The TCA-TLZ reporter line labels thalamocortical axons during development
The TCA-TLZ transgenic reporter line was created fortuitously by pronuclear injection and random genomic insertion of a transgene containing the golli
promoter driving the tau-lacZ
reporter gene. This reporter fuses the axonal tau
microtubule binding protein to beta-galactosidase to localize it to axons [33
]. The golli
promoter is a portion of the myelin basic protein promoter that was shown to promote expression in deep cortical layer neurons [34
]. Surprisingly, in this line, the tau-lacZ
was expressed not in the cortex but instead in dorsal thalamus. The unexpected pattern is presumably due to positional effects of unknown enhancers at the insertion site, mapped to an 8.5-Mb interval of about 45 genes on chromosome 3 (data not shown). The insertion does not appear deleterious: homozygotes are viable and fertile, with no detectable abnormal phenotypes in brain morphology or TCA patterning at birth (n > 40).
The TCA-TLZ reporter line expresses the axonal reporter tau
-beta-galactosidase in the cell bodies and axons of dorsal thalamic neurons. These neurons are born between E10 and E13 in mice [35
]. Expression of the TCA-TLZ transgene is detectable from E13.5 onwards, allowing visualization of TCAs during prenatal development, as they project through vTel and innervate the cortex (Figure ). (In this paper, the term 'ventral telencephalon' or vTel refers to the region extending from the ventral surface to the lateral ventricle, including the ganglionic eminences, and the forming basal ganglia and amygdala.) No cortical axons are labeled, although scattered cell bodies in the cortex stain postnatally (Figure , postnatal day (P)9.5). The tau
-beta-galactosidase labels axons strongly enough to be visible to the naked eye in whole brains (Figure ). Importantly, the transgene is expressed in the same pattern consistently across different individuals, generations, and genetic backgrounds: in dorsal thalamus, not ventral thalamus (Additional file 1
), and in a small number of other neuronal tracts and populations, including the optic tract (Figure , ot), the accessory olfactory bulb and accessory lateral olfactory tract (LOT), the pontocerebellar tract (PCT), and the outer external granular layer of early cerebellum (Figure ).
The TCA-TLZ reporter can reveal the TCA pathfinding and cortical lamination defects found in the reeler mutant
To test whether the TCA-TLZ reporter can reveal TCA guidance and cortical morphogenesis phenotypes, it was crossed to the well-known cortical lamination mutant reeler
(Figure ). In reeler
mutant brains, cortical layers are roughly inverted, and the subplate cells remain superficial [36
]. The TCA-TLZ reporter shows that in control brains at P0 (Figure ), the TCAs had entered the cortex and could be seen as a dark blue bundle growing in a restricted zone defined by the subplate (Figure , sp), above the mitotic layer and beneath the cortical plate. The collateral branches, thin perpendicular offshoots from the axon shafts, were seen as a lighter blue haze in the deep half of the cortical plate (Figure , br). By contrast, in reeler
mutant brains, the TCAs did not extend beneath the cortical plate, but grew obliquely across it (Figure ), to reach the displaced subplate (sometimes called the superplate, sp*). The appearance of the TCAs in these reeler
mutants matched that seen with dye tracing previously [28
]. This experiment demonstrates that the TCA-TLZ reporter can readily reveal both the abnormal TCA pattern and the aberrant cortical layering in the reeler
mutant, and may be an extremely useful readout of forebrain development abnormalities and tool for analysis of other thalamocortical projection mutants.
Figure 2 The TCA-TLZ reporter line reveals the TCA pathfinding and cortical lamination defects of the reeler mutant. (A, A') In a P0 control brain, the TCAs elongate (dark blue) in the subplate (sp) layer beneath the cortical plate, and extend collateral branches (more ...)
A genetic screen focused on thalamocortical development
To discover novel genes and phenotypes in thalamocortical development and forebrain morphogenesis, we employed an efficient screening and mapping strategy previously used to identify mouse models of human birth defects [30
]. First, a three generation breeding strategy of two intercrosses followed by a backcross allowed for efficient collection of recessive mutants and concurrent mapping (Figure ). Second, screening was performed on the day before birth so that all of prenatal cortical development could be assayed, but mutations causing postnatal lethality could still be collected. This was important since several mouse knockouts affecting thalamocortical development die at birth. Third, initial mapping was accomplished relatively rapidly through the use of an autosomal genome panel of SNP markers [32
]. Finally, incorporating the TCA-TLZ reporter into the scheme enhanced detection and description of prenatal thalamocortical phenotypes.
Figure 3 Mutants found in the thalamocortical screen display a variety of defects in the TCA projection. (A) Intercross breeding scheme for recessive thalamocortical mutant screen. ENU, N-ethyl-N-nitrosourea; wt, wild type. (B) A control E18.5 brain stained with (more ...)
Males carrying the TCA-TLZ transgene on a C57BL/6 background were mutagenized with N-ethyl-N-nitrosourea (ENU) and mated to wild-type females of the FVB/N strain. G1 males carrying the TCA-TLZ transgene were bred to wild-type FVB/N females, and the resulting G2 daughters were backcrossed to their fathers and sacrificed at embryonic day E18.5 to harvest G3 embryos for screening (Figure and Materials and methods). Embryo brains were cut in half coronally, stained for beta-galactosidase, and examined as whole-mounts. All brains were checked for morphology, and those carrying the transgene (approximately 63%) were examined for abnormalities in the TCA pattern. Five to eight litters from each G1 line were screened. The repeated observation of a specific phenotype in independent litters, followed by faithful transmission after further outcrosses, indicated a high likelihood the abnormality was caused by a monogenic mutation [30
We screened 57 G1 lines, each representing an independently mutagenized haploid autosomal genome derived from a single G0 sperm. The X chromosome was not assayed in this screen because males were mutagenized and only their male progeny were bred. Seven independent recessive brain development mutants were found, and six of these showed defects in thalamic axons (Table and following sections). Several mutations caused pleiotropic phenotypes, affecting more than one tissue, and three additional mutant lines had only non-brain phenotypes (see Materials and methods). Mutant lines not selected for analysis included a few with exencephaly or embryonic lethal phenotypes. Only those lines that behaved as recessive Mendelian, highly penetrant phenotypes were mapped.
Mutants found in thalamocortical development screen
Since the screen was done as an intercross between the inbred strains C57BL/6 and FVB/N (Figure ), genetic mapping by analysis of meiotic chromosomal recombination could be done directly with DNA from affected progeny. By genotyping mutants for a genome-wide panel of up to 768 SNPs that are polymorphic between C57BL/6 and FVB/N, analysis of small numbers of mice resulted in mutation localization to chromosomal intervals of approximately 40 Mb [32
]. Microsatellite (simple repeat) markers were then used to confirm and narrow the SNP intervals.
Mutants display defects at various steps in the TCA projection
The thalamocortical screen revealed several mutant lines with TCA defects visible at low magnification in stained E18.5 brains. TCAs were disrupted at various steps along their pathway (Figure ). Additionally, several mutants had morphological defects (Table ), and all were postnatal lethal. The ND21 mutant had normal TCA patterning but a small brain, and is described elsewhere as a mutant in the Golgi protein GMAP210 [37
Three mutants were found with similar TCA phenotypes at Step 2 of the pathway, in which a subset of TCAs failed to turn laterally upon crossing the DTB (Figure ). In fuddle, magoo, and wanderer mutants, a single bundle of TCAs was oriented ventrally from the internal capsule just after crossing the DTB, while the remainder of TCAs appeared to navigate normally to the cortex. In the fuddle mutant line, the misrouted fascicles were very thin (Figure , arrow), while those observed in magoo and wanderer appeared thicker with presumably more axons (Figure , arrows). These turning errors could represent failures to detect or respond to ventral repellents, or defects in the interactions with internal capsule guidepost cells. All fuddle mutants displayed enlarged lateral ventricles and hippocampal hypoplasia, suggesting other defects in forebrain development. About one-fourth had a TCA defect, and about one-fourth also displayed subtle eye abnormalities, such as irregular irises. The fuddle phenotypes co-segregated across generations and mapped to the same region of chromosome 19, indicating that they are all caused by the same mutation.
Steps 3 and 4, in which TCAs spread through vTel and cross the CSB, appeared abnormal in sprawl and baffled mutants. In both of these mutants, some TCAs were overfasciculated and stalled (Figure ). Fewer axons entered the cortex. This phenotype could represent a defect in axon defasciculation from one large bundle to many smaller bundles, or a problem with recognizing the corridor cells or other cues that guide the TCAs through the ventral telencephalon. The baffled mutant defect was more dramatic, and seemed most suggestive of a defect in step 4, crossing the CSB (also called the pallial-subpallial boundary). The baffled thalamic axons appeared disorganized in the lateral vTel and most failed to enter the cortex (Figure , red arrows).
mutant phenotype may represent an indirect effect on step 5, the restriction of TCAs beneath the cortical plate. The surface of the bumpy
mutant forebrain had ectopic lumps of cells outside the pia (Figure , red arrows). Cortical lamination appeared disorganized beneath these ectopia. This phenotype is reminiscent of the human brain malformation known as cobblestone (type II) lissencephaly, also called Walker-Warburg syndrome, which is due to cortical neuron over-migration past the marginal zone [38
]. Interestingly, a small number of TCAs crossed the cortical plate to invade the 'cobblestones' (Figure , upper red arrow; zoomed in Figure ). Thin sections through cortical ectopia showed they contained both cells erupted through the marginal zone (Figure , red arrow) and axonal fibers (Figure , red arrow). This finding suggests that the misplaced cells may express substrates attractive to TCAs, or that the same mechanism that normally prevents neuron overmigration also acts on TCAs to keep them from invading the cortical plate inappropriately. Axonal innervation of cobblestone-type ectopia has not been shown before in human patients or mouse models, but aberrant cortical wiring could help explain varying seizure phenotypes of some type II lissencephaly patients [39
magoo mutants have small brains and craniofacial defects along with a TCA ventral misrouting defect
magoo mutants showed a ventral misrouting defect of TCAs. Out of ten magoo mutant embryos stained and expressing the TCA-TLZ reporter, three displayed a small subset of TCAs turned ventrally out of the internal capsule (Figure , arrow). The misrouted bundle appeared to turn ventrally just after the DTB, and then curve slightly rostrally and stop. L1 antibody, which marks several forebrain tracts, including TCAs and corticothalamic axons (Figure ), appeared to confirm the ventral misrouting, showing an aberrant thick bundle of axons extending ventrally from the internal capsule along the vTel side of the DTB (Figure , red arrow). The TCAs that did grow to the cortex in the mutant showed no other apparent abnormalities, although the mutant cortex was thinner, with a thinner axonal layer (Figure ). Indeed, the entire forebrain was reduced in size in every homozygous magoo mutant, but severity varied (Figure ). Seventy-one percent (35 of 49) of magoo brains were categorized as mild, with only slightly small forebrains (for example, Figure , middle), and the other 29% (14 of 49) were categorized as severe, with hypoplasia of all brain regions (extreme example in Figure , right). Interestingly, the two cortices or olfactory bulbs in a given individual mutant brain were sometimes asymmetric in size (for example, see olfactory bulb asymmetry in Figure , middle). This variability within two halves of one organ suggests that stochastic events underlie the phenotypes. It is not clear whether the TCA defect is cell autonomous or secondary to changes in the forebrain pathway.
Figure 4 magoo mutants have small malformed brains and craniofacial defects. (A, B) L1 immunolabels TCAs and corticothalamic axons in E16.5 brains. The approximate position of the DTB is indicated by a black arrowhead. In the magoo mutant brain, an abnormal axon (more ...)
In addition to small forebrain size, magoo mutants often had craniofacial and eye abnormalities (Figure ). The snout was usually shortened (76%; 35 of 46), often with cleft palate (24%; 10 of 42). Most mutants had eye phenotypes on one or both sides ranging from iris coloboma to microphthalmia (83%; 38 of 46). Internal organs below the neck appeared normal and proportional to body size, but digits were abnormal in 13% of homozygous mutants (7 of 53). Heterozygotes were indistinguishable from wild types. The phenotypes could indicate a primary defect in patterning, proliferation, or cell migration. However, even in the mildest magoo mutants with no craniofacial defects, the forebrain was still slightly small, suggesting that forebrain tissue is the most vulnerable to the loss of this gene.
The magoo mutant gene appears to be novel. The mutation was mapped using SNP and microsatellite markers to a small region on chromosome 19 between D19Mit135 and D19Mit12, which does not contain any known thalamocortical development genes. In addition, since the magoo map interval overlapped with the larger fuddle interval on chromosome 19, and both had a ventral misrouting TCA defect, we tested whether they were allelic by complementation. In five intercross litters, none of 40 embryos showed brain morphology or TCA phenotypes, suggesting that the magoo and fuddle mutations are in different genes.
The baffled mutant shows severely reduced thalamocortical innervation
In contrast to the partial TCA defect seen in magoo, the baffled mutant showed a fully penetrant and severe TCA phenotype (Figure and ). As seen from dorsal views of whole brains, wild-type cortices stained blue from innervating TCAs (Figure , left), but baffled mutant cortices had greatly reduced cortical staining, suggesting less TCA innervation (Figure , right). While the cortex and olfactory bulbs of baffled mutants usually appeared slightly smaller than littermates', they were not misshapen.
Figure 5 baffled mutants have the most severe deficit in thalamocortical innervation. (A) Compared to a wild-type E18.5 forebrain (left), a baffled mutant littermate forebrain (right) has a slightly smaller cortex (ctx) and olfactory bulbs (OB), and shows severely (more ...)
Coronal cuts revealed why baffled mutants had fewer TCAs in the cortex. Control brains showed a parallel array of TCAs traveling through the lateral vTel, and curving neatly to cross the CSB into the cortex (Figure ). By contrast, in baffled mutant brains (Figure ), the TCAs appeared tangled in the lateral vTel, and some seemed to be stalled in masses near the CSB (Figure , red arrow). Lipophilic dye placements into dorsal thalamus (dTh) to trace TCAs showed a very similar result (Figure ). Similarly, L1 antibody staining (Figure ) confirmed that axon fascicles were disorganized in lateral vTel (5G, arrows), and thickened near the CSB (5G, arrowheads). To examine these axon bundles in cross-section, thin sagittal sections were taken from the lateral cortex of control and mutant brains and stained with hematoxylin and eosin (Figure ). In the lateral cortex near the CSB, the control brain intermediate zone (Figure , bracket) contains evenly dispersed small fascicles, whereas the baffled brain intermediate zone contained large swollen bundles of axons (Figure , bracket and red arrowheads). It is uncertain whether these oversized bundles contain only thalamocortical axons, or corticothalamic axons as well.
The thickened disorganized axon fascicles could signify a loss of the ability of TCAs to defasciculate or to interact with the appropriate substrate. The problem appears to arise before the TCAs reach the CSB, but may result in stalling at the CSB. Therefore, the baffled
mutant may represent a disruption in both step 3, defasciculating and fanning out within the ventral telencephalon, and step 4, crossing the CSB. This unusual TCA phenotype most closely resembles the TCA defects reported in the Tbr1
transcription factor knockouts [22
]. However, baffled
mapped to an independent locus on chromosome 2, which does not contain a known TCA guidance gene, between D2Mit203 and a marker '58-3' we designed (see Materials and methods).
Aside from the dramatic and very consistent TCA defect, baffled mutants had other highly penetrant phenotypes. baffled mutants had small kidneys (100%; 27 of 27), hematoma under the nose (95%; 20 of 21), and those collected after birth died within hours (100%; 15 of 15). Heterozygotes appeared normal, indicating a recessive mutation. All phenotypes segregated together and were mapped to the same interval, indicating that they are caused by the same genetic lesion. Candidate genes in the interval are under investigation.
wanderer mutants display TCA fascicles misrouted onto the ventral forebrain surface
The wanderer mutant forebrains had a consistent distinctively abnormal shape. The small oval cortical hemispheres barely touched at the midline, and the olfactory bulbs were short and conical (Figure ). The hippocampus was also reduced, but the midbrain and hindbrain appeared normal. Other than a slightly flattened forehead, craniofacial features were normal. The kidneys were small, and any mutants born died within a few hours. The forebrain and kidney phenotypes were fully penetrant (n > 30 mutants), and heterozygotes appeared identical to wild types, indicating a fully penetrant recessive mutation.
Figure 6 wanderer mutants misroute TCA fascicles onto the ventral surface of the forebrain. (A) Dorsal views of a normal littermate (left) and wanderer mutant (right) show the wanderer mutant has reduced cortex (ctx) and olfactory bulb (ob) size, and reduced blue (more ...)
In addition to the abnormal forebrain morphology, a striking TCA pathfinding phenotype was observed in wanderer mutant brains. A significant reduction of thalamocortical innervation was suggested by reduced beta-galactosidase staining in dorsal cortex (Figure ). Coronal views revealed that a subset of TCAs was misrouted ventrally out of the internal capsule (Figure and ). Staining for neurofilament-M, which labels many axons, including TCAs and corticothalamic axons, showed a similar aberrant fascicle adjacent to the DTB (Figure , red arrow). However, in contrast to fuddle and magoo mutants, in which ventrally misrouted axons stalled within ventral telencephalon, the wanderer misrouted TCA fascicle grew down onto the ventral forebrain surface and continued in a lengthy rostral trajectory (Figure ). The fascicle sometimes diverged into multiple bundles (Figure , short red arrows). Aberrant ventral surface axons were always observed in both hemispheres in mutants, but never in heterozygotes or wild types (n = 12 -/- and n > 50 +/+ and +/- brains). However, the proportion, number, and precise pathways of the misrouted axons varied. In most mutant hemispheres about half of the axons were misrouted, but one mutant hemisphere had a complete misrouting of all TCAs ventrally with none innervating the cortex (data not shown). Usually the aberrant fascicles grew in a rostro-lateral trajectory; the longest grew all the way to the olfactory bulbs to terminate in bouton structures on the ventral side (Figure , long red arrows). Occasionally a misrouted fascicle stayed in the diencephalon and grew medially along the optic tract (Figure , wide red arrow), but none grew caudally.
The optic tract axons, which are labeled by the TCA-TLZ transgene as well as neurofilament, grow from the optic chiasm up the side of the diencephalon near the point where the wanderer misrouted TCAs surface on the medial margin of the ventral forebrain (Figure , ot). To ascertain whether the optic tract axons were normal or might contribute to the aberrant fascicles in wanderer mutants, the caudal cortex overlying dorsal thalamus was removed to expose the lateral side of the diencephalon (Figure ). The optic tract could be seen coursing up from the optic chiasm on the side of the thalamus to the dorsolateral geniculate nucleus in both control and mutant brains (6 H, I, black arrows). TCAs were seen in both control and mutant exiting dorsal thalamus rostral to the optic tract and then curving toward the neocortex. However, the wanderer mutants also showed a TCA fascicle that extended ventrally from the internal capsule and curved rostro-laterally on the ventral surface (Figure , red arrows). A similar pattern was observed in 6 of 6 dissected wanderer mutant hemispheres. These data suggest that the optic tract axons are guided normally in wanderer mutants and that the aberrant ventral surface fascicles contain only TCAs.
wanderer mutants are homozygous for a nonsense mutation in Emx2
To determine whether wanderer
represented a known or a new thalamocortical development gene, the mutation was mapped and found on distal chromosome 19 beyond D19Mit1. This 6.3-Mb interval contains the well-known cortical development gene Emx2
(Figure , left). Since Emx2
knockout mice have a small cortex, small olfactory bulbs, ventral TCA misrouting, kidney dysgenesis, and perinatal lethality, Emx2
was a prime candidate gene. Also, the distinctive shape of the wanderer
mutant forebrain was remarkably similar to that of the Emx2
knockout (compare Figure here to Figure in [41
]). Sequencing of Emx2
from genomic DNA of wanderer
mutants identified a T to A mutation near the end of the first coding exon (Figure , right). This mutation is predicted to convert residue 130 (Tyr) to a stop codon and result in nonsense-mediated decay, or a protein truncated before the homeodomain and unable to bind DNA. Either way, this allele should act as a null.
Figure 7 wanderer is a nonsense allele of the cortical transcription factor Emx2. (A) The wanderer mutation was mapped to the distal end of chromosome 19 in a region containing the Emx2 gene. Sequencing revealed a T-to-A change (red asterisk) in the first coding (more ...)
To prove that this nonsense mutation in Emx2
indeed causes the wanderer
mutant phenotype, we performed a genetic complementation test between wanderer
mutants and Emx2
knockouts, which have a deletion/insertion in the homeodomain [41
]. Heterozygotes for the two alleles were crossed, and progeny were analyzed at day E18.5. Trans-heterozygotes had a visible phenotype indistinguishable from either of the single homozygous mutants, including the distinctively shaped small cortex and olfactory bulbs, and kidney hypoplasia (n = 8 out of 8 trans-heterozygotes). Three of them also carried the TCA-TLZ transgene, which showed long TCA fascicles growing on the ventral forebrain surface (Figure ). The failure of the two alleles to complement confirmed that the wanderer
point mutation in Emx2
is causative for the wanderer
phenotype, and that it behaves as a recessive loss of function mutation.
Surprisingly, the long TCA fascicles on the forebrain ventral surface that appeared striking to us had not been reported in prior studies of the TCA guidance defect in Emx2
knockout mutants [42
]. To ascertain whether this phenotype was present in the homozygous Emx2
knockout brains independent of the wanderer
mutation, we crossed the TCA-TLZ reporter into the Emx2
deletion line. Indeed, in brains from homozygous knockout (ko
) animals carrying the reporter, TCA-TLZ fascicles were seen growing in a rostral trajectory on the ventral surface of the forebrain, just as in wan/wan or wan/ko
brains (n = 8 of 8 ko
To control for the further possibility that the TCA ventral surface fascicles could be synthetically caused by the tau-lacZ transgene, carbocyanine dye tracing was used as an independent method to trace the TCAs. Dye crystal placement in the dorsal thalamus was performed in both Emx2 knockout animals that had not been crossed to the TCA-TLZ reporter line, and wanderer mutants that did not carry the reporter. In nearly all mutant cases, aberrant dye-labeled fascicles extended rostrally on the ventral surface of the forebrain (Figure , arrows; 6 out of 6 knockout hemispheres, 5 out of 6 wanderer hemispheres). The longest mutant TCAs labeled with DiI were just barely detectable curving toward the olfactory bulb on the whole-mounts. By contrast, heterozygous and wild-type littermate brains (Figure ) never exhibited the ventral surface fascicles (for Emx2 knockout, 0 of 16 ko/+ hemispheres, 0 of 12 +/+ hemispheres; for wanderer, 0 of 10 +/wan hemispheres, 0 of 6 +/+ hemispheres). These findings confirm that loss of Emx2 results in some TCAs growing to distant aberrant targets on the ventral forebrain surface, and that wanderer has the same TCA phenotype as the Emx2 knockout.