Modeling a model: Mouse genetics, 22q11.2 Deletion Syndrome, and disorders of cortical circuit development

Abstract

Understanding the developmental etiology of autistic spectrum disorders, attention deficit/hyperactivity disorder and schizophrenia remains a major challenge for establishing new diagnostic and therapeutic approaches to these common, difficult-to-treat diseases that compromise neural circuits in the cerebral cortex. One aspect of this challenge is the breadth and overlap of ASD, ADHD, and SCZ deficits; another is the complexity of mutations associated with each, and a third is the difficulty of analyzing disrupted development in at-risk or affected human fetuses. The identification of distinct genetic syndromes that include behavioral deficits similar to those in ASD, ADHC and SCZ provides a critical starting point for meeting this challenge. We summarize clinical and behavioral impairments in children and adults with one such genetic syndrome, the 22q11.2 Deletion Syndrome, routinely called 22q11DS, caused by micro-deletions of between 1.5 and 3.0 MB on human chromosome 22. Among many syndromic features, including cardiovascular and craniofacial anomalies, 22q11DS patients have a high incidence of brain structural, functional, and behavioral deficits that reflect cerebral cortical dysfunction and fall within the spectrum that defines ASD, ADHD, and SCZ. We show that developmental pathogenesis underlying this apparent genetic "model" syndrome in patients can be defined and analyzed mechanistically using genomically accurate mouse models of the deletion that causes 22q11DS. We conclude that "modeling a model", in this case 22q11DS as a model for idiopathic ASD, ADHD and SCZ, as well as other behavioral disorders like anxiety frequently seen in 22q11DS patients, in genetically engineered mice provides a foundation for understanding the causes and improving diagnosis and therapy for these disorders of cortical circuit development.

Keywords: ADHD; Animal models; Autism; Cortex; Schizophrenia.

Fig. 1

22q11.2 Deletion Syndrome (22q11DS) provides a model for disorders of cortical circuit development that can be analyzed mechanistically in genetically engineered mice. In this and all other figures, human data is indicated beneath green header boxes, and mouse data is provided beneath blue header boxes. Key clinical features of the syndrome in infants (top left) and in adolescents (top right) are summarized. The clinical features in infants are consistent with disrupted developmental/morphogenetic processes during embryonic/fetal life; those in adolescents are focused on disorders of cortical circuit development. We note that adolescents and adults with 22q11DS face a broad range of additional clinical challenges, some related to the early morphogenetic issues. Others that are seen variably across the population of individuals with the disease may reflect additional vulnerabilities that arise in maturity. Phenotypes in genetically engineered mouse models at birth (lower left) and in adulthood (lower right) are potentially parallel to the key clinical features of infants and children with 22q11DS. Thus, if these phenotypic features in mouse models are truly comparable to the clinical features of 22q11DS, it may be possible to model 22q11DS, which itself is a model genetic syndrome for understanding disorders of cortical connectivity, in genetically engineered mice. This modeling of a “model” syndrome can give insight into developmental, cellular and molecular mechanisms that cannot be easily gained working solely with human patients. This developmental/mechanistic insight is likely to be key for developing new diagnostic and therapeutic approaches for 22q11DS and more broadly for disorders of cortical circuit development including autism and schizophrenia.

Fig. 2

The genomic organization of human 22q11.2 has been compared with that of orthologous regions in several mammalian species. The extent of the most common 22q11.2 deletions is noted (top). Low copy repeats (LCR) are indicated in beige. Open reading frames are indicated in gray, and non-conserved genes are indicated in yellow. Most deleted individuals (85%) carry deletions spanning approximately 3 Mb, while a subset (10%) carry deletions a shorter 1.5 Mb deletion. The low copy repeats (LCRs) that mediate the chromosomal deletion are evident in humans and chimpanzees, but are absent from the more distantly related primates (galago, squirrel monkey) and other mammals (Dog and mouse are shown here). In the Mouse, the minimal critical deleted region (1.5 MB deletion) has undergone a partial 3′ to 5′ inversion (left, near bottom). Key mouse models of the 1.5 MB minimal critical deletion, the LgDel, Df(16)A, and Df1 are indicated with arrows at bottom.

Fig. 3

The LgDel mouse models cardiovascular 22q11DS pathology and underlying developmental mechanisms. (Top): Simplified schematic of cardiac development, illustrating the remodeling of the branchial arch arteries to form the arteries of the aortic arch. (Top left) The cardiac vasculature arises from the arteries of the pharyngeal/branchial arches, 1–6. Each arch, except the fifth (which is only present as a transient structure, not shown here) has its own artery that connects the outflow of the heart tube (truncus arteriosis, yellow, and aortic sac, orange) to the aorta (red). (Top right) As heart development proceeds, the branchial arch arteries are remodeled to produce the aortic arch arteries. (Second row) Developing vasculature in wild-type control (WT) and LgDel embryos at E10.5, visualized by immunofluorescent staining for a vascular marker (PECAM). Brackets outline the region containing the 3rd, 4th, and 6th pharyngeal arch arteries. Most LgDel embryos have a constricted 4th arch artery (stenosis). A schematic version of the arch vasculature is provided as an inset for additional clarity. (Third row) Cardiac development in LgDel embryos is highly sensitive to even modest changes of embryonic retinoic acid (RA) signaling that are benign for wild type (WT) littermates. Center panels show WT and LgDel vasculature under normal conditions. A modest reduction in RA levels in the Raldh2 heterozygote (Raldh2^+/−) does not cause detectable changes in aortic arch morphology by itself, but it severely disrupts arch development in a compound LgDel:Raldh2^+/− embryo. Likewise, injection of low-levels of RA does not significantly impair arch development by itself, but causes catastrophic anomalies in LgDel embryos. (Bottom row): These results suggest that RA signaling is dysregulated in the LgDel embryo, and normal mechanisms that compensate for modest fluctuations in RA levels are impaired. Morphology schematics adapted from Larsen’s Human Embryology, 4th edition (2009), Figs. 13–16. LgDel illustrations and schematic adapted from Maynard et al. (2013).

Fig. 4

The consequences of pediatric dysphagia—feeding and swallowing difficulties during early life—in 22q11DS are also seen in mouse models of 22q11 deletion. (Upper left) Growth curves for male and female 22q11DS patients from birth through 3 years of age show that birth weights are comparable to typically developing infants/toddlers, and then diverge so that 22q11DS children weigh less (data re-graphed from Tarquinio et al., 2012). Some of this lack of normal weight gain is attributed to feeding/swallowing difficulties. (Upper middle) Growth curves for female and male LgDel mice as well as wild type (WT) littermate controls from birth through 30 days of age. Similar to 22q11DS patients, birth weights for both female and male LgDel and WT littermate pups are equal. The weights, however, diverge shortly after birth, with a similar deficit in weight gain seen for LgDel pups as is seen in 22q11DS children. (Top right) Cranial nerves in humans that are critical for feeding and swallowing. These include the trigeminal (V), facial (VII), glossopharyngeal (IX), vagus (X) and hypoglossal (XII) nerves. Coordinated activity of sensory and motor divisions of these nerves (and the related peripheral ganglia and brainstem motor nuclei) insures optimal feeding and swallowing. In the panel below, these cranial nerves are visualized clearly in the developing mouse embryo at E10.5 using whole embryo immuno-staining. (Middle left) LgDel pups aspirate when swallowing milk during nursing. We developed the equivalent of the “barium swallow” test used in human infants and children to monitor the distribution of milk once ingested in WT and LgDel pups at post-natal day (P) 7. P7 WT pups swallow milk so that the bolus is seen in the stomach (St), with only remnants of fluorescent milk adhering to the tongue (To) after ingesting milk labeled with fluorescent microspheres via a feeding pipette. In contrast, LgDel pups aspirate into the nasal pharynx (NP). (Middle right) Aspiration results in protein inclusions (consisting of murine milk protein, data not shown) infiltrated with leukocytes, in the olfactory turbinates/nasal sinuses and lungs. (Lower left) Normal patterning of the hindbrain and constituent rhombomeres (r2–6), demonstrated here by expression of the RA regulated gene Cyp26b1, is key for the normal cranial nerve (CN) development. For CN V this includes a highly branched ophthalmic division (op), a robust maxillary division with multiple axon fascicles (mx) and a bifurcating mandibular branch (md). (Lower middle) In the LgDel retinoic acid (RA) dependent patterning is disrupted so that RA-sensitive genes, including Cyp26b1, expand in the hindbrain. CN V and IX/X development is disrupted in parallel. Lower right: Genetically lowering RA signaling by crossing in one null allele of Raldh2, the key RA synthetic gene for RA signaling in the hindbrain, “rescues” the disrupted patterning in anterior rhombomeres, demonstrated by Cyp26b1 expression, and also the anterior CN V phenotype.

Fig. 5

Altered cortical Activity, disrupted axonal projections and neuronal changes are associated with disorders of cortical connectivity. (Top row) Functional magnetic imaging (FMRI) and other non-invasive physiological assessments in ASD, ADHD, and SCZ patients show localized and/or network related changes in cortical activity, especially in association regions. (Upper middle row) Diffusion tensor imaging (DTI) studies indicate that white matter tracts can be reduced in their density and topographical projections. (Lower middle row) MRI studies have shown both reduced cortex, particularly in schizophrenia, and thickened cortex, particularly in autism. (Bottom row) Activity and connectivity changes in patients may reflect reduced projection neuron density, location and inappropriate projection neuron cell fate (left). Density, distribution and cell fate of interneurons can also accompany these changes (middle). Associated with these changes to projection neurons and interneurons are altered neurite complexity and dendritic spine density (right).

Fig. 6

Expression, localization and genetic analysis of individual 22q11 genes during development. (Top row) Dynamic expression of 22q11 genes from the minimal critical deleted region analyzed in 13 weeks post conception fetal human and adult human cortex as well as embryonic and post-natal mouse brain. In most cases, there is agreement between mouse and human in the direction of change of gene expression between fetal and adult cortex. Human gene expression levels were assessed from the brainspan database (brainspan.org) and averaged across ventrolateral prefrontal cortex, anterior cingulate cortex and dorsolateral prefrontal cortex at both time points. Gene expression levels in mouse brain between embryonic and post-natal time periods were assessed by qPCR and derived from data in Meechan et al. (2006, and Maynard et al. (2008). (Middle row) At left, in situ hybridization on E14.5 coronal brain sections indicate selectivity of 22q11 gene expression in the progenitor zones (the ventricular zone, VZ and subventricular zone, SVZ) as well as the location of newly generated neurons (the cortical plate, CP) in the cortex. Cdc45l and Ranbp1 are expressed most robustly in the progenitor zones that contain the apical and basal progenitor cells. Trmt2a, Hira and Ufd1l display a more ubiquitous expression pattern, while Sept5 is more robustly present in the cortical plate occupied by post-mitotic neurons. At right: In maturing post-natal cortex, Zdhhc8 and Mrpl40 displays widespread expression across all layers, while T10 shows robust and selective expression in layer 5 (modified from Maynard et al., 2008; Meechan et al., 2009). (Bottom row) Targeted inactivation of 22q11 genes in the mouse result in embryonic/perinatal lethality in 10 of the 16 loci that have been deleted thus far, indicating the importance of genes in this region for embryonic development and survival.

Fig. 7

Neuronal proliferation defects during development in the LgDel model prefigure changes to the mature cortex. (Top row) A schematic of cortical neurogenesis, migration, and projection neuron differentiation. Cortical neuroepithelial progenitors (far left) give rise to apical/radial glial progenitors, the committed stem cells of the cortex. Radial glia can directly generate neurons (and at later stages, astrocytes), or an intermediate/transit amplifying precursor called the basal progenitor. Basal progenitors give rise to projection neurons, with a bias toward generating layer 2/3 projection neurons. After terminal divisions, newly generated cortical neurons. (Middle row) Basal progenitor proliferation, but not apical progenitor proliferation in the embryonic LgDel cortex is disrupted. At left: labeling for the S-phase marker BrdU and the basal progenitor specific marker, Tbr2, show reduced co-labeling in the LgDel embryonic cortex. Arrowheads indicate double-labeled cells (modified from Meechan et al., 2009). At right: quantification of Tbr2-positive basal progenitors in S-phase based on BrdU labeling. There is a statistically significant, approximately 25% decrease in actively proliferating Tbr2-labeled basal progenitors. (Bottom row) Neuronal markers in post-natal LgDel cortex reveal reduced numbers in layer 2/3. At left: Immunofluorescent staining for the pan-neuronal marker, NeuN, displays reduced cell numbers in layer 2/3. At right: the layer 2/3 projection neuron marker, Cux1, is also reduced in LgDel cortex (modified from Meechan et al., 2009).

Fig. 8

Disrupted migration and placement of subsets of GABAergic interneurons reflects altered function of the Cxcr4 receptor caused by 22q11 deletion. (Top row) Schematic of interneuron migration from their site of neurogenesis in the medial ganglionic eminence (MGE) to the neocortical rudiment. The drawing indicates the migratory disruption seen for subsets of GABAergic interneurons in the LgDel fetus, as well as the final consequence for interneuron distribution in the LgDel adult cortex. (Upper middle row) Live imaging of migrating GABAergic interneurons, labeled with a Dlx5/6:Cre:eGFP transgene, as they enter the cortex at E14.5 shows that LgDel cells have diminished velocities as well altered trajectories. (Lower middle row) Top panel: heterozygous deletion of Cxcr4 in interneurons, using the Dlx5/6:Cre:eGFP, does not have a detectable effect on interneuron migration. Middle panel: In LgDel fetuses, migrating interneurons accumulate in the SVZ. Bottom panel: When 1 copy of Cxcr4 is excised using the Dlx5/6:Cre:eGFP, the accumulation of migrating interneurons in the SVZ is enhanced specifically. (Bottom row) In mature LgDel cortex, laminar distribution of one specific interneuron sub-type, those expressing parvalbumin, is altered in layers 2/3 and 5/6. The distribution of several other subtypes of interneurons is not altered, as shown by the schematics. Images and data are from Meechan et al. (2012).

Fig. 9

Ranbp1, a 22q11 gene implicated in cell cycle control, is necessary for normal cortical development. (Top left) Ranbp1^−/− embryos are dysmorphic at E10.5. Anomalies include a shortened telencephalic vesicle (bracket) and hypoplastic branchial arches. In addition, a subset of Ranbp1^−/− embryos is exencephalic. (Bottom left) At E14.5 Ranbp1^−/− embryos are visibly dysmorphic, including a smaller head and frequent eye defects. The brain, and particularly the cortex of Ranbp1^−/− embryos is substantially smaller at E14.5, as shown in dorsal view. (Right) Cortical precursor proliferation and neurogenesis is disrupted in the Ranbp1^−/− embryo. Proliferation of rapidly dividing neuroepithelial progenitors in the E10.5 Ranbp1^−/− embryo is diminished, shown here using PH3 labeling of mitotically active precursors (top right). At later stages proliferation of rapidly dividing Tbr2+ basal progenitors in the E14.5 cortex is diminished, as evidenced by decreased numbers of Tbr2+/BrdU+ double-labeled cells (middle right). These proliferative defects prefigure an overall loss in cortical size, as well as a selective loss of layer 2/3 neurons (bottom right). This change is seen clearly in the E17.5 cortex. The frequency of layer 2/3 projection neurons (labeled with Cux1) is diminished; however, the frequency of layer 5/6 projection neurons (labeled with Ctip2) is not significantly changed. Figure adapted from Paronett et al. (2014).

Fig. 10

Disruption of circuits in LgDel mice that engage the medial frontal association cortex (mFAC) is associated with cognitive deficits that rely on the integrity of mFAC. (Top row) Dorsal view of the mouse brain with the location of the mFAC highlighted in red. At right, the comparable area is indicated in a representation of coronal section of adult mouse cortex at mid-anterior levels (the level of the anterior commissure). (Middle row) At left, a touchscreen reversal task measures aspects of cognitive flexibility in WT as well as LgDel mice. During acquisition, the animals must correctly choose between horizontal or vertical bars to elicit a food reward. During the reversal phase, the reinforced stimulus is changed, and the animals have to learn that the previously reinforced contingency no longer elicits a food reward. At right: In LgDel mice, reversal learning takes significantly longer than in WT mice. There is, however, significant variability in performance in the LgDel animals that is not seen in WT. A subset of LgDel animals (indicated with a bracket) performs particularly poorly during the learning phase of the reversal task. (Bottom row) From the behaviorally tested animals, we observed that neuron density in upper layers of the mFAC (bin 1) correlated with reversal learning performance. A subset of LgDel animals had lower neuron density in bin 1 and performed particularly poorly during the task. At right, This ‘behavioral subset’ of LgDel animals is reported as its own group compared to normally performing LgDel animals and wild-type animals for NeuN density across 5 bins spanning all layers of the mFAC. There is a significant NeuN reduction in bin 1 for the behaviorally compromised LgDel animals. Tissue staining of the cortex of wild-type, normally performing, and poorly performing LgDel animals with an antibody for the neuronal marker, NeuN. Bin 1 density for neurons (upper layers) was notably reduced in the poorly performing LgDel animals. Modified from Meechan et al. (2013).

Fig. 11

A hypothesis of disrupted development that could underlie disorders of cortical circuit development associated with 22q11DS. (Left) A schematic of normal development indicating normal mechanisms of basal progenitor proliferation that insure appropriate numbers of layer 2/3 projection neurons as well as migratory mechanisms that mediate appropriate placement of key subsets of GABAergic interneurons. (Left middle) Changes in layer 2/3 projection neuron frequency lead to diminished corticocortical connectivity normally made by these neurons, while local alterations in excitatory/inhibitory balance due to disrupted migration and placement of GABAergic interneurons modifies local processing of information in layer 2/3. (Right middle) These changes lead to under-connectivity, especially between cortical association regions in the LgDel mouse. (Right) Similar changes may lead to underconnectivity in frontal, parietal and temporal association cortical regions in 22q11DS patients.