DEVELOPMENTAL DYNAMICS 218:94 –101 (2000) Severe Peripheral Sensory Neuron Loss and Modest Motor Neuron Reduction in Mice With Combined Deficiency of Brain-Derived Neurotrophic Factor, Neurotrophin 3 and Neurotrophin 4/5 XIN LIU1* AND RUDOLF JAENISCH2,3 1 Department of Pathology and Molecular Medicine, Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, California 2 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 3 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts ABSTRACT Neurotrophins are a family of structurally and functionally related proteins that regulate neuronal survival during development. In the peripheral nervous system (PNS), both in vitro and in vivo studies have shown that neurotrophins are potent factors for the survival of various sensory neurons and sympathetic neurons. However, it is not clear whether all PNS neurons are neurotrophin-dependent. In the central nervous system (CNS), studies using injury models show that neurotrophins promote the survival of CNS neurons. But mice lacking individual neurotrophins or a combination of BDNF and NT4 did not show significant CNS neuronal loss. Here we derived mice lacking three neurotrophins, brain-derived growth factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT4) to study the effect of triple neurotrophin deficiency on peripheral and central neurons. These triple-deficient mice did not nurse and died within 12 hours after birth. Neuronal cell counts showed that triple mutant pups lacked most of their peripheral sensory neurons and had a statistically significant reduction of motor neurons in several motor nuclei. Our results suggest that neurotrophins are essential for the survival of most peripheral sensory neurons and affect the survival of a small portion of motor neurons during embryogenesis. Dev Dyn 2000;218:94 –101. © 2000 Wiley-Liss, Inc. Key words: PNS; CNS neuron neurotrophin dependence in embryogenesis INTRODUCTION In mammals, the neurotrophin family consists of nerve growth factor (NGF), brain-derived growth factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT4) (Berkemeier et al., 1991; Hohn et al., 1990; Ibaanez 1994; Ip et al., 1992; Jones and Reichardt, 1990; Leibrock et al., 1989; Lewin and Barde, 1996; Maisonpierre et al., 1990). The mature forms of NGF, BDNF, NT3, and NT4 share a greater than 80% iden© 2000 WILEY-LISS, INC. tity at the amino acid level. These neurotrophins bind two types of receptors, the trks which belong to the receptor tyrosine kinase superfamily, and p75, a member of the tumor necrosis factor (TNF) receptor family (Barbacid, 1995; Bothwell, 1995; Chao, 1994). NGF is a ligand of trkA (Cordon-Cardo et al., 1991) and BDNF is a ligand of trkB (Klein et al., 1991; Soppet et al., 1991; Squinto et al., 1991). NT3 in addition to being a ligand of trkC (Lamballe et al., 1991), also binds trkA and trkB and induces phosphorylation of these receptors (Cordon-Cardo et al., 1991; Klein et al., 1991; Soppet et al., 1991; Squinto et al., 1991; Tessarollo et al., 1997). NT4 binds trkA and induces the phosphorylation of trkA in addition to its function as a ligand of trkB (Berkemeier et al., 1991; Ip et al., 1992). All neurotrophins bind with low affinity to the non-catalytic p75 receptor. The expression of the neurotrophins and the receptors often overlaps, both in tissue and developmental stage distribution (Eide et al., 1993; Korsching, 1993). For example, in the CNS, while trkA expression is restricted to only a few populations of neurons (Holtzman et al., 1992), trkB and trkC as well as their ligands have very broad patterns of expression (Ernfors et al., 1990; Escandon et al., 1994; Ip et al., 1992; Lamballe et al., 1994; Tessarollo et al., 1993; Wetmore et al., 1990). Since most CNS neurons appear to express trkB and trkC, it is likely that many classes of neurons in the CNS may respond to BDNF, NT3, and NT4. Ample studies have shown that neurotrophins are potent factors that promote the survival of peripheral sensory neurons, sympathetic neurons and various types of CNS neurons in cell culture and animal injury models (Eide et al., 1993; Korsching, 1993). In recent years, the in vivo functions of neurotrophins were under intense study using gene targeting approaches Grant sponsor: NIH/NCI; Grant number: R35-CA 44339. *Correspondence to Dr. Xin Liu, CHS 23-263, Department of Pathology and Molecular Medicine, Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, California 90095-1735. E-mail: email@example.com Received 21 October 1999; Accepted 13 January 2000 95 PNS AND CNS NEUROTROPHIN DEPENDENCE IN DEVELOPMENT TABLE 1. Genotypes of 250 Newborn Offspring of BDNFⴙ/ⴚ, NT3ⴙ/ⴚ, NT4ⴚ/ⴚ Mice ⫹/⫹ ⫹/⫹ ⫺/⫺ 17 6.8 6.25 V BDNF NT3 NT4 Number of offspring Actual % Theoretical % Adult viabilitya ⫹/⫹ ⫹/⫺ ⫺/⫺ 36 14.4 12.5 V ⫹/⫹ ⫺/⫺ ⫺/⫺ 16 6.4 6.25 NV ⫹/⫺ ⫹/⫹ ⫺/⫺ 29 11.6 12.5 V ⫹/⫺ ⫹/⫺ ⫺/⫺ 66 26.4 25 V ⫹/⫺ ⫺/⫺ ⫺/⫺ 36 14.4 12.5 NV ⫺/⫺ ⫹/⫹ ⫺/⫺ 11 4.4 6.25 NV ⫺/⫺ ⫹/⫺ ⫺/⫺ 27 10.8 12.5 NV ⫺/⫺ ⫺/⫺ ⫺/⫺ 12 4.8 6.25 NV a V, viable; NV, non-viable. (Conover and Yancopoulos, 1997; Snider, 1994). Mice carrying null mutations in individual neurotrophin or neurotrophin receptor genes were derived (Conover et al., 1995; Crowley et al., 1994; Ernfors et al., 1994a; Ernfors et al., 1994b; Farianas et al., 1994; Jones et al., 1994; Klein et al., 1994; Klein et al., 1993; Lee et al., 1992; Liu et al., 1995; Smeyne et al., 1994; Tessarollo et al., 1994). Consistent with previous studies in the PNS, these mutant mice show various degrees of neuronal reduction in the peripheral sensory ganglia and superior cervical ganglion (SCG) during late embryogenesis. These results support the hypothesis that neurotrophins control peripheral nervous system development by selectively promoting survival of specific groups of neurons. However, it is not entirely clear whether most of PNS sensory and sympathetic neurons or just overlapping subgroups of them depend on neurotrophins to survive during development. In contrast to the PNS, studies focused on the CNS of these mutant mice are not consistent with previous studies. In vitro studies have shown that neurotrophins promote the survival of motor neurons, dopaminergic neurons and other types of CNS neurons (Alderson et al., 1990; Hyman et al., 1991; Hyman et al., 1994; Hynes et al., 1994; Oppenheim et al., 1992; Sendtner et al., 1992; Yan et al., 1992). However, in mice carrying null mutations in neurotrophin genes, no significant CNS neuron loss was detected (Conover and Yancopoulos, 1997; Snider, 1994). In fact, removal of any single neurotrophic factor alone, including glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF), did not result in significant reduction of CNS neurons during embryogenesis (Masu et al., 1993; Moore et al., 1996; Pichel et al., 1996; Sanchez et al., 1996). These results may imply that multiple, functionally redundant neurotrophins may deliver the signals for CNS neurons to survive. To address these issues, we derived triple mutant mice deficient for BDNF, NT3, and NT4. RESULTS Mutant mice were consecutively crossed to derive offspring carrying different combinations of mutant alleles. We found that mice lacking NT4 and carrying only one wild-type (wt) allele of the BDNF and NT3 genes survived to adulthood and were fertile. Also, mice deficient in all three neurotrophin genes or any combination of two of the three neurotrophin genes, BDNF, NT3, or NT4, survived to birth (Table 1). However, none of the double- or triple-deficient pups survived for longer than 12 to 48 hours post delivery. Close observation of triple-deficient mutant newborns, revealed: 1) difficulty with breathing but no gross anatomical abnormalities; 2) failure to nurse as none of the animals had milk in their stomachs. To examine whether neurons were lost in the mutant animals, live newborns (within 12 hr from birth) were collected. The head and spine of each animal were processed as described in Experimental Procedures. First inspection indicated that the peripheral sensory ganglia of the triple-mutant mice were dramatically reduced in size compared to those of wt animals. Figure 1 A to D shows that the dorsal root (B) and nodose ganglia (D) of [BDNF⫺/⫺, NT3⫺/⫺, NT4⫺/⫺] mice were significantly smaller than those (A and C) of wt mice. To quantify the reduction of neurons in ganglia of the triple mutant mice, neurons in peripheral sensory ganglia and the superior cervical ganglion (SCG) were counted. Setting the cell numbers from wt mice as 100%, we found that approximately 90% of the neurons in nodose, trigeminal, and dorsal root ganglia were lost in [BDNF⫺/⫺, NT3⫺/⫺, NT4⫺/⫺] mice, whereas geniculate and vestibular neurons were completely absent (Table 2). These data provide strong evidence that survival of most, if not all, PNS sensory neurons depends on the presence of neurotrophins during embryogenesis. In contrast to the sensory neurons, sympathetic neurons in the SCG of [BDNF⫺/⫺, NT3⫺/⫺, NT4⫺/⫺] mice were reduced by 50%, a deficit which is comparable to the reduction seen in mice lacking only NT3 (Ernfors et al., 1994b; Jones et al., 1994). This result is consistent with the previous conclusions that losses of BDNF and NT4 have little or no effect on sympathetic neuron survival (Conover et al., 1995; Liu et al., 1995). To study the role of neurotrophins in promoting CNS neuronal survival during development, we examined several motor nuclei in the brain stem and motor neurons in the spinal cord. We found a small (⬃20%) but statistically significant reduction of motor neurons in the hypoglossal, facial, and trigeminal nuclei of tripledeficient mutants (Table 2). The remaining motor neurons in these nuclei of mutant animals are often smaller in size and stain darker compared to their counterparts in wt animals (Fig. 1, E and F). To docu- 96 LIU AND JAENISCH Fig. 1. Histochemistry and immunohistochemistry of wt and neurotrophin triple deficient (TD) mice. A–F: Cresyl violet stained paraffin sections. A and B are cross sections of lumbar spinal column 4 (L4) from wt and TD neonates, respectively. Arrows point to DRG on each side of the spinal cord. Scale bar ⫽ 0.4 mm. C and D are coronal sections of nodose ganglia from wt and TD neonates, respectively. Nodose ganglia are circled with dashed lines. E and F are coronal sections of facial motor nuclei of wt and TD, respectively. Arrows point to individual motor neurons. G and H are tyrosine hydroxylase immunohistochemistry of coronal sections of the midbrain of wt and TD neonates, respectively. Cytoplasm and neurite are stained. Arrows point to some neuronal cell bodies in substantia nigra. Scale bars ⫽ 0.12 mm for C–H. ment this change, we compared the size of facial motor neurons between wt and mutant mice. Figure 2 shows that, in the facial motor nuclei of mutant mice, the percentage of small neurons (⬍ 10 m in diameter) increased and the percentage of large neurons (⬎ 10 m in diameter) decreased compared to that of wt animals. In the same section level, when the sizes of neurons adjacent to the facial motor neurons are compared, there are no detectable changes. In addition to motor nuclei, we also examined dopaminergic neurons in the substantia nigra. Anti-tyrosine hydroxylase antibodies were used to identify these neurons. Staining was detected in the cytoplasm (Fig. 1, G and H, arrows) of dopaminergic neurons in the substantia nigra of PNS AND CNS NEUROTROPHIN DEPENDENCE IN DEVELOPMENT 97 TABLE 2. Neuronal Cell Counts in Sensory and Sympathetic Ganglia and Motor Nuclei of Wild-Type and Mutant Mice Wild-type Trigeminal ganglion Vestibular ganglion Superior cervical ganglion Nodose-petrosal ganglia Geniculate ganglion Dorsal root (L4) ganglion Facial motor neurons Hypoglossal motor neurons Trigeminal motor neurons Spinal motor column Mean number of neuronsa 29,796 ⫾ 860 (n ⫽ 4) 3,061 ⫾ 169 (n ⫽ 3) 15,226 ⫾ 1122 (n ⫽ 4) 4,175 ⫾ 173 (n ⫽ 3) 912 ⫾ 8 (n ⫽ 3) 6,744 ⫾ 272 (n ⫽ 3) 3565 ⫾ 136 (n ⫽ 6) 1673 ⫾ 70 (n ⫽ 6) 839 ⫾ 37 (n ⫽ 6) 821 ⫾ 80 (n ⫽ 4) % of control 100 ⫾ 3 100 ⫾ 6 100 ⫾ 7 100 ⫾ 4 100 ⫾ 1 100 ⫾ 5 100 ⫾ 4 100 ⫾ 4 100 ⫾ 5 100 ⫾ 10 BDNF⫺/⫺/NT3⫺/⫺/NT4⫺/⫺ Mean number of neuronsa % of control 3,623 ⫾ 376 12 ⫾ 1*** (n ⫽ 6) 0 0 (n ⫽ 6) 8,043 ⫾ 333 53 ⫾ 2*** (n ⫽ 6) 165 ⫾ 22 4 ⫾ 1*** (n ⫽ 6) 0 0 (n ⫽ 6) 558 ⫾ 24 8 ⫾ 1*** (n ⫽ 4) 2781 ⫾ 148 78 ⫾ 4* (n ⫽ 8) 1362 ⫾ 42 81 ⫾ 2* (n ⫽ 8) 682 ⫾ 19 81 ⫾ 2* (n ⫽ 8) 664 ⫾ 28 80 ⫾ 4 (n ⫽ 2) All are neuronal counts of newborn mice (P0). Counts are displayed as number ⫾ s.e.m. *P ⬍ 0.05; ***P ⬍ 0.001 for student’s t-test (unpaired). a DISCUSSION Fig. 2. Comparison of facial motor (fm) neuron sizes between wt and neurotrophin triple deficient (TD) mice. There is higher percentage of small fm neurons (⬍10 m) in the TD mice (69%) than that (44%) in wt mice (P ⫽ 0.0056). There is lower percentage of large fm neurons (10 –15 m) in the TD mice (31%) than that (54%) of wt mice (P ⫽ 0.0058). In wt but not TD mice, there is also a small percentage (ⵒ2%) of fm neurons that are larger than 15 m in diameter. When the sizes of the neurons located adjacent to fm neurons were compared between wt and TD mice, no size difference was detected (⬍ 7.5 m, P ⫽ 0.591; 7.5–10 m, P ⫽ 0.995; ⬎ 10 m, P ⫽ 0.088). both wt and [BDNF⫺/⫺, NT3⫺/⫺, NT4⫺/⫺] mice suggesting that development or survival of these neurons was not substantially affected by the lack of neurotrophins. In this study we have generated mutant mice that carry deletions in all or in different combinations of the genes coding for the three neurotrophins, BDNF, NT-3, and NT-4. The [BDNF⫹/⫺, NT3⫹/⫺, NT4⫺/⫺] mice are viable and fertile suggesting that a substantial reduction of neurotrophin dose does not hamper the overall development. The [BDNF⫺/⫺, NT3⫺/⫺, NT4⫺/⫺] embryos survived to term but the pups died within 12 hrs: that is the earliest of all known neurotrophin mutants (Conover and Yancopoulos, 1997; Snider, 1994). When compared to wild-type pups, no gross anatomical differences were detected in the mutants. However, the mutant pups appeared to have difficulties breathing and it is possible that the dysfunction of the respiratory system may contribute to the death of these animals. Consistent with this possibility, a recent study showed that BDNF⫺/⫺ mice have a deficit in neurons that control breathing (Erickson et al., 1996). We speculate that additional loss of neurotrophins may exasperate deficits in neurons that control breathing, and therefore results in the early postnatal death of the triple neurotrophin deficient mutants. Our histological analyses revealed a profound deficit of sensory neurons with not more than 5–10% neurons present in the various ganglia of the peripheral nervous system of triple mutants. In addition, about 50% of the SCG neurons were absent. The phenotypes of animals deficient for a single neurotrophin, for BDNF and NT-3, for BDNF and NT-4, and for trkB and trkC 98 LIU AND JAENISCH TABLE 3. Summary of the Percent of Neuronal Loss in the Various Ganglia of Neurotrophin and trk Mutants Compared to Wild-Type Mice NGF⫺/⫺ Trigeminal Vestibular SCG Nodose Geniculate DRG Motor (Facial) Spiral 82b 70b BDNF⫺/⫺ 44d/27e 82d/87e ⫹14*d 66d/43e 40e 30d/34e 3*d NT3⫺/⫺ 64d/61c 34d/23*c 53d/48c 47d/30c 25c 55d/78c 2*f NT4⫺/⫺ 2*f/5*a 21*f ⫹12*f 59f/56a 50f 14*f 8*f BDNF⫺/⫺ NT3⫺/⫺ 99d 66h 100d BDNF⫺/⫺ NT4⫺/⫺ 30f/34a 82f 4*f 90f/79a 94f 11*f trkB/C⫺/⫺ 58g/100i 95i 98i 41g,i 5i BDNF⫺/⫺ NT3⫺/⫺ NT4⫺/⫺ 88 100 47 96 100 92 20 *Not statistically significant. Previously published results, aConover et al., bCrowley et al., cFarianas et al., dErnfors et al., 1994a,b, 1995, eJones et al., fLiu et al., 1995, gMinichiello et al., h ElShamy and Ernfors, and iSilos-Santiago et al. ⫹ Shows an increase instead of loss. double-mutants have been described previously (Conover et al., 1995; Ernfors et al., 1994a; Ernfors et al., 1994b; Ernfors et al., 1995; Farianas et al., 1994; Jones et al., 1994; Liu et al., 1995; Minichiello et al., 1995; Silos-Santiago et al., 1997). The neuronal deficits of these mutants and our results are summarized in Table 3. Our results are consistent with the notion that, in addition to neurons in the vestibular and spiral ganglia (Ernfors et al., 1995), almost all the neurons in every PNS sensory ganglion that we examined depend on neurotrophin to survive during embryogenesis. It is possible that, if the triple mutant animals live long enough, we may observe the complete PNS sensory neuron loss. How do these neurotrophins cooperate to regulate the neuronal survival in each ganglion? Comparing our results with previous data (see Table 3), it seems that there are three major patterns. In vestibular, and geniculate ganglia, the extent of PNS neuronal loss in animals with multiple neurotrophin mutations is additive reflecting the combined deficits caused by each individual neurotrophin mutation. This pattern favors the hypothesis that each type of neurotrophin is required by largely non-overlapping subpopulations of neurons in a ganglion. In the nodose ganglion, deficiency in either BDNF, NT3, or NT4 lead to about 50% of neuron reduction. In DRG, deficiency in NGF leads to 70% neuronal reduction whereas our triple mutants suffered a 92% neuronal reduction. This second pattern suggests that there are subpopulations of neurons in these ganglia, which require more than one type of neurotrophin to survive. Indeed, Pinon and colleagues have shown that trigeminal neurons may require BDNF in early stage and later shift to NGF (Pinon et al., 1996). ElShamy and Ernfors have shown that BDNF and NT3 may be required sequentially for the same group of nodose neurons (ElShamy and Ernfors, 1997). The last pattern is seen in the SCG where the neuronal loss in triple mutants is similar to that in NT3 single mutants (Table 3). Taken together, we suggest that, during embryogenesis, neurotrophins regu- late the survival of most PNS sensory neurons, that subpopulations of PNS neurons require more than one type of neurotrophin to survive, and that BDNF and NT4 are not required for SCG neuronal survival. The most surprising result of our study is the modest but statistically significant neuronal reduction in several motor nuclei of the triple mutants. We do not know whether the severe loss of PNS sensory neurons can affect the differentiation and survival of motor neurons and cause the reduction that we detect. In previous studies, we and others have observed between a 2 to 11% loss of facial motor neurons, that is not statistically significant, in single neurotrophin mutants and BDNF and NT4 double mutants (Ernfors et al., 1994a; Ernfors et al., 1994b; Liu et al., 1995). It is possible that the accumulation of the small but additive reductions of motor neurons in single and double neurotrophin mutants made the motor neuron loss in triple mutants detectable. Alternatively, any one of these three neurotrophins, BDNF, NT3, or NT4 may deliver the signal for a small but distinct group of cells in these motor nuclei to survive. This later possibility is in agreement with a recent study showing that [trkB⫹/⫺, trkC⫺/⫺] or [trkB⫺/⫺, trkC⫹/⫺] mice have similar hippocampal and cerebellar granule neuron reduction suggesting that CNS neurons are capable of using more than one neurotrophin/trk receptor signal transduction pathway for survival (Minichiello and Klein, 1996). In this study, Minichiello and colleagues did not detect motor neuron loss in trkB and trkC double-mutant mice (Minichiello and Klein, 1996). We speculate that lack of BDNF, NT3, and NT4 may lead to a more severe phenotype than that of [trkB⫺/⫺, trkC⫹/⫺] and [trkB⫹/⫺, trkC⫺/⫺] mice because these receptor mutant mice are not deficient for both trkB and trkC receptors. Moreover, the truncated form of trkB may exist in these mice (Klein et al., 1993). It has been demonstrated that truncated forms of trkB are capable of mediating BDNF-induced signal transduction (Baxter et al., 1997). These speculations may also explain the excessive neuronal loss in vestibular ganglia and DRG (see PNS AND CNS NEUROTROPHIN DEPENDENCE IN DEVELOPMENT 99 Fig. 3. Genotyping of mutant mice. A: PCR analysis of BDNF and NT3 wt and knockout (KO) alleles. B: Southern analysis of NT4 wt and KO alleles. For details, please see Experimental Procedures. Table 3) of the triple mutants compared to that of [trkB⫺/⫺, trkC⫺/⫺] mice (Minichiello et al., 1995; Silos-Santiago et al., 1997). Alternatively, NT3 and NT4 may signal through trkA in the DRG neurons of [trkB⫺/⫺, trkC⫺/⫺] mice. It is not known that trkA is expressed in facial or trigeminal motor neurons. We also observed the neuronal size reduction in the remaining motor neurons of the triple mutants suggesting a substantial percentage of motor neurons are affected by neurotrophin deficiency. However, we do not know the precise nature of changes that take place in these neurons. In our study, we have not observed gross change in the hippocampus or cerebellum of triple mutants. Since Minichiello and colleagues’ results were from postnatal day 14 animals, it could be that neurotrophins are required for the survival of these granule neurons in postnatal development and there is no detectable deficit of these cells in P0 mutant mice. It would be interesting to examine if there are reductions in the granule neurons of viable [BDNF⫹/⫺, NT3⫹/⫺, NT4⫺/⫺] mice. EXPERIMENTAL PROCEDURES Mouse Mutants The generation of mice carrying targeted null mutations in BDNF, NT3, or NT4 genes has been described previously (Ernfors et al., 1994a; Ernfors et al., 1994b; Liu et al., 1995). Triple mutant mice [BDNF⫺/⫺, NT3⫺/⫺, NT4⫺/⫺] were derived by several steps of breeding. BDNF or NT3 heterozygous mutant mice were bred with NT4 homozygous mutant mice to obtain [BDNF⫹/⫺, NT4⫹/⫺] and [NT3⫹/⫺, NT4⫹/⫺] mice. Then, [BDNF⫹/⫺, NT4⫹/⫺] mice were bred with [NT3⫹/⫺, NT4⫹/⫺] mice to obtain [BDNF⫹/⫺, NT3⫹/⫺, NT4⫺/⫺] mice. Finally, the triple-deficient [BDNF⫺/⫺, NT3⫺/⫺, NT4⫺/⫺] mice were obtained from breeding of [BDNF⫹/⫺, NT3⫹/⫺, NT4⫺/⫺] mice. To identify the genotypes of these mice, a small amount of skin from each animal was taken to harvest DNA. Polymerase chain reaction (PCR) was used to genotype BDNF and NT3 mutants (Fig. 3A). The primers used for BDNF are BL-1: ATGAAAGAAGTAAACGTCCAC, BL-2: CCAGCAGAAAGAGTAGAGGAG, and PGK: GGGAACTTCCTGACTAGGGG. The primers used for NT3 are NT3-3: CCTGGCTTCTTTACATCTCG, NT3-4: TGGAGGATTATGTGGGCAAC, and PGK as described above. PCR using primers BL-1 and BL-2 or primers NT3-3 and NT3-4 amplify DNA fragments only from the wt allele of BDNF and NT3, respectively. PCR using BL-1 and PGK or NT3-3 and PGK amplify DNA fragments only from the targeted allele of BDNF and NT3, respectively. Southern blot analysis was used to genotype NT4 mutants (Fig. 3B) as described previously (Liu, et al., 1995). Cresyl Violet Staining and Neuron Counting Heads and lumbar spinal column 3 (L3) to lumbar spinal column 5 (L5) from newborn mice were collected. Skulls of each head were carefully removed to expose the brain. These tissues were fixed in formalin for 24 hr, embedded in paraffin, sectioned at 5 m thickness, and stained with cresyl violet. Neurons with cytoplasm, clear nucleus, and nucleoli were counted in every fourth section as previously described (Ernfors et al., 1994b). The sizes of neurons are measured by using a grid inserted in one side of the ocular lens. Based on the sizes, the facial motor neurons are divided into three groups, ⬍ 10, 10 –15, and ⬎ 15 m in diameter and counted. The neurons adjacent to facial motor neurons 100 LIU AND JAENISCH are defined as neurons located in a 250 m diameter circular area between the motor nuclei and the midline of the brain. These neurons are also divided into three groups, ⬍ 7.5, 7.5–10, and ⬎ 10 m in diameter and counted. If the neuron has an oval shape, the smaller of the two diameters is used to classify the neuron. Facial motor neurons and neurons adjacent to facial motor neurons in every eighth section of the serial brain sections are measured and counted. Serial brain sections from three wt and four triple-deficient mice are used in the measuring and counting. The student’s t-test is used for the statistical analysis of the data. Tyrosine Hydroxylase Immunohistochemistry Brains from newborn mice were dissected, fixed in 4% paraformaldehyde, equilibrated with 30% sucrose, and sectioned at 10 m thickness. Sections were preincubated in dilution buffer (0.1 M NaCl, 0.01 M phosphate buffer, pH 7.4, 3% bovine serum albumin and 0.3% Triton X-100) for 1 hr followed by overnight incubation with rabbit anti-tyrosine hydroxylase antiserum (Peel-Freeze) diluted at 1:200 in dilution buffer containing 5% goat serum at 4°C. After 4 washes in PBS, sections were incubated with biotinylated anti rabbit IgG secondary antibody and avidin-peroxidase (Vectastain ABC kit, Vector Laboratories) as well as with 3,3⬘-diaminobenzidine (DAB, Sigma Chemical Company, St. Louis, MO) as previously described (Liu, et al., 1991). ACKNOWLEDGMENTS This work is supported by NIH/NCI grant R35-CA 44339 to RJ and Beatrice Kolliner Fellow award to XL. We thank Hong Wu, Samson Chow, Inbal Israely, and Brian Bates for reviewing the manuscript. REFERENCES Alderson RF, Alterman AL, Barde YA, Lindsay RM. 1990. Brainderived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture. Neuron 5:297–306. Barbacid M. 1995. Neurotrophic factors and their receptors. Curr Opin Cell Biol 7:148 –155. Baxter GT, Radeke MJ, Kuo RC, Makrides V, Hinkle B, Hoang R, Medina-Selby A, Coit D, Valenzuela P, Feinstein SC. 1997. Signal transduction mediated by the truncated trkB receptor isoforms, trkB.T1 and trkB.T2. J Neurosci 17:2683–2690. Berkemeier LR, Winslow JW, Kaplan DR, Nikolics K, Goeddel DV, Rosenthal A. 1991. Neurotrophin-5: a novel neurotrophic factor that activates trk and trkB. Neuron 7:857– 866. Bothwell M. 1995. Functional interactions of neurotrophins and neurotrophin receptors. Annu Rev Neurosci 18:223–253. Chao MV. 1994. The p75 neurotrophin receptor. J Neurobiol 25:1373– 1385. Conover JC, Yancopoulos GD. 1997. Neurotrophin regulation of the developing nervous system: analyses of knockout mice. Rev Neurosci 8:13–27. Conover JC, Erickson JT, Katz DM, Bianchi LM, Poueymirou WT, McClain J, Pan L, Helgren M, Ip NY, Boland P, et al. 1995. Neuronal deficits, not involving motor neurons, in mice lacking BDNF and/or NT4. Nature 375:235–238. Cordon-Cardo C, Tapley P, Jing SQ, Nanduri V, O’Rourke E, Lamballe F, Kovary K, Klein R, Jones KR, Reichardt LF, et al. 1991. The trk tyrosine protein kinase mediates the mitogenic properties of nerve growth factor and neurotrophin-3. Cell 66:173–183. Crowley C, Spencer SD, Nishimura MC, Chen KS, Pitts-Meek S, Armanini MP, Ling LH, MacMahon SB, Shelton DL, Levinson AD, et al. 1994. Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. Cell 76:1001–1011. Eide FF, Lowenstein DH, Reichardt LF. 1993. Neurotrophins and their receptors— current concepts and implications for neurologic disease. Exp Neurol 121:200 –214. ElShamy WM, Ernfors P. 1997. Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 complement and cooperate with each other sequentially during visceral neuron development. J Neurosci 17:8667– 8675. Erickson JT, Conover JC, Borday V, Champagnat J, Barbacid M, Yancopoulos G, Katz DM. 1996. Mice lacking brain-derived neurotrophic factor exhibit visceral sensory neuron losses distinct from mice lacking NT4 and display a severe developmental deficit in control of breathing. J Neurosci 16:5361–5371. Ernfors P, Wetmore C, Olson L, Persson H. 1990. Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family. Neuron 5:511–526. Ernfors P, Lee KF, Jaenisch R. 1994a. Mice lacking brain-derived neurotrophic factor develop with sensory deficits. Nature 368:147– 150. Ernfors P, Lee KF, Kucera J, Jaenisch R. 1994b. Lack of neurotrophin-3 leads to deficiencies in the peripheral nervous system and loss of limb proprioceptive afferents. Cell 77:503–512. Ernfors P, Van De Water T, Loring J, Jaenisch R. 1995. Complementary roles of BDNF and NT-3 in vestibular and auditory development. Neuron 14:1153–1164. Escandon E, Soppet D, Rosenthal A, Mendoza-Ramairez JL, Szeonyi E, Burton LE, Henderson CE, Parada LF, Nikolics K. 1994. Regulation of neurotrophin receptor expression during embryonic and postnatal development. J Neurosci 14:2054 –2068. Farianas I, Jones KR, Backus C, Wang XY, Reichardt LF. 1994. Severe sensory and sympathetic deficits in mice lacking neurotrophin-3. Nature 369:658 – 661. Hohn A, Leibrock J, Bailey K, Barde YA. 1990. Identification and characterization of a novel member of the nerve growth factor/ brain-derived neurotrophic factor family. Nature 344:339 –341. Holtzman DM, Li Y, Parada LF, Kinsman S, Chen CK, Valletta JS, Zhou J, Long JB, Mobley WC. 1992. p140trk mRNA marks NGFresponsive forebrain neurons: evidence that trk gene expression is induced by NGF. Neuron 9:465– 478. Hyman C, Hofer M, Barde YA, Juhasz M, Yancopoulos GD, Squinto SP, Lindsay RM. 1991. BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Nature 350:230 –232. Hyman C, Juhasz M, Jackson C, Wright P, Ip NY, Lindsay RM. 1994. Overlapping and distinct actions of the neurotrophins BDNF, NT-3, and NT-4/5 on cultured dopaminergic and GABAergic neurons of the ventral mesencephalon. J Neurosci 14:335–347. Hynes MA, Poulsen K, Armanini M, Berkemeier L, Phillips H, Rosenthal A. 1994. Neurotrophin-4/5 is a survival factor for embryonic midbrain dopaminergic neurons in enriched cultures. J Neurosci Res 37:144 –154. Ibaanez CF. 1994. Structure-function relationships in the neurotrophin family. J Neurobiol 25:1349 –1361. Ip NY, Ibaaanez CF, Nye SH, McClain J, Jones PF, Gies DR, Belluscio L, Le Beau MM, Espinosa Rd, Squinto SP, et al. 1992. Mammalian neurotrophin-4: structure, chromosomal localization, tissue distribution, and receptor specificity. Proc Natl Acad Sci USA 89:3060 – 3064. Jones KR, Reichardt LF. 1990. Molecular cloning of a human gene that is a member of the nerve growth factor family. Proc Natl Acad Sci USA 87:8060 – 8064. Jones KR, Farianas I, Backus C, Reichardt LF. 1994. Targeted disruption of the BDNF gene perturbs brain and sensory neuron development but not motor neuron development. Cell 76:989 –999. Klein R, Nanduri V, Jing SA, Lamballe F, Tapley P, Bryant S, CordonCardo C, Jones KR, Reichardt LF, Barbacid M. 1991. The trkB PNS AND CNS NEUROTROPHIN DEPENDENCE IN DEVELOPMENT tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. Cell 66:395– 403. Klein R, Smeyne RJ, Wurst W, Long LK, Auerbach BA, Joyner AL, Barbacid M. 1993. Targeted disruption of the trkB neurotrophin receptor gene results in nervous system lesions and neonatal death. Cell 75:113–122. Klein R, Silos-Santiago I, Smeyne RJ, Lira SA, Brambilla R, Bryant S, Zhang L, Snider WD, Barbacid M. 1994. Disruption of the neurotrophin-3 receptor gene trkC eliminates la muscle afferents and results in abnormal movements [see comments]. Nature 368:249–251. Korsching S. 1993. The neurotrophic factor concept: a reexamination. J Neurosci 13:2739 –2748. Lamballe F, Klein R, Barbacid M. 1991. trkC, a new member of the trk family of tyrosine protein kinases, is a receptor for neurotrophin-3. Cell 66:967–979. Lamballe F, Smeyne RJ, Barbacid M. 1994. Developmental expression of trkC, the neurotrophin-3 receptor, in the mammalian nervous system. J Neurosci 14:14 –28. Lee KF, Li E, Huber LJ, Landis SC, Sharpe AH, Chao MV, Jaenisch R. 1992. Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. Cell 69:737–749. Leibrock J, Lottspeich F, Hohn A, Hofer M, Hengerer B, Masiakowski P, Thoenen H, Barde YA. 1989. Molecular cloning and expression of brain-derived neurotrophic factor. Nature 341:149 –152. Lewin GR, Barde YA. 1996. Physiology of the neurotrophins. Annu Rev Neurosci 19:289 –317. Liu X, Yu QA, Huang ZS, Zwiebel LJ, Hall JC, Rosbash M. 1991. The strength and periodicity of D. melanogaster circadian rhythms are differentially affected by alterations in period gene expression. Neuron 6:753–766. Liu X, Ernfors P, Wu H, Jaenisch R. 1995. Sensory but not motor neuron deficits in mice lacking NT4 and BDNF. Nature 375:238 – 241. Maisonpierre PC, Belluscio L, Squinto S, Ip NY, Furth ME, Lindsay RM, Yancopoulos GD. 1990. Neurotrophin-3: a neurotrophic factor related to NGF and BDNF. Science 247:1446 –1451. Masu Y, Wolf E, Holtmann B, Sendtner M, Brem G, Thoenen H. 1993. Disruption of the CNTF gene results in motor neuron degeneration. Nature 365:27–32. Minichiello L, Klein R. 1996. TrkB and TrkC neurotrophin receptors cooperate in promoting survival of hippocampal and cerebellar granule neurons. Genes Dev 10:2849 –2858. Minichiello L, Piehl F, Vazquez ES, T., Hokfelt T, Represa J, Klein R. 1995. Differential effects of combined trk receptor mutations on dorsal root ganglion and inner ear sensory neurons. Development 121:4067– 4075. Moore MW, Klein RD, Farianas I, Sauer H, Armanini M, Phillips H, Reichardt LF, Ryan AM, Carver-Moore K, Rosenthal A. 1996. Renal and neuronal abnormalities in mice lacking GDNF. Nature 382:76 – 79. Oppenheim RW, Yin QW, Prevette D, Yan Q. 1992. Brain-derived neurotrophic factor rescues developing avian motoneurons from cell death. Nature 360:755–757. 101 Pichel JG, Shen L, Sheng HZ, Granholm AC, Drago J, Grinberg A, Lee EJ, Huang SP, Saarma M, Hoffer BJ, Sariola H, Westphal H. 1996. Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 382:73–76. Pinon LG, Minichiello L, Klein R, Davies AM. 1996. Timing of neuronal death in trkA, trkB and trkC mutant embryos reveals developmental changes in sensory neuron dependence on Trk signalling. Development 122:3255–3261. Sanchez MP, Silos-Santiago I, Frisaen J, He B, Lira SA, Barbacid M. 1996. Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 382:70 –73. Sendtner M, Holtmann B, Kolbeck R, Thoenen H, Barde YA. 1992. Brain-derived neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section. Nature 360:757–759. Silos-Santiago I, Fagan AM, Garber M, Fritzsch B, Barbacid M. 1997. Severe sensory deficits but normal CNS development in newborn mice lack TrkB and TrkC tyrosine protein kinase receptors. Euro J Neurosci 9:2045–2056. Smeyne RJ, Klein R, Schnapp A, Long LK, Bryant S, Lewin A, Lira SA, Barbacid M. 1994. Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene [see comments]. Nature 368:246 –249. Snider WD. 1994. Functions of the neurotrophins during nervous system development: what the knockouts are teaching us. Cell 77:627– 638. Soppet D, Escandon E, Maragos J, Middlemas DS, Reid SW, Blair J, Burton LE, Stanton BR, Kaplan DR, Hunter T, et al. 1991. The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor. Cell 65:895–903. Squinto SP, Stitt TN, Aldrich TH, Davis S, Bianco SM, Radziejewski C, Glass DJ, Masiakowski P, Furth ME, Valenzuela DM, et al. 1991. trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor. Cell 65:885– 893. Tessarollo L, Tsoulfas P, Martin-Zanca D, Gilbert DJ, Jenkins NA, Copeland NG, Parada LF. 1993. trkC, a receptor for neurotrophin-3, is widely expressed in the developing nervous system and in nonneuronal tissues [published erratum appears in Development 1993 Aug; 118: following 1384]. Development 118:463– 475. Tessarollo L, Vogel KS, Palko ME, Reid SW, Parada LF. 1994. Targeted mutation in the neurotrophin-3 gene results in loss of muscle sensory neurons. Proc Natl Acad Sci USA 91:11844 –11848. Tessarollo L, Tsoulfas P, Donovan MJ, Palko ME, Blair-Flynn J, Hempstead BL, Parada LF. 1997. Targeted deletion of all isoforms of the trkC gene suggests the use of alternate receptors by its ligand neurotrophin-3 in neuronal development and implicates trkC in normal cardiogenesis. Proc Natl Acad Sci USA 94:14776 –14781. Wetmore C, Ernfors P, Persson H, Olson L. 1990. Localization of brain-derived neurotrophic factor mRNA to neurons in the brain by in situ hybridization. Exp Neurol 109:141–152. Yan Q, Elliott J, Snider WD. 1992. Brain-derived neurotrophic factor rescues spinal motor neurons from axotomy-induced cell death. Nature 360:753–755.