The C‐type lectin domain family 12, member A (CLEC12A) receptor has emerged as a leukaemia‐associated and cancer stem cell marker in myeloid malignancies. However, a detailed delineation of its expression in normal haematopoiesis is lacking. Here, we have characterized the expression pattern of CLEC12A on the earliest stem‐ and myeloid progenitor subsets in normal bone marrow. We demonstrate distinct CLEC12A expression in the classically defined myeloid progenitors, where on average 39.1% (95% CI [32.5;45.7]) of the common myeloid progenitors (CMPs) expressed CLEC12A, while for granulocyte‐macrophage progenitors and megakaryocyte‐erythroid progenitors (MEPs), the average percentages were 81.0% (95% CI [76.0;85.9]) and 11.9% (95% CI [9.3;14.6]), respectively. In line with the reduced CLEC12A expression on MEPs, functional assessment of purified CLEC12A^+/? CMPs and MEPs in the colony‐forming unit assay demonstrated CLEC12A⁺ subsets to favour non‐erythroid colony growth. In conclusion, we provide evidence that the earliest CLEC12A⁺ cell in the haematopoietic tree is the classically defined CMP. Furthermore, we show that CLEC12A‐expressing CMPs and MEPs are functionally different than their negative counterparts. Importantly, these data can help determine which cells will be spared during CLEC12A‐targeted therapy, and we propose CLEC12A to be included in future studies of myeloid cancer stem cell biology.     

Reduced expression of SRC family kinases decreases PI3K activity in NBS1-/- lymphoblasts

SRC family kinases (SFKs) are involved in the activation of phosphatidylinositol-3-kinase (PI3K). In addition, the activity of this lipid kinase can be regulated by the DNA repair protein NBS1. Here, we describe a disturbed expression of some members of the non-receptor tyrosine kinase family in lymphoblastoid cell lines generated from cells of Nijmegen breakage syndrome (NBS) patients. Especially, only minor amounts of the kinases LCK and HCK are expressed in the NBS1^−/− cell lines as compared to the consanguineous NBS1^+/− cells. We demonstrate that SFK activity is important for a proper activation of PI3K in these cells and that it is reduced in NBS1^−/− cells. We provide evidence that the observed reduced PI3K activity in NBS lymphoblasts is caused by an impaired expression of the SFKs LCK and/or HCK. Thus, our data establish a new function for the NBS1 protein as a regulator of PI3K activity via SFK members.     

Neural-Cadherin Influences the Homing of Terminally Differentiated Memory CD8 T Cells to the Lymph Nodes and Bone Marrow

Memory T (T_M) cells play an important role in the long-term defense against pathogen reinvasion. However, it is still unclear how these cells receive the crucial signals necessary for their longevity and homeostatic turnover. To understand how T_M cells receive these signals, we infected mice with lymphocytic choriomeningitis virus (LCMV) and examined the expression sites of neural cadherin (N-cadherin) by immunofluorescence microscopy. We found that N-cadherin was expressed in the surroundings of the white pulps of the spleen and medulla of lymph nodes (LNs). Moreover, T_M cells expressing high levels of killer cell lectin-like receptor G1 (KLRG1), a ligand of N-cadherin, were co-localized with N-cadherin⁺ cells in the spleen but not in LNs. We then blocked N-cadherin in vivo to investigate whether it regulates the formation or function of T_M cells. The numbers of CD127^hiCD62L^hi T_M cells in the spleen of memory P14 chimeric mice declined when N-cadherin was blocked during the contraction phase, without functional impairment of these cells. In addition, when CD127^loKLRG1^hi T_M cells were adoptively transferred into anti–N-cadherin–treated mice compared with control mice, the number of these cells was reduced in the bone marrow and LNs, without functional loss. Taken together, our results suggest that N-cadherin participates in the development of CD127^hiCD62L^hi T_M cells and homing of CD127^loKLRG1^hi T_M cells to lymphoid organs.     

Identification of CLEC12B, an inhibitory receptor on myeloid cells

Activation of immune cells has to be tightly controlled to prevent detrimental hyperactivation. In this regulatory process molecules of the C-type lectin-like family play a central role. Here we describe a new member of this family, CLEC12B. The extracellular domain of CLEC12B shows considerable homology to the activating natural killer cell receptor NKG2D, but unlike NKG2D, CLEC12B contains an immunoreceptor tyrosine-based inhibition motif in its intracellular domain. Despite the homology, CLEC12B does not appear to bind NKG2D ligands and therefore does not represent the inhibitory counterpart of NKG2D. However, CLEC12B has the ability to counteract NKG2D-mediated signaling, and we show that this function is dependent on the immunoreceptor tyrosine-based inhibition motif and the recruitment of the phosphatases SHP-1 and SHP-2. Using monoclonal anti-CLEC12B antibodies we found de novo expression of this receptor on in vitro generated human macrophages and on the human myelo-monocytic cell line U937 upon phorbol 12-myristate 13-acetate treatment, suggesting that this receptor plays a role in myeloid cell function.     

Comparative gene expression analysis of the engulfment and cell motility (ELMO) protein family in the mouse brain

Engulfment and cell motility (ELMO) proteins bind to Dock180, a guanine nucleotide exchange factor (GEF) of the Rac family, and regulate GEF activity. The resultant ELMO/Dock180/Rac module regulates cytoskeletal reorganization responsible for the engulfment of apoptotic cells, cell migration, and neurite extension. The expression and function of Elmo family proteins in the nervous system, however, are not yet fully understood. Here, we characterize the comparative gene expression profiles of three Elmo family members (Elmo1, Elmo2, and Elmo3) in the brain of C57BL/6J mice, a widely used inbred strain, together with reeler mutant mice to understand gene expression in normal laminated brain areas compared with abnormal areas. Although all three Elmo genes showed widespread mRNA expression over various mouse tissues tested, Elmo1 and Elmo2 were the major types expressed in the brain, and three Elmo genes were up-regulated between the first postnatal week (infant stage) and the third postnatal week (juvenile, weaning stage). In addition, the mRNAs of Elmo genes showed distinct distribution patterns in various brain areas and cell-types; such as neurons including inhibitory interneurons as well as some non-neuronal cells. In the cerebral cortex, the three Elmo genes were widely expressed over many cortical regions, but the predominant areas of Elmo1 and Elmo2 expression tended to be distributed unevenly in the deep (a lower part of the VI) and superficial (II/III) layers, respectively, which also changed depending on the cortical areas and postnatal stages. In the dentate gyrus of the hippocampus, Elmo2 was expressed in dentate granule cells more in the mature stage rather than the immature-differentiating stage. In the thalamus, Elmo1 but not the other members was highly expressed in many nuclei. In the medial habenula, Elmo2 and Elmo3 were expressed at intermediate levels. In the cerebellar cortex, Elmo1 and Elmo2 were expressed in differentiating-mature granule cells and mature granule cells, respectively. In the Purkinje cell layer, Elmo1 and Elmo2 were expressed in Purkinje cells and Bergmann glia, respectively. Disturbed cellular distributions and laminar structures caused by the reeler mutation did not severely change expression in these cell types despite the disturbed cellular distributions and laminar structures, including those of the cerebrum, hippocampus, and cerebellum. Taken together, these results suggested that these three Elmo family members share their functional roles in various brain regions during prenatal-postnatal development.