Mouse Hoxb8 mutants show unexpected behavior manifested by compulsive grooming and hair removal, similar to behavior in humans with the obsessive-compulsive disorder spectrum disorder trichotillomania. As Hox gene disruption often has pleiotropic effects, the root cause of this behavioral deficit was unclear. Here we report that, in the brain, Hoxb8 cell lineage exclusively labels bone marrow-derived microglia. Furthermore, transplantation of wild-type bone marrow into Hoxb8 mutant mice rescues their pathological phenotype. It has been suggested that the grooming dysfunction results from a nociceptive defect, also exhibited by Hoxb8 mutant mice. However, bone marrow transplant experiments and cell type-specific disruption of Hoxb8 reveal that these two phenotypes are separable, with the grooming phenotype derived from the hematopoietic lineage and the sensory defect derived from the spinal cord cells. Immunological dysfunctions have been associated with neuropsychiatric disorders, but the causative relationships are unclear. In this mouse, a distinct compulsive behavioral disorder is associated with mutant microglia.

Unfortunately, animal models that directly associate distinct behavioral deficits with defective microglia have been lacking. Here we provide such a model, which should allow interrogation at the molecular genetic and cellular levels, the roles of microglia in promoting normal behavior, and how perturbation of microglia leads to pathological behavior.

The above experiments strongly support the hypothesis that the excessive pathological grooming behavior observed in Hoxb8 mutant mice originates from defective microglia, thus directly connecting hematopoietic function to mouse behavior. The extensive role of microglia, as the brain's monitor and responder of immune activity, in the normal function of our brain is becoming increasingly apparent. As examples, immunological dysfunctions have been widely linked to many psychiatric disorders including OCD, major depression, bipolar disorder, autism, schizophrenia, and Alzheimer's disease (). In addition, results from genome-wide association studies suggest that genes whose dysfunction have been implicated in immune dysfunction and/or signaling contribute to increased susceptibility to the above mentioned mental disorders ().

Since implementation of grooming in rodents is rooted within the brain, we anticipated that Hoxb8 would be expressed in a neural circuit that modulates grooming behavior. Instead, as we report here, we have surprisingly found that, in the brain, the site that generates and implements grooming behavior, the only detectable cells derived from Hoxb8 cell lineage are microglia. Second, we demonstrate that normal bone marrow transplantation into lethally irradiated Hoxb8 mutant mice rescues the excessive pathological grooming behavior, without correcting the spinal cord defects. Third, conditional restriction of Hoxb8 deletion to the hematopoietic system results in mice with the excessive grooming and hair removal behavioral defects, without induction of the nociceptive/spinal cord defects. Finally and conversely, conditional deletion of Hoxb8 in the spinal cord generates mice with the spinal cord sensory defects, but with normal grooming behavior.

It was quite unexpected that disruption of a Hox gene should result in a distinct behavioral deficit such as excessive and pathological grooming (). Hox genes are normally involved in establishing body plans by providing positional values along the major axes of the embryo (). However, Hox genes also have direct roles in the formation of multiple tissues and organs, including the formation of the hematopoietic system, and, with respect to Hoxb8, maintenance and differentiation of myeloid progenitor cells, one of the two known sources of microglia ().

Hoxb8 mutant mice also show altered response to nociceptive and thermal stimuli, which have been attributed to deficiencies in the formation and organization of interneurons in the dorsal spinal cord laminae I and II that receive the majority of nociceptive inputs ().further suggested that the excessive and pathological grooming defects previously described in Hoxb8 mutants result from to these sensory spinal cord defects.

Mice homozygous for a loss of function mutation in Hoxb8 show excessive grooming. The syntax of grooming appears normal, but the number of incidences per unit time and the duration of grooming bouts are increased (). However, the behavior is pathological, for it leads to hair removal and self-inflicted open skin lesions at the over groomed sites. This behavior is very similar to that described for humans with the obsessive-compulsive disorder (OCD) spectrum disorder trichotillomania, where compulsive removal of hair is also a hallmark. This disorder is quite common in humans with an occurrence ranging from 1.9 to 2.5 per 100 in seven separate international communities (). Curiously, these mutant mice also excessively groom their wild-type cage mates. This aspect of the phenotype suggested that the peripheral nervous system is not likely responsible for the excessive grooming behavior ().

Grooming in mammals is an innate, stereotypic behavior with a well defined syntax (). The head is invariably groomed first, followed by body regions, and finally the anogenital region and tail. This cephalocaudal progression of grooming is defined as the “syntactic groom chain.” Previous studies have shown that multiple regions of the rodent brain, notably the brainstem, striatum, and cortex are used to implement the syntactic groom chain ().

Next we wanted to determine if conditional deletion of Hoxb8 in the spinal cord recapitulates the nociceptive defects and whether or not they would be associated with the pathological grooming defects. Hoxc8 cell lineage in the spinal cord has a broad pattern of expression very similar to that of Hoxb8 ( Figure 7 A ). However, relative to Hoxb8, Hoxc8 is poorly expressed in the hematopoietic system. Most importantly, the number of Hoxc8-ICre/Rosa26-YFP-labeled microglia in the brain is much lower (i.e., more than 10-fold lower) when compared to Hoxb8-labeled cell lineage (compare, for example, Figure 7 B to Figure 1 F, both derived from adult cortical brain sections). Quantitation of YFPIba1+ cell counts relative to Iba1+ cells, which label microglia, using the respective Hoxb8-ICre and Hoxc8-ICre drivers is shown in Figure 7 C. Since Hoxc8 is strongly expressed in the spinal cord, but poorly represented within microglia, the Hoxc8-ICre mouse can be used to determine whether or not induction of the nociceptive defects are sufficient to initiate the pathological grooming defects. Ten such animals (i.e., Hoxc8-ICre; Hoxb8c/c) were collected. None of these mutant animals developed the hair removal and skin lesion patterns typical of Hoxb8 mutant mice ( Figure 7 D). Also, the time engaged in grooming by these mice was not significantly greater than in their control siblings ( Figure 7 E). However the latency of their response to heat was significantly longer than control siblings and was comparable to that of Hoxb8 mutant mice ( Figure 7 F). Further, examination of these mutants by immunohistochemistry with calbindin and calretinin antibodies showed that ablating Hoxb8 in the Hoxc8 expression domain fully recapitulated the dorsal horn spinal cord defects typical of Hoxb8 mutant mice ( Figures 7 G–7L). Thus, conditional deletion of Hoxb8 in the Hoxc8 expression domain (i.e., the spinal cord) cleanly separated the nociception/dorsal spinal cord defects from the excessive pathological grooming defects. Selective induction of the Hoxb8-associated nociceptive/spinal cord defects (Hoxc8-ICre) is not sufficient to induce the pathological grooming defects.

(G–L) The numbers of interneurons in laminae I and II are decreased in Hoxc8-ICre-conditional Hoxb8 mutants. Spinal cord sections at L4–L5 levels were collected from wild-type mice (G and J), Hoxb8 mutant mice (H and K), and Hoxc8-ICre-conditional Hoxb8 mutants (I and L) and stained for calbindin (G–I) and calretinin (J–L). The positive regions are highlighted with a white dashed line.

(C) Compared to the Hoxb8 lineage, only a small percentage (<3%) of ramified microglia were labeled by the Hoxc8 lineage. The percentage of YFP-positive cells to total ramified microglia (Iba1-positive cells) was determined from sections through the cerebral cortex derived from three Hoxb8-ICre/Rosa26-YFP (black column) and three Hoxc8-ICre/Rosa26-YFP mice (white column). The values are mean ± SEM. ∗ p < 0.05.

(A) Hoxc8 lineage is present in all laminae of the spinal cord. X-gal staining was performed in spinal cord sections collected from mice carrying both Hoxc8-ICre and ROSA26-LacZ alleles.

In order to perform tissue-specific deletion of Hoxb8, we constructed a conditional allele in which the entire coding sequence of this gene was flanked by Lox511 sites ( Figure S1 C). Mice carrying the Hoxb8 conditional allele were crossed to Tie2Cre males to produce conditional mutants. Five conditional mutants carrying Tie2Cre and homozygous for the Hoxb8 conditional mutation were collected, and their grooming behavior was analyzed. Four out of the five mice developed hairless patches very similar to the patterns observed in Hoxb8 mutant mice ( Figure 6 B). The grooming times, measured on the Laboras platforms, were significantly longer than control siblings and comparable to those observed in Hoxb8 mutant mice (n = 5; Figure 6 C). However, these mice did not exhibit heat insensitivity ( Figure 6 D), and immunohistochemical analysis of their dorsal spinal cord laminae I and II labeled for calbindin and calretinin appeared normal when compared to Hoxb8 mutants ( Figures 6 E–6J). Thus, restricted deletion of Hoxb8 in the hematopoietic system is sufficient to induce pathological grooming in mice and, importantly, the presence of the nociceptive defects is not observed or required for induction of the aberrant grooming behavior.

To further explore the causality of the Hoxb8 mutant phenotype, we used Tie2 Cre/loxP-based conditional mutagenesis. Tie2 was originally considered an endothelial cell marker. However,showed that a Tie2Cre transgenic line displayed loxP-mediated recombination in all hematopoietic cells, as well as in endothelial cells. In the brain, Tie2 lineage is present in blood vessels and microglia, but not in interneurons of the dorsal spinal cord ( Figure 6 A and data not shown).

(E–J) No histological defects analogous to those in Hoxb8 mutant mice were found in dorsal spinal laminae of Tie2Cre conditional mutants. Spinal cord sections at L4–L5 levels were collected from wild-type mice, Hoxb8 mutant mice, and Tie2Cre conditional Hoxb8 mutants. Interneurons in laminae I and II were stained for calbindin (E–G) and calretinin (H–J). The regions with neurons positively staining for these markers are circumscribed with a white dashed boundary. Scale bar: (A) 50 μM; (E–J) 100 μM.

(A) Tie2 lineage is present in CNS microglia. Brains and spinal cords from mice carrying the Tie2Cre transgene and ROSA26-YFP allele were stained with anti-GFP antibodies. The microglial identity was confirmed by double staining with CD11b (data not shown).

The first insight that the excessive grooming and hair removal behavior could be separated from the sensory spinal cord defects in our Hoxb8 mutant mice was gained from the transplantation experiments. Irradiated Hoxb8 mutant mice transplanted with normal bone marrow show normal grooming behavior, but retained their thermal stimuli insensitivity spinal cord defects ( Figure 5 ). The latency of response to thermal stimuli in the bone marrow-rescued animals was comparable to that observed in Hoxb8 mutant mice and significantly longer than in wild-type mice. Thus, while normal bone marrow transplants into irradiated Hoxb8 mutant mice efficiently rescued the pathological grooming defect, it did not restore the defective pain response.

Hoxb8 mutant mice, whose pathological grooming defects were rescued by normal bone marrow transplants, still exhibit significantly longer latency of response to heat (53°C Hot Plate Test), comparable to the Hoxb8 mutant mice. White column, wild-type controls (n = 14); black column, Hoxb8 mutants (n = 11); gray column, Hoxb8 mutants phenotype rescued with normal bone marrow (n = 6). Data were collected 4–5 months after bone marrow transplantation. Columns represent the mean ± SEM. ∗ p < 0.05 versus wild-type.

Consistent with the localized scratching caused by a response to chronic itching,reported that their behavior could be alleviated by localized injection of lidocaine. We tested whether our Hoxb8 mutant mice would respond to lidocaine treatment. Eight Hoxb8 mutant mice and eight control sibling mice were injected with lidocaine into regions where hairless patches had developed and placed on Laboras platforms to measure their grooming periods. Lidocaine treatment did not alter the grooming behavior of either the Hoxb8 mutant or wild-type mice ( Figure S4 B).

Scratching in rodents is a rather simple movement made by the hind limbs () that can be distinguished from grooming by the Laboras platforms. To determine if our Hoxb8 mutants exhibited excessive scratching, eight Hoxb8 mutants and eight wild-type mice were placed on the Laboras platforms set to score scratching. No significant differences in time spent scratching were detected between the Hoxb8 mutant and wild-type controls ( Figure S4 A ). Only in mutant animals that had developed severe lesions as a consequence of compulsive, excessive grooming could increased scratching begin to be observed. For comparison we provide measurements of time spend grooming over the same duration of time.

(C) Animals homozygous for the Hoxb8-CreERTM knockin allele develop lesions in the posterior body. In this mouse line, the majority of Hoxb8 exon 1 coding sequence was replaced with the CreERfusion polypeptide (see Figure S2 D). The CreERknockin effectively generates a Hoxb8 null allele, and expands the length of the Hoxb8 gene by approximately 1.5 kb. The Hoxb8-CreERTM mutants develop lesions in the rump and perigenital regions with a penetrance of ∼10%.

(B) Lidocaine treatment does not alter the grooming behavior in Hoxb8 mutant mice. Subcutaneous administration of lidocaine was performed to determine if local anesthesia would reduce the excessive grooming defect observed in Hoxb8 mutant mice. However, the treated Hoxb8 mutants (black column) spent approximately twice as much time grooming relative to the wild-type mice (white column), which is very similar to the differential observed in untreated Hoxb8 mutant. Columns represent the mean ± SEM. ∗ p < 0.05.

(A) Grooming and scratching can be separately analyzed by Laboras platforms. Time spent scratching in eight pairs of Hoxb8 mutants (black columns) and wild-type mice (white) was measured in a 4 hr period. Grooming times of Hoxb8 mutants and wild-type mice during the same period is shown for comparison. Columns represent the mean ± SEM. ∗ p < 0.05.

A puzzling aspect of the hair removal phenotype described byis that it appears quite different from the excessive grooming and hair removal phenotype that we observe in our Hoxb8 mutant mice. In their study, the hairless patches and skin lesions are very localized to the dorsal rump () and appear more consistent with the consequences of scratching a chronic itch. We observe a gradual progression of hair removal along most of the ventral surface of the mouse and extending to the lateral surfaces, which correlates with the consequences of an excessive normal grooming pattern (see, for example, Figure 3 A). The hair is removed from the overgroomed areas in our mutant mice by the use of their teeth and accumulates in between their incisors, reflecting an extension of normal grooming behavior rather than scratching with their hind paws (). What we observe and have reported in our Hoxb8 mutant mice is that the grooming syntax does not appear to be altered, but rather the number and duration of grooming bouts are increased. This aspect of the grooming phenotype has not been reported by. The marked difference in the pattern of hair removal and the excessive normal grooming observed in our mutant animals, rather than excessive scratching, suggests that the two groups might be studying different behavioral paradigms in their respective Hoxb8 mutant mice.

have reported that mice mutant for Hoxb8 show attenuated responses to noxious and thermal stimuli and display a reduction and disorganization of interneurons in laminae I and II of the dorsal horn of the spinal cord, which receive the majority of the nociceptive and thermal sensations. We have observed very similar insensitivity to noxious and thermal stimuli, as well as dorsal horn spinal cord defects in our Hoxb8 mutant mice ( Figure 4 ). Figures 4 A and 4B show intact cervical spinal cord sections from wild-type and Hoxb8 mutant mice stained with a general neuronal marker anti-Neu N. It is apparent from these histological sections that the number of neuronal cell bodies present in the dorsal horn of Hoxb8 mutant mice is significantly decreased relative to those from wild-type mice. Immunohistochemical analysis of the spinal cord (shown at lumbar levels 4 and 5) further illustrate that in Hoxb8 mutant mice, relative to control mice, disorganization and reduction in numbers are apparent in both the input sensory fibers, labeled with CGRP, as well as interneurons in laminea I and II, labeled for calbindin and calretinin ( Figures 4 D–4I). Nociceptive insensitivity was demonstrated by significantly greater latency time required by Hoxb8 mutant mice to respond to heat, relative to wild-type control mice ( Figure 4 C).further suggested that the nociceptive/spinal cord defects account for the excessive grooming and hair removal phenotypes that we previously described.

(D–I) Anatomical defects in dorsal spinal cord of Hoxb8 mutant mice. Spinal cord sections shown at L4–L5 from wild-type mice (D–F) and Hoxb8 mutant mice (G–I). The spinal cord sections were labeled with a marker for nociceptive sensory fibers (CGRP) and with interneuron markers for lamina I and II (calbindin and calretinin). The numbers of interneurons in laminae I and II are decreased and disorganized in Hoxb8 mutant mice relative to wild-type mice. Scale bar: 100 μM.

(A and B) Spinal cord sections at cervical levels in wild-type mice (A) and Hoxb8 mutants (B) stained with anti-NeuN antibody. These sections are representative of wild-type and Hoxb8 mutant sections taken along the spinal cord from C4 through the lumbar region L5. Neuron counts are decreased and the remaining interneurons are noticeably disorganized in the mutant spinal laminae.

The role of Hoxb8 in hematopoiesis has not been fully elucidated. In the literature, based on experiments involving Hoxb8 overexpression, a case has been made for Hoxb8 involvement in the maintenance and differentiation of the myeloid progenitor pool (). The very early expression of Hoxb8 during hematopoiesis and our bone marrow transplantation competition studies suggest a broader role for Hoxb8 in hematopoiesis, affecting both the myeloid and lymphoid lineages. Do T and B cells contribute to the compulsive grooming phenotype observed in Hoxb8 mutant mice? To test this possibility, bone marrow transplantations using bone marrow derived from RAG2 mice, which do not produce T and B cells, into irradiated Hoxb8 mutant mice were carried out. These experiments show that RAG2 bone marrow can rescue the excessive pathological grooming phenotype. However, the robustness of rescue is not as high as with wild-type bone marrow either in terms of the extent of hair replenishment of the hairless patches ( Figures S3 D and S3E) or in time spent grooming by these animals ( Figure S3 F). These experiments suggest that T and B cells do not have a primary role in the induction of the Hoxb8 mutant pathological grooming phenotype, but that T and B cell deficiencies may contribute to the severity of the phenotype.

The above experiments demonstrate that transplantation of normal bone marrow can efficiently rescue the Hoxb8 mutant grooming phenotypes, including restoration of the hairless patches, healing of open lesions, and reduction of excessive grooming times back to baseline. In separate experiments we have shown that CAG-GFP-labeled bone marrow () transferred into wild-type-irradiated mice can be detected in brain microglia 4 weeks after transplantation ( Figure S3 A ) and increases by 12 weeks after transplantation ( Figure S3 B). The lower efficiency of conferring the Hoxb8 mutant phenotype to irradiated wild-type mice following transplantation of Hoxb8 mutant bone marrow, compared to efficient rescue of the mutant phenotype by transplantation of normal bone marrow into irradiated Hoxb8 mutant mice, appears to be a consequence of the lower robustness of Hoxb8 mutant bone marrow. Reconstitution of lethally irradiated wild-type mice with bone marrow containing 50% GFP labeled wild-type cells and 50% unlabeled Hoxb8 mutant cells showed that myeloid and T cells derived from Hoxb8 mutant bone marrows were at a measurable disadvantage relative to the normal GFP-labeled wild-type cells blood lineages ( Figure S3 C). In contrast, B cells derived from Hoxb8 mutant bone marrow were at a competitive advantage relative to the wild-type cells.

(D–F) RAG2 Mutant Bone Marrow Transplantations Rescue Hairless Patches and Reduce Excessive Grooming, Related to Figure 3 . (D and E) Due to a defect in DNA rearrangement, RAG2 mutants are B cell and T cell-deficient. When RAG2 mutant bone marrow was transplanted into Hoxb8 mutant mice, three out of 7 irradiated Hoxb8 mutants exhibited hair re-growth after 3.5-months (A) or 4.5-months (B). (F) Grooming times of these bone marrow chimeras were measured on Laboras platforms 4 to 5 months after bone marrow transplantation relative to Hoxb8 mutants (black bar) and wild type controls (white bar). Grey bar represents the grooming phenotype of Hoxb8 mutant mice rescued with normal bone marrow. Stippled bar shows the grooming times of Hoxb8 mutant mice rescued with RAG2 mutant bone marrow. Columns represent the mean ± SEM.p < 0.05 versus mutant.

(C) Competitive repopulation test with bone marrow from Hoxb8 mutant mice. Bone marrow containing 50% of CAG-GFP-labeled normal bone marrow cells and 50% of non-GFP Hoxb8 mutant bone marrow cells were transplanted into irradiated wild-type recipients. Peripheral blood samples were collected 2 months after bone marrow transplantation and the percentage of GFP positive cells was determined in granulocyte/monocyte, B cell and T cell lineages. White columns represent the relative cell counts of GFP-positive cells derived from normal bone marrow. Black columns show the fractions of GFP negative cells derived from Hoxb8 mutant bone marrow (n = 3). Columns represent the mean ± SEM. ∗ p < 0.05.

(A and B) Infiltration of microglia in the cerebral cortex after bone marrow transplantation. Bone marrow cells were collected from CAG-GFP mice and transplanted to irradiated wild-type recipients. Brains of these bone marrow chimeras were dissected (A) 4 weeks or (B) 12 weeks after bone marrow transplantation, sectioned, stained with anti-GFP antibodies and analyzed with confocal microscopy. (A) Four weeks after bone marrow transplantation, only a few cells were found in the cerebral cortex, and they did not display ramified microglia morphology. CC, cerebral cortex; CP, choroid plexus. (B) Twelve weeks after bone marrow transplantation, ∼30% of total microglia were GFP positive and showed ramified microglial morphology. Scale bar: 100 μM.

We showed above that the only cells derived from Hoxb8 cell lineage detectable in the adult mouse brain are microglia, likely of bone marrow origin. To investigate whether a dysfunction in the hematopoietic system is responsible for the Hoxb8-excessive grooming and hair removal phenotypes, bone marrow transplantation experiments were conducted. Four different experimental groups were included. Bone marrow cells were collected from both wild-type or Hoxb8 mutant adult mice and transplanted into either irradiated Hoxb8 mutant or wild-type mice at 2 months of age, respectively. The development or regrowth of hairless patches, as well as grooming times, were monitored in all mice receiving transplants over a 5 month period. In the control group, in which irradiated wild-type mice received wild-type bone marrow cells, the grooming behavior remained normal, and no hairless patches were detected for the duration of the experiment (n = 10; data not shown). In the second control group, irradiated Hoxb8 mutant mice received Hoxb8 mutant bone marrow. These animals showed deteriorating health, and 6 out of 11 animals in this group died before the observation period was completed, presumably due to difficulty to reestablish bone marrow with a mutant erythropoietic lineage. The surviving recipients continued to exhibit very severe grooming and self-mutilation phenotypes, without detectable hair regrowth during the observation period. Figure 3 A shows one of the Hoxb8 mutants 4 weeks after transplantation with normal bone marrow. In the group of irradiated Hoxb8 mutant mice receiving normal, wild-type bone marrow cells, the hairless patches continued to develop for the first few weeks after transplantation. However, starting from 3 months after bone marrow transplantation, six of ten animals showed extensive regrowth of hair in the hairless areas and healing of open lesions. Four of them fully recovered and were indistinguishable from wild-type mice ( Figures 3 B and 3C). Grooming time was assessed on the Laboras platforms 4 months after bone marrow transplantation. The grooming times of these animals (n = 6) decreased significantly to levels comparable to those of control mice ( Figure 3 D). Finally, among the irradiated wild-type mice that were transplanted with bone marrow collected from Hoxb8 mutant mice, two of ten animals developed hairless patches very similar to the phenotype observed in Hoxb8 mutant mice ( Figure 3 E). These mice (n = 2) also showed increased grooming times relative to wild-type animals, but not increases as high as normally shown in Hoxb8 mutant mice ( Figure 3 F).

(F) Grooming times of two wild-type mice transplanted with mutant bone marrow that developed hairless patches. These experimental animals (gray column, n = 2) showed elevated grooming times, although not as long as the average observed in a large cohort of Hoxb8 mutants. Error bars represent SEM. ∗ p < 0.05 versus wild type.

(D) Laboras data collected over a 24 hr period with Hoxb8 mutant mice transplanted with wild-type bone marrow cells show significant decrease in grooming times relative to Hoxb8 mutant mice. White bar represents wild-type controls (n = 22) relative to Hoxb8 mutants (n = 25). Gray bar indicates the grooming time of Hoxb8 mutant mice rescued by normal bone marrow transplants (n = 6). All values are mean ± standard error of the mean SEM. ∗ p < 0.05 versus mutant.

The presence of Hoxb8 lineage in the hematopoietic compartment was directly tested. Peripheral blood was collected and sorted by size and then by fluorescently labeled antibodies to cell surface antigens that distinguish the different types of white blood cells. All tested hematopoietic lineages, including platelets, granulocytes/monocytes, B cells, and T cells, exhibited Hoxb8-YFP signal ( Figures 2 A–2F ). Observation of the YFP signal in both myeloid and lymphoid lineages is suggestive of Hoxb8 expression in stem cells or multipotent progenitor cells. To further support this hypothesis, bone marrow cells from Hoxb8-ICre/Rosa26-YFP mice were collected and examined by fluorescence microscopy. Most of the bone marrow cells were YFP positive ( Figure 2 G). In addition, the Hoxb8 -YFPsignal was present in Sca1+C-kit+ cells, consistent with Hoxb8 expression in hematopoietic stem cells ( Figure 2 H). It has been reported that Hoxb8 is not expressed in mature hematopoietic cells (). The above results suggest that Hoxb8 is expressed at early stages of hematopoiesis but subsequently downregulated in fully differentiated blood cells.

(H) Most of the cells in the hematopoietic stem cell and multipotent progenitor cell domain are YFP positive. (Left) FACS analysis with Sca-1 and c-kit markers. The cells shown in the rectangle were further analyzed for YFP fluorescence. The black histogram (top right) represents YFP fluorescence detected in cells collected from ROSA26-YFP reporter mice in the absence of the Hoxb8-ICre driver, whereas the white histogram (bottom right) represents cells collected from ROSA26-YFP reporter mice carrying the Hoxb8-ICre driver.

(D–F) Analysis of blood samples collected from ROSA26-YFP reporter mice combined with the Hoxb8-ICre driver. Markers used were as follows: (A and D) Mac1/Gr-1 and YFP; (B and E) CD19 and YFP; (C and F) CD4/CD8 and YFP.

To assess if the Hoxb8 mutation affects the number of microglia present in the adult brain, comparable sections of the brain were evaluated for the presence of Iba1-positive cells in six Hoxb8 mutants and six control mice. We consistently observe an approximate 15% reduction of total number of microglia present in Hoxb8 mutant versus control mice ( Figure S2 ). Currently, we cannot specifically label Hoxb8 mutant (i.e., Hoxb8) microglial lineage because our Hoxb8-ICre driver is a “knockin” IRES-Cre driver, which does not itself affect Hoxb8 function. Therefore, the above count for reduction of microglia in Hoxb8 mutant mice may represent an underestimate of the actual reduction of the hematopoietic bone marrow-derived microglia subpopulation, should such loss be partially compensated by an increase in the resident non-Hoxb8-expressing microglial subpopulation.

Microglial counts per area unit were determined in 4- to 6-month-old wild-type mice and Hoxb8 mutants in comparable brain regions, including the orbital cortex, cingulate cortex, and dorsal striatum. These brain regions are known for their role in compulsive behavior.

Although the origin of microglia is still debated, there is general agreement that at least one subpopulation is of bone marrow origin, (i.e., derived from circulating monocytes;). The time of first appearance and the site of entry of Hoxb8-labeled microglia is consistent with this subpopulation being of hematopoietic, bone marrow-derived origin.

In newborn mice, very few Hoxb8-labeled cells are observed in the brain and these cells are found predominantly in the choroid plexus ( Figure 1 D), meninges, and the ventricular lining, with their numbers declining with increased distance from the ventricular zone. This gradient suggests migration of YFP-positive cells from the ventricular zone into the forebrain areas. Between P2 and P14, YFP-positive cell count in the mouse brain increases dramatically and is then maintained at this high level ( Figures 1 E and 1F and data not shown).

The expression pattern of Hoxb8 in the adult brain is broad (). However, the expression level is very low and dispersed in the brain, making it difficult to identify the cell type(s) expressing Hoxb8. To identify the Hoxb8 cell lineage in the mouse brain, we generated a Hoxb8-IRES-Cre (Hoxb8-ICre onwards) driver that could be used to activate Cre-dependent LacZ or YFP reporter genes targeted to the ubiquitously expressed ROSA26 locus ( Figure S1 B;). In mice carrying both the Hoxb8-ICre driver and ROSA26-YFP reporter alleles, activation of Hoxb8 expression also triggers YFP production. Brains of such mice were collected at pre- and postnatal stages and examined by immunohistochemistry. In adult brains, YFP-positive cells can be found throughout the brain, but consistent with previous results, predominantly in the cerebral cortex, striatum, olfactory bulb, and brainstem (data not shown). These cells appear morphologically to be microglia and indeed coexpress the general microglia marker CD11b and Iba1, a marker of activated microglia ( Figures 1 A–1C and data not shown), indicating that Hoxb8 is expressed in microglia or their progenitor cells. Notably, not all microglia in the brain are YFP positive, suggesting that Hoxb8 expression was present only in a subpopulation of microglia or their progenitors (∼40% of total, see Figure 7 C).

(D) Cortical microglia originating from the Hoxb8 cell lineage first appear in the brain during the first two postnatal days (P2), in the choroid plexus, and in association with the ventricular lining.

(A–F) Analysis of Hoxb8 lineage in mice heterozygous for the Hoxb8-ICre and ROSA26-YFP alleles. To determine if cells of Hoxb8 lineage in the brain are microglia, the identity of YFP-positive cells was examined by immunohistochemistry. Sagittal sections of the adult cerebral cortex were costained with anti-GFP antibody (A) and anti-CD11b antibody (B).

Hoxb8 mutant behavior is characterized by excessive pathological grooming. Previously, we determined the number and duration of grooming bouts from continuous video recording of mouse activity (). This procedure was robust but very labor intensive. More recently, we have been using technology developed by B.V. Metris based on the use of very sensitive vibration detectors (Laboras platforms). Each activity such as drinking, eating, rearing, climbing, locomotion, immobility, grooming, and scratching is associated with characteristic patterns of vibration, which are continuously recorded. A computer algorithm then interprets specific vibration patterns as individual behaviors. The advantage of this approach is that behavior is classified automatically and collected unobtrusively over any chosen period of time. We typically monitor activity over 24 hr periods. We have evaluated the Laboras platforms for their assessment of time spent grooming by co-monitoring mouse activity using our video camera system. The Laboras platforms are remarkably accurate, comparable to human classification and far less labor intensive. Figure S1 A (available online) shows the average time spent grooming for 25 Hoxb8 mutant mice and 22 controls over 24 hr periods as measured by the Laboras platforms. These results compare very well with those previously obtained by analyzing continuous video recordings, illustrating that on average Hoxb8 mutant mice spend approximately twice as much time grooming as their wild-type littermates (). The penetrance for excessive grooming in Hoxb8 mutant mice is 100%.

(E) Hoxb8-CreERTM allele was prepared by replacing the Hoxb8 exon 1 (ATG to BamHI site) with CreER TM fusion that enables temporal control of lox-specific recombination in the Hoxb8 expression domain. The external probes used in screening for accurate recombination events are indicated. All neo selection markers were flanked with FRT sites and subsequently removed by breeding with FLPe deleter mice. Selected landmark restriction sites are shown. pA, polyadenylation site.

Discussion

Herein we provide strong support for the hypothesis that the pathological grooming behavior observed in Hoxb8 mutant mice results from a deficiency in microglia. In support of this hypothesis we have shown that the only detectable Hoxb8-labeled cell lineage in the brain (the source of the complex, innate behavioral grooming syntax) is microglia. Second, disruption of Hoxb8 function results in the reduction of the total number of microglia in adult mouse brains (i.e., a microglia phenotype). Further, the excessive pathological grooming behavior in Hoxb8 mutant mice can be rescued by transplantation with normal bone marrow. Finally, restricted deletion of Hoxb8 in the hematopoietic system (Tie2Cre) recapitulates the excessive pathological grooming behavior in these mice, while restricted disruption of Hoxb8 in the spinal cord (Hoxc8-ICre) does not.

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Perry V.H. Microglial physiology: unique stimuli, specialized responses. There appear to be two principle sources of microglia in the mouse, a resident population that is present in the brain early during embryogenesis prior to vascularization () and a second population of bone marrow origin, derived from circulating monocytes, that migrate into the brain through the vascular system shortly after birth (). The kinetics of infiltration of Hoxb8-labeled microglia into the brain is consistent with this population being the bone marrow-derived subpopulation. As such, the Hoxb8 lineage provides a useful molecular marker for distinguishing between these two microglial subpopulations. Do they have similar or different roles in the brain? Molecular markers allow genetic interrogation of the system. What would be the consequences of selectively ablating only one population? Interestingly, although Hoxb8-labeled microglia represent only 40% of the total microglial population present within the adult brain, selective inactivation of Hoxb8 in this subpopulation is sufficient to induce the pathological grooming behavior. This fact would favor the hypothesis that the two microglial subpopulations present in the brain are performing distinguishable roles.

Frade and Barde, 1998 Frade J.M.

Barde Y.A. Microglia-derived nerve growth factor causes cell death in the developing retina. Marin-Teva et al., 2004 Marin-Teva J.L.

Dusart I.

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Mallat M. Microglia promote the death of developing Purkinje cells. Wake et al. (2009) Wake H.

Moorhouse A.J.

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Nabekura J. Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. Microglia could affect neuronal activity and behavior by a number of mechanisms, including the secretion of cytokines that stimulate or inhibit neuronal activity, and work in parallel with neurotransmitters. Microglia have also been reported to function in regulating neuronal cell death during embryogenesis (). Absence of appropriate cell death during neurogenesis could manifest itself later as aberrant behavior. Finally, the experiments ofillustrating that microglia processes are very dynamic and engage in intimate contacts with synapses are particularly intriguing. They observed that the duration of contact at synapses is dependent on neuronal activity. From the above, it is becoming apparent that due to their mobility and dynamic contacts with synapses, microglia could represent an additional system for stabilizing and managing neural networks. By virtue of their high abundance in the cortex, including the frontal orbital regions and basal ganglia, the microglia of Hoxb8 lineage are positioned in close proximity to the pathways controlling repetitive behavior.

Graybiel and Rauch, 2000 Graybiel A.M.

Rauch S.L. Toward a neurobiology of obsessive-compulsive disorder. Huey et al., 2008 Huey E.D.

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Grafman J. A psychological and neuroanatomical model of obsessive-compulsive disorder. Berridge, 1989 Berridge K.C. Progressive degradation of serial grooming chains by descending decerebration. Berridge and Whishaw, 1992 Berridge K.C.

Whishaw I.Q. Cortex, striatum and cerebellum: control of serial order in a grooming sequence. Taylor et al., 2010 Taylor J.L.

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Aldridge J.W. Dopamine receptor modulation of repetitive grooming actions in the rat: potential relevance for Tourette syndrome. Welch et al. (2007) Welch J.M.

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et al. Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice. OCD in human patients is associated with three principal brain regions: the prefrontal cortex, particularly the orbitofrontal cortex and anterior cingulated cortex; basal ganglia, including dorsal striatum and globus pallidus; and thalamus, namely, the dorsal medial nucleus (). Excessive grooming in rodents is widely believed to mimic the key traits of OCDs. Although the syntactic grooming chain can be fully executed in rats decerebrated at various mesencephalic levels, indicating that neural circuits specifying the basic sequential structure are all present within the brainstem (), cortex and striatum play important roles in modulating the initiation and completion of grooming bouts (). Dopamine is a prominent neurotransmitter for implementation of the grooming pattern (). However,have reported OCD-like behaviors in Sapap3 mutant mice that have reduced synaptic transmission in glutamanergic corticostriatal circuits, and they further showed that the compulsive grooming in these mutants is alleviated with serotonin reuptake blockers. Thus, it is apparent that multiple brain regions and signaling pathways control the frequency of repetitive behaviors.

Holstege et al., 2008 Holstege J.C.

de Graaff W.

Hossaini M.

Cano S.C.

Jaarsma D.

van den Akker E.

Deschamps J. Loss of Hoxb8 alters spinal dorsal laminae and sensory responses in mice. An alternative hypothesis has been put forward that the pathological grooming observed in Hoxb8 mutant mice is due to the sensory defects resulting from impaired formation of the spinal cord (). However, all of the experiments that we have presented contradict this hypothesis and have instead identified a defect in the hematopoietic system and more specifically a deficiency in microglia as likely causative for the aberrant grooming behavior. Our experiments have clearly separated the pathological grooming behavior from the sensory spinal cord defects to distinct cellular compartments. However, our experiments do not rule out the possibility that at later stages of the pathology the spinal sensory defects could exacerbate the consequences of excessive grooming.

Some of the apparent differences in the interpretation of Hoxb8 mutant phenotype by the Deschamps and our laboratories may result from monitoring different behavioral features. We monitor the time spent grooming (on Laboras platforms), whereby excesses in Hoxb8 mutant mice leads to pathological behavior and very broad hair removal and skin lesion patterns. Grooming can be distinguished from scratching by the Laboras platforms. We did not observe increased scratching in our Hoxb8 mutant mice except modest increases at the very late stages of the pathology when lesions become apparent and the animals would normally be euthanized. The Deschamps laboratory reports very localized hair removal and skin lesions that appear more consistent with a response to a localized chronic itch. What accounts for the differences in phenotypic outcomes between the two Hoxb8 mutant mice? Differences in genetic backgrounds is not likely to be a strong contributor since both lines have been crossed to a predominantly C57Bl/6J background.

Mainguy et al., 2007 Mainguy G.

Koster J.

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Durston A. Extensive polycistronism and antisense transcription in the Mammalian Hox clusters. Greer and Capecchi, 2002 Greer J.M.

Capecchi M.R. Hoxb8 is required for normal grooming behavior in mice. Holstege et al. (2008) Holstege J.C.

de Graaff W.

Hossaini M.

Cano S.C.

Jaarsma D.

van den Akker E.

Deschamps J. Loss of Hoxb8 alters spinal dorsal laminae and sensory responses in mice. Notably, the Hoxb8 mutant alleles are different. This is a concern because the density of genes is very high within the Hox complex, and there are many noncoding RNA transcripts as well as protein-encoding transcripts transcribed within this complex (). The Deschamps allele is a LacZ knockin into the first exon of Hoxb8. Our allele was generated by introduction of a nonsense codon in the first exon and a loxP site into the second. Both inserts are small relative to the LacZ insert. We have shown that a knockin allele of neor into the Hoxb8 locus shows additional phenotypes due to perturbation of neighboring Hox gene expression, which disappear upon removal of the neor gene (). Similarly, the LacZ gene could perturb neighboring RNA and/or protein expression and thereby alter phenotypes. Consistent with this interpretation, in mice homozygous for a knockin allele of CreERTM inserted into the first exon of Hoxb8 ( Figure S1 E), we have observed the localized hair removal and skin lesion phenotype described byin their Hoxb8 mutant ( Figure S4 D). This new phenotype shows up at a low penetrance (∼10%) in addition to our characteristic Hoxb8 mutant grooming phenotype.

We have demonstrated that a deficiency in hematopoiesis of Hoxb8 mutant mice is causal to the pathological grooming deficit observed in these mutant animals. This deficit is correctable by normal bone marrow transplants. We have argued that the grooming malbehavior is primarily manifested by a deficit in microglia derived from bone marrow. However, a deficiency in T and/or B cells may also contribute to the severity of the behavioral pathology. Also, we have not ruled out other cellular members of the hematopoietic system as potential contributors to Hoxb8 pathological grooming.

Why couple behavior such as grooming to the host's immune system? From an evolutionary perspective it may make perfect sense to couple a behavior such as grooming, whose purpose is to reduce pathogen count with the cellular machinery, the innate and adaptive immune systems, used to eliminate pathogens.

In summary, we have provided strong support for the hypothesis that the excessive pathological grooming behavior exhibited by Hoxb8 mutant mice is caused by a defect in microglia. That a behavioral deficit could be corrected by bone marrow transplantation is indeed surprising. The therapeutic implications of our study on amelioration of neurological behavioral deficits in humans have not escaped us. This mouse model provides an opportunity to determine how impaired microglia results in generating such a distinct compulsive behavioral anomaly. Further, since the Hoxb8 lineage specifically marks the microglia subpopulation derived from bone marrow, this mouse can also be used to genetically interrogate this cell subpopulation relative to the resident microglial subpopulation.