Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice
1998; Springer Nature; Volume: 17; Issue: 15 Linguagem: Inglês
10.1093/emboj/17.15.4213
ISSN1460-2075
AutoresRosanna Dono, Gemma Texidó, R. Dussel, Heimo Ehmke, Rolf Zeller,
Tópico(s)Genetic Syndromes and Imprinting
ResumoArticle3 August 1998free access Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice Rosanna Dono Rosanna Dono EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Gemma Texido Gemma Texido EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Rudolf Dussel Rudolf Dussel Institute of Physiology, University of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany Search for more papers by this author Heimo Ehmke Heimo Ehmke EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Institute of Physiology, University of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany Search for more papers by this author Rolf Zeller Rolf Zeller EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Rosanna Dono Rosanna Dono EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Gemma Texido Gemma Texido EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Rudolf Dussel Rudolf Dussel Institute of Physiology, University of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany Search for more papers by this author Heimo Ehmke Heimo Ehmke EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Institute of Physiology, University of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany Search for more papers by this author Rolf Zeller Rolf Zeller EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Author Information Rosanna Dono1, Gemma Texido1, Rudolf Dussel2, Heimo Ehmke1,2 and Rolf Zeller1 1EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany 2Institute of Physiology, University of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany The EMBO Journal (1998)17:4213-4225https://doi.org/10.1093/emboj/17.15.4213 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Fibroblast growth factor-2 (FGF-2) has been implicated in various signaling processes which control embryonic growth and differentiation, adult physiology and pathology. To analyze the in vivo functions of this signaling molecule, the FGF-2 gene was inactivated by homologous recombination in mouse embryonic stem cells. FGF-2-deficient mice are viable, but display cerebral cortex defects at birth. Bromodeoxyuridine pulse labeling of embryos showed that proliferation of neuronal progenitors is normal, whereas a fraction of them fail to colonize their target layers in the cerebral cortex. A corresponding reduction in parvalbumin-positive neurons is observed in adult cortical layers. Neuronal defects are not limited to the cerebral cortex, as ectopic parvalbumin-positive neurons are present in the hippocampal commissure and neuronal deficiencies are observed in the cervical spinal cord. Physiological studies showed that FGF-2-deficient adult mice are hypotensive. They respond normally to angiotensin II-induced hypertension, whereas neural regulation of blood pressure by the baroreceptor reflex is impaired. The present genetic study establishes that FGF-2 participates in controlling fates, migration and differentiation of neuronal cells, whereas it is not essential for their proliferation. The observed autonomic dysfunction in FGF-2-deficient adult mice uncovers more general roles in neural development and function. Introduction Members of the fibroblast growth factor (FGF) family and their high affinity tyrosine kinase FGF receptors (FGFR) have been implicated in embryonic growth and patterning and, in particular, during central nervous system (CNS) development (reviewed by Eckenstein, 1994). Studies using Xenopus laevis embryos revealed roles for FGF signaling during establishment of initial posterior neural tube identities (reviewed by Sasai and De Robertis, 1997). Similarly, FGFR1-deficient mouse embryos display neural tube defects, which most likely arise from incorrect specification of neuroectodermal cell fates (Yamaguchi et al., 1994). During initial patterning of the brain vesicle, FGF-8 is expressed by the isthmus, the organizer which separates mid- from hindbrain (Crossley et al., 1996). Ectopic rostral FGF-8 application or expression results in transformation of fore- into midbrain structures, indicating that FGF-8 determines midbrain identity (Crossley et al., 1996; Lee et al., 1997). The spatial distribution of FGF-3 in the developing hindbrain is consistent with roles in establishment of rhombomere boundaries (Wilkinson et al., 1988), but no changes in rhombomere identities are observed in FGF-3-deficient embryos (Mansour et al., 1993). Roles for FGF-2 during CNS morphogenesis have been postulated as high levels are detected from neurulation onwards (Dono and Zeller, 1994; Riese et al., 1995; reviewed by Eckenstein, 1994). Furthermore, overexpression of specific FGF-2 isoforms in the neural tube of transgenic mouse embryos provided evidence for growth-regulatory functions during spinal cord morphogenesis (Zuniga Mejia Borja et al., 1996). Such a proposed mitogenic role in neurogenesis was also supported by studies using cultured neural progenitors. These studies indicated that FGF-2 levels regulate proliferation of specific neural cell types (reviewed by Vescovi et al., 1993; Temple and Qian, 1995). In contrast, other studies provided evidence that FGF-2 participates in promoting differentiation and long-term survival of neurons in culture. These in vitro studies suggested that FGF-2 functions in a concentration-dependent manner to regulate mainly proliferation, but also differentiation and survival of neural progenitors (Petroski et al., 1991; Ray et al., 1993; Ghosh and Greenberg, 1995; Vicario-Abejon et al., 1995; Qian et al., 1997). In addition to the CNS, FGF-2 is expressed during other developmental processes such as limb, kidney and cardiovascular morphogenesis (e.g. Dono and Zeller, 1994). Of particular interest for the present study is expression by vascular smooth muscle cells, endothelial cells and post-mitotic cardiomyocytes (Kardami and Fandrich, 1989; Lindner and Reidy, 1993; Cuevas et al., 1996). Both acute (Cuevas et al., 1991) and chronic (Lazarous et al., 1995) intravenous infusion of recombinant FGF-2 in normotensive animals induces a significant reduction in blood pressure (hypotension), which is mediated partly by release of nitric oxide and activation of ATP-sensitive K+ channels (Cuevas et al., 1991). In the spontaneously hypertensive rat, endogenous levels of FGF-2 in endothelial cells are chronically reduced and blood pressure can be lowered by chronic infusion of FGF-2 (Cuevas et al., 1996). These studies suggested that FGF-2 functions in an antihypertensive vasodilator cascade. In contrast, other studies indicated that FGF-2 acts as a potent mitogen for smooth muscle cells in vitro and regulates blood vessel growth in vivo (Lazarous et al., 1995; Davis et al., 1997). Furthermore, FGF-2 is released from cardiomyocytes upon increased mechanical load (Clarke et al., 1995) and induces adaptive myocardial growth (Kaye et al., 1996). These studies suggested that FGF-2 signaling functions in the cardiovascular adaptations leading to a chronic blood pressure increase (hypertension). To study the essential in vivo functions of FGF-2 during embryogenesis and in adult physiology, the FGF-2 gene was inactivated in mice using homologous recombination in embryonic stem (ES) cells. FGF-2-deficient mice show neuronal defects in the cerebral cortex, hippocampal commissure and spinal cord. In particular, a fraction of neuronal precursors fail to colonize their normal target layers II and III during cerebral cortex development. Furthermore, adult FGF-2-deficient mice have a reduced blood pressure. Continued angiotensin II infusion induced hypertension of a similar magnitude as in wild-type litter mates, demonstrating an intact responsiveness of their cardiovascular system. In contrast, infusion of isoproterenol into FGF-2-deficient mice uncovered an impairment of their baroreceptor reflex. Therefore, blood pressure regulation is affected by an autonomic dysfunction. Results Mice homozygous for a loss-of-function FGF-2 allele are viable and fertile The first coding exon (e1, Figure 1A) of the murine FGF-2 gene encodes three protein isoforms (Figure 1D), which are translated by alternative initiation. Therefore, the complete first FGF-2 coding exon and parts of the 5′- and 3′-flanking intronic sequences were replaced by the neomycin resistance gene (neo, Figure 1A) in the opposite transcriptional orientation using homologous recombination in ES cells. Chimeric mice were generated from correctly targeted ES cells and crossed to C57BL/6J mice for germline transmission. Subsequently, homozygous FGF-2−/− mice were obtained in a mixed C57BL/6J×129/Sv genetic background. Genotyping of their offspring at birth and weaning age (Figure 1B and C) showed that FGF-2−/− mice are born alive, mature normally to adulthood and are fertile (data not shown). As FGF-2 is expressed from gastrulation onwards (Riese et al., 1995), it was essential to determine that a complete loss-of-function FGF-2 allele had been generated. Normalized amounts of proteins from brains of newborn mice (Figure 1D) and embryos (data not shown) of all three genotypes were analyzed using specific FGF-2 antibodies (Dono and Zeller, 1994). Western blot analysis showed that levels of all three FGF-2 isoforms are reduced to about half in heterozygous mice (Figure 1D, compare lane 10 with 9), whereas no FGF-2 proteins are detected in homozygous litter mates (Figure 1D, lanes 11 and 12, see also Figure 2B). Further analysis confirmed that no truncated proteins are expressed and that there is no embryonic rescue by maternal FGF-2 protein (data not shown). In summary, expression of all three FGF-2 isoforms is abolished and a complete loss-of-function allele has been generated. Figure 1.Generation of a loss-of-function FGF-2 allele by gene targeting. (A) Using homologous recombination in ES cells, the first coding exon (e1) of FGF-2 and flanking intronic sequences were replaced by the PGK-neo cassette in the opposite transcriptional orientation (arrows indicate transcriptional orientation). Three FGF-2 protein isoforms normally are synthesized by alternative translation from an ATG and two upstream in-frame CTG start codons (see D). Probe: a DNA fragment located 3′ to the short arm is used to discriminate the wild-type and mutated alleles by Southern blotting (see C). E. EcoRI; B, BamHI; S, SalI; P: PstI; X, XbaI. (B) PCR analysis of genomic DNA using specific oligos. A 0.5 kb PCR fragment is derived from intronic sequences of the wild-type allele, whereas a 1 kb fragment is amplified from the recombined allele with the neo cassette. (C) Southern blot analysis of genomic DNA to detect correct 3′ recombination (using the probe shown in A). A 3.5 kb PstI fragment marks the wild-type allele, whereas a 3 kb PstI fragment indicates the disrupted locus (see A). (D) Immunoblot analysis of protein extracts prepared from brains of newborn mice. All extracts were checked for integrity and normalized for total protein content. Affinity-purified polyclonal FGF-2 antibodies were used to detect the murine FGF-2 proteins. Two larger Leu-initiated isoforms (21 and 20 kDa) are synthesized in addition to the Met isoform (18 kDa) in wild-type mice (lane 9). Note that protein levels are halved in heterozygous mice (lane 10), whereas no FGF-2 proteins are detected in homozygous mice (lanes 11 and 12). The weak band in lane 12 is not detected reproducibly and is of a size (∼20.5 kDa) different from any of the three FGF-2 isoforms. Download figure Download PowerPoint Figure 2.Spatial distribution of FGF-2 and its cognate receptor isoforms during patterning of the cerebral cortex. (A–D) FGF-2 protein distribution in the CNS at embryonic day 14.5. (A) The isolated brain and attached spinal cord of a wild-type embryo were cut along the midline and FGF-2 proteins were detected by whole-mount antibody staining. The dashed white line indicates the approximate level of the coronal section shown in (C). (B) Whole-mount antibody staining of a CNS isolated from an age-matched FGF-2−/− litter mate. No staining is detected. (C) A rostral coronal cryosection (60 μm) of one cerebral hemisphere as indicated in (A). The rectangle indicates the approximate position of the enlargement shown in (D). (D) Magnification of the developing cortical plate and ventricular zone. FGF-2 levels are highest in the ventricular zone, where neuronal progenitors are born. (E) Distribution of transcripts encoding FGFR1 isoform IIIc in the ventricular zone and cortical plate of a wild-type embryo (embryonic day 16). (F) FGFR2 isoform IIIc transcripts in a section adjacent to the one shown in (E). The rostral positions of both sections (E and F) are similar to (C). Serial coronal sections (7 μm) were used to detect transcripts by non-radioactive RNA in situ hybridization (blue staining). Note that both FGF receptor isoforms are expressed at high levels in the ventricular and subventricular zones, whereas only FGFR2-IIIc expression persists in the cortical plate. Cb, cerebellum; CC, cerebral cortex; CP, cortical plate; HF, hippocampal formation; LGE, lateral ganglionic eminence; LV, lateral ventricle; MB, mid brain; SC, spinal cord; Th, thalamus; VZ, ventricular zone. Download figure Download PowerPoint Development of the cerebral cortex is impaired in FGF-2-deficient mice FGF-2 is expressed in the CNS from its earliest developmental stages onwards. In the forebrain, FGF-2 proteins are first detected during embryonic day 9.5 (Nurcombe et al., 1993). Figure 2 shows the distribution of FGF-2 proteins (Figure 2A–D) and transcripts of their high affinity FGF receptor isoforms [FGFR1-IIIc (Figure 2E) and FGFR2-IIIc (Figure 2F); reviewed by Miller and Rizzino (1994)] during embryonic days 14.5 and 16, respectively. At this developmental stage, the highest levels of FGF-2 proteins are present in the cerebral hemispheres, whereas levels are lower in more caudal brain regions and in the spinal cord (Figure 2A). As expected from molecular analysis (Figure 1D), no FGF-2 proteins are detected in FGF-2-deficient embryos (Figure 2B). In cerebral hemispheres, FGF-2 protein levels are highest in the ventricular and sub-ventricular zones of the lateral ventricles (Figure 2C and D), the region where neural progenitors are born and determined (Bayer and Altman, 1991). In contrast, cells within the cortical plate express much lower amounts of FGF-2 (Figure 2D). Similarly, transcripts encoding FGFR1-IIIc (Figure 2E) and FGFR2-IIIc (Figure 2F) isoforms are co-expressed in both ventricular and sub-ventricular zones. FGFR2-IIIc isoform transcripts persist in the cortical plate, whereas FGFR1-IIIc expression is much lower (compare Figure 2F with E). No changes in FGFR transcripts were observed in brains of FGF-2-deficient embryos (data not shown). To determine whether FGF-2 is necessary for embryonic CNS morphogenesis, brains of wild-type and FGF-2-deficient newborn litter mates were analyzed by comparative histology (Figure 3). Serial coronal sections of FGF-2-deficient mice showed no gross morphological abnormalities (compare Figure 3A with B and C). In newborn mice, differentiation of the cerebral cortex progresses from deep to superficial layers (VI–I, see Figure 3D; Bayer and Altman, 1991) and large pyramidal neurons are apparent (Figure 3D and G). No differences were observed between wild-type (+/+) and heterozygous (+/−) mice (data not shown). However, FGF-2-deficient litter mates display defects in organization and differentiation of their cerebral cortex. In particular, the thickness of the cerebral cortex in homozygous (−/−) mice is reduced by ∼10% in comparison with their control litter mates at birth (+/+; +/−, Table IA and B; P <0.05). At the cellular level, all differentiating cortical layers appear compressed and less distinct (compare Figure 3D with E and F). Differentiated, large pyramidal neurons are apparent in newborn wild-type mice (Figure 3D and G), whereas only a few are observed in FGF-2-deficient litter mates (Figure 3E and F and H and I). This phenotype reveals a requirement for FGF-2 for embryonic cerebral cortex morphogenesis. Figure 3.Compression of cortical layers and pyramidal neuron deficiencies in FGF-2-deficient mice at birth. (A–C) Serial coronal sections (7 μm paraffin) of the rostral brain (somato-sensory cerebral cortex, only one hemisphere shown) were analyzed by Nissl staining. (A), (D) and (G) Brain of a wild-type newborn mouse. (B), (E) and (H) Brain of an FGF-2-deficient mouse with only few differentiated pyramidal neurons (see H). (C), (F) and (I) Brain of another FGF-2-deficient mouse. (D–F) Enlargement of the parietal region of the cerebral cortex. The arrow indicates the polarity of the cerebral cortex layers (VI, deepest layer; I, most superficial layer). (G) Enlargement of the pyramidal neuron layer (arrowheads) in the wild-type. (H) Enlargement of the corresponding region in an FGF-2-deficient cerebral cortex. The arrowhead indicates the only cell within the field reminiscent of a pyramidal neuron. The small nuclei most likely correspond to cells located normally more superficially (compare D with E), which reflects the compression and mixing of the cortical layers. (I) Enlargement of the homozygous cerebral cortex shown in (F). (H) and (I) are representative of the range of pyramidal neuron deficiencies detected at birth. CC, corpus callosum; Co, differentiating cortex; Fo, fornix; LV, lateral ventricle; Sp, subplate; St, striatum. Download figure Download PowerPoint Table 1. Neuronal cell migration is affected during cerebral cortex development in FGF-2-deficient mice Early patterning and development of the embryonic brain appear normal in FGF-2-deficient embryos, as neither spatial nor temporal changes were detected in molecular markers during embryonic days 11–16 (BF1, Emx1, Otx1, Otx2, mtll; data not shown). After compartmentalization of the brain vesicle, the neurons of the cerebral cortex are generated by the pseudo-stratified ventricular epithelium that lines the lateral ventricles in the telencephalon. Cortical neurogenesis is marked by the appearance of the first post-mitotic neurons during embryonic day 11 and continues until embryonic day 17. Determined neurons leave the ventricular zone through the intermediate zone and migrate outwards along radial glial tracts. In the cortical plate, they colonize their respective target layers that form successively from deep to superficial (layer VI–II) as cortical development proceeds. After completing migration, cortical neurons undergo terminal differentiation which continues after birth (reviewed by Bayer and Altman, 1991; Hatten, 1993). To determine whether proliferation of cortical progenitors is affected in FGF-2-deficient embryos, nuclei of proliferating cells were pulse-labeled for 1 h with 5-bromodeoxyuridine (BrdU). Quantitative analysis (Table IC) shows that proliferation of progenitor cells within the ventricular zone of FGF-2-deficient embryos (Figure 4B) is indistinguishable from that of wild-type litter mates (Figure 4A) during embryonic days 14.5–15. Analysis of brains from embryonic days 13 (n = 2; data not shown) and 18 (n = 4; data not shown) confirmed that FGF-2 is not essential for cell proliferation during cerebral cortex development. Figure 4.A fraction of neuronal progenitors fail to colonize their target layers during cerebral cortex morphogenesis. (A) and (B) Proliferation of neuronal progenitors during embryonic day 14.5–15 as visualized by BrdU incorporation. Coronal sections show proliferating cells in the ventricular zone of the lateral ventricle (positions are similar to those shown in Figures 2C–F). (A) Wild-type embryo, (B) homozygous litter mate. Insets in (A) and (B) show an enlargement of the lateral aspects of the dorsal ventricular zone as indicated by rectangles. (C–F) Migration of neuronal precursors to cortical layers III and II was studied by injecting pregnant females with BrdU between embryonic days 14.5 and 15. The distribution of BrdU-labeled neuronal cells was analyzed 4–5 weeks after birth on serial coronal cryosections using specific antibodies. Sections from the somato-sensory part of the rostral cerebral cortex are shown. (C) Rostral cortex section of a heterozygous brain. (D) Enlargement to show the deeper cortical layers and corpus callosum. Note that very few labeled cells are present in this region of the control cortex. (E) Rostral cortex section of an FGF-2-deficient brain with ∼27% of all BrdU-positive cells remaining in layers IV–VI and the corpus callosum. (F) Enlargement to show BrdU-labeled cells remaining in the deeper cortical layers and corpus callosum of the FGF-2-deficient brain. The rectangles in (C) and (E) indicate the approximate position of (D) and (F). (G–I) Distribution of parvalbumin-positive neurons in a control (G and H) and FGF-2-deficient cerebral cortex (I). The rectangle in (G) indicates the approximate position of the enlargements shown in (H) and (I). II, cortex layer II; III, cortex layer III; CC, corpus callosum; CP, cortical plate; LGE, lateral ganglionic eminence; LV, lateral ventricle; St, striatum; VZ, ventricular zone. Download figure Download PowerPoint Possible defects in cortical layer morphogenesis were studied using an in vivo cell labeling system, which allows one to examine the migratory history of individual cells (reviewed by Bayer and Altman, 1991). This system takes advantage of the fact that the prospective target layer of a neuron normally is linked directly to its time of birth in the embryonic ventricular zone. Therefore, pregnant females were injected with BrdU at precise developmental time points to label pre-migratory neuronal cells. The fate of BrdU-positive neurons was analyzed ∼1 month after birth on serial coronal sections of the rostral cerebral cortex (for details, see Material and methods). Pulse labeling at embryonic day 14.5–15 marks predominantly neuronal cells that colonize cortical layers III and II (Figure 4C), whereas cortical layers VI–IV are mostly devoid of labeled cells in heterozygous adult mice (Figure 4C and D). In contrast, about twice as many labeled neuronal cells are located in the deeper cortical layers and corpus callosum of FGF-2-deficient mice (Table ID and E; P <0.05; Figure 4E and F), indicative of their failure to reach target layers III and II. This defect is variable, as many more BrdU-positive cells (25–36%) are detected in corpus callosum and deep layers of the more severely affected FGF-2-deficient mice (n/total = 4/12; Figure 4E and F), whereas a few others are indistinguishable from controls (for details, see scatter plot, Table ID). This could be either a consequence of incomplete penetrance of the embryonic FGF-2 phenotype (see Discussion) or variable elimination of mislocalized cells by cellular apoptosis (see, for example, review by D'Mello, 1998). Concerning the latter, quantitative TUNEL analysis showed no significant differences in cell survival in either the embryonic (E16; n = 5), newborn (n = 4) or adult (n = 4) cerebral cortex (data not shown). These defects should alter the distribution of specific neuronal populations. In particular, the distribution of the calcium-binding protein parvalbumin, which marks a significant number of neurons in several cortical layers (Celio, 1990), was analyzed in adult brains (Figure 4G–I). An average reduction of ∼25% in parvalbumin-positive neurons is seen in FGF-2-deficient mice in comparison with controls (compare Figure 4H with I; see Table IF). This reduction affects all layers (Figure 4I and data not shown) and is comparable with the proportion of neuronal cells which fail to reach their target layers as estimated by BrdU pulse labeling (Table IE). In contrast, no differences in the distribution of glial fibrillary acidic protein (GFAP), which marks astroglial cells, were detected (data not shown). Furthermore, no striking behavioral abnormalities such as seizures (Chae et al., 1997) or reeler-like phenotypes (e.g. D'Arcangelo et al., 1995) were observed in FGF-2-deficient mice, although further tests are necessary to detect possible subtle defects. Neuronal defects are not limited to the cerebral cortex The hippocampal commissure, a brain region located beneath the cerebral cortex (Figure 5A and B), is devoid of parvalbumin-positive neurons in wild-type and heterozygous adult mice (Figure 5C; n = 8). However, FGF-2-deficient adult mice (n/total = 8/14) show varying degrees of ectopic parvalbumin-positive neurons in this region (Figure 5D). In contrast, the distributions of calbindin-positive neurons (Celio, 1990) and GFAP-positive glial cells were not altered (data not shown). Analysis of serial coronal sections of the adult spinal cord also revealed neuronal abnormalities throughout the mantle layer of the cervical region (Figure 5E–H; n/total = 5/12). Fewer differentiated large neurons (compare Figure 5H with G; arrowheads in Figure 5H) were observed, whereas all other neural cell types appear normal (arrows in Figure 5H). These studies indicate that FGF-2 also functions in subcortical and spinal cord neurons in agreement with its expression by the hippocampal formation (Figure 2C) and embryonic spinal cord (Figure 2A, see also Dono and Zeller, 1994). Figure 5.Neuronal defects in other CNS areas of FGF-2-deficient mice. (A–D) Ectopic parvalbumin-positive neurons are present in the region of the hippocampal commissure in FGF-2-deficient adult brains. Parvalbumin-positive neurons were detected on serial coronal cryosections using specific antibodies. (A) Low magnification to show the area of the hippocampal commissure in a wild-type brain. This section is located rostral to the hippocampus. (B) A comparable area of an FGF-2-deficient brain. The rectangles in (A) and (B) indicate the approximate positions of (C) and (D). (C) The wild-type hippocampal commissure does not contain parvalbumin-positive neurons. (D) In contrast, many parvalbumin-positive neurons are present in the corresponding region of the FGF-2-deficient brain. (E–H) Neuronal deficiencies in the spinal cord of adult FGF-2-deficient mice. Serial transversal sections of the cervical spinal cord (C2) were analyzed following Nissl staining. (E) Wild-type spinal cord with well differentiated neurons in its mantle layer. (F) A comparable section of a homozygous mutant spinal cord with a less distinct mantle layer containing fewer neurons. The rectangles indicate the approximate positions of (G) and (H). (G) Enlargement showing many large and well differentiated neurons in the wild-type spinal cord. (H) Only small clusters of differentiated large neurons (arrowheads) are present in the mutant spinal cord. Other neural cell types appear normal (arrows). CC, corpus callosum; Co, cortex; dors., dorsal; Fi, fimbria; LV, lateral ventricle; ML, mantle layer; Th, thalamus; vent., ventral; VHC, ventral hippocampal commissure; WM, white matter. Download figure Download PowerPoint Reduced blood pressure and impaired baroreflex function in FGF-2-deficient mice Exogenous administration of FGF-2 induces hypotensive effects in rats, rabbits and dogs (Cuevas et al., 1991; Lazarous et al., 1995), suggesting possible antihypertensive actions of endogenous FGF-2 proteins (Cuevas et al., 1996). Therefore, the resting blood pressure of adult FGF-2-deficient mice was determined and compared with that of wild-type (+/+) and heterozygous (+/−) litter mates to uncover possible FGF-2 functions in blood pressure control. Initial statistical evaluation of resting blood pressure recordings showed that no differences are observed between wild-type and heterozygous mice (data not shown). Therefore, these two genotypes (+/+; +/−) served as phenotypic controls. However, studies by Krege et al. (1995) revealed a gender difference in blood pressure levels using wild-type mice of a similar genetic background to the present studies (mixed C57BL/6J×129/Sv). In agreement with these studies, male control mice display on average an ∼14 mmHg higher blood pressure than females (compare Figure 6B with A). Figure 6.Analysis of resting blood pressure levels. (A) Scatter plots showing the resting blood pressures in awake female control (+/+ and +/−; ○) and FGF-2-deficient (−/−; ●) mice. Group mean values are indicated by lines. Table: group mean values (± SD) of control and FGF-2-deficient females. *P <0.05. (B) Scatter plots showing resting blood pressures in awake male control (+/+ and +/−; ○) and FGF-2-deficient (−/−; ●) mice. Group mean values are indicated by lines. Table: group mean values (± SD) in control and FGF-2-deficient males. *P <0.2. BP, mean arterial blood pressure. (C) Histogram showing the distribution of individual blood pressure differences (Δ blood pressure) in control mice of both sexes (+/+ and +/−; open bars). (D) Histogram showing the distribution of individual blood pressure differences in FGF-2-deficient mice of both sexes (−/−; filled bars). For details of statistical evaluation, see Results. Download figure Download PowerPoint Analysis of the primary recordings of female FGF-2-deficient mice (Figure 6A) reveals a significant decrease in resting blood pressure in comparison with wild-type and heterozygous female litter mates (Figure 6B). 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