Limits...
The transcription factor Nfix is essential for normal brain development.

Campbell CE, Piper M, Plachez C, Yeh YT, Baizer JS, Osinski JM, Litwack ED, Richards LJ, Gronostajski RM - BMC Dev. Biol. (2008)

Bottom Line: On standard lab chow Nfix-/- animals show a decreased growth rate from ~P8 to P14, lose weight from ~P14 to P22 and die at ~P22.A fraction of the animals survive to adulthood and are fertile.The weight loss correlates with delayed eye and ear canal opening and suggests a delay in the development of several epithelial structures in Nfix-/- animals.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept. of Biochemistry and New York State Center of Excellence in Bioinformatics and Life Sciences, State University of New York at Buffalo, 3435 Main St,, Buffalo, NY 14214, USA. cc59@buffalo.edu

ABSTRACT

Background: The Nuclear Factor I (NFI) multi-gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects; Nfib-deficient mice have defects in lung maturation and show callosal agenesis and forebrain defects resembling those seen in Nfia-deficient animals, while Nfic-deficient mice have defects in tooth root formation. Recently the Nfix gene has been disrupted and these studies indicated that there were largely uncharacterized defects in brain and skeletal development in Nfix-deficient mice.

Results: Here we show that disruption of Nfix by Cre-recombinase mediated excision of the 2nd exon results in defects in brain development that differ from those seen in Nfia and Nfib KO mice. In particular, complete callosal agenesis is not seen in Nfix-/- mice but rather there appears to be an overabundance of aberrant Pax6- and doublecortin-positive cells in the lateral ventricles of Nfix-/- mice, increased brain weight, expansion of the cingulate cortex and entire brain along the dorsal ventral axis, and aberrant formation of the hippocampus. On standard lab chow Nfix-/- animals show a decreased growth rate from ~P8 to P14, lose weight from ~P14 to P22 and die at ~P22. If their food is supplemented with a soft dough chow from P10, Nfix-/- animals show a lag in weight gain from P8 to P20 but then increase their growth rate. A fraction of the animals survive to adulthood and are fertile. The weight loss correlates with delayed eye and ear canal opening and suggests a delay in the development of several epithelial structures in Nfix-/- animals.

Conclusion: These data show that Nfix is essential for normal brain development and may be required for neural stem cell homeostasis. The delays seen in eye and ear opening and the brain morphology defects appear independent of the nutritional deprivation, as rescue of perinatal lethality with soft dough does not eliminate these defects.

Show MeSH

Related in: MedlinePlus

Aberrant cortex depth and ventricular zone cells in Nfix-/- mice. Brains from WT (+/+) and Nfix-/- animals were harvested, fixed in 4% PFA and imaged before (A) or after (B-E) sectioning, staining and mounting. A) Gross structure of P22 brains of WT (+/+) and Nfix-/- animals. Note difference in cortex anterior-posterior length (double-headed arrows). B) Cresyl violet-stained coronal sections of P22 WT (+/+) and Nfix-/- brains. Arrows denote normal ventricular region in Nfix+/+ and aberrant ventricular zone cells in Nfix-/- brains. C) Immunostaining of Pax6 in coronal sections of P16 WT (+/+) and Nfix-/- brains. Arrow shows Pax6 expression in aberrant ventricular zone cells of an Nfix-/- brain and in the ventricular zone of a +/+ littermate brain. D)Pax6 and phospho-histone H3 staining of the ventricular region. Pax6 (α-Pax6, panels 1&3) and phospho-histone H3 (α-pH3, panels 2&4) immuno-staining of ventricular regions of P16 WT (+/+, panels 1&2) and Nfix-/- (-/- panels 3&4) brains. Arrows in panels 2&4 denote some pH3-positive cells. Note that most of the Pax6 positive ventricular cells in the -/- brains do not react with α-pH3. Bar in panel 1 = 100 μm for D1-4 and 500 μm for both panels of C. E) Doublecortin (DCX) immuno-staining in p69 WT (+/+) and Nfix-/- brains. Panels 2&4 are higher magnification images of the boxed regions of panels 1&2, respectively. Arrows show the locations of some DCX-positive cells. Bars in panels 2&4 = 50 μm and in panel 3 = 2 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2414869&req=5

Figure 3: Aberrant cortex depth and ventricular zone cells in Nfix-/- mice. Brains from WT (+/+) and Nfix-/- animals were harvested, fixed in 4% PFA and imaged before (A) or after (B-E) sectioning, staining and mounting. A) Gross structure of P22 brains of WT (+/+) and Nfix-/- animals. Note difference in cortex anterior-posterior length (double-headed arrows). B) Cresyl violet-stained coronal sections of P22 WT (+/+) and Nfix-/- brains. Arrows denote normal ventricular region in Nfix+/+ and aberrant ventricular zone cells in Nfix-/- brains. C) Immunostaining of Pax6 in coronal sections of P16 WT (+/+) and Nfix-/- brains. Arrow shows Pax6 expression in aberrant ventricular zone cells of an Nfix-/- brain and in the ventricular zone of a +/+ littermate brain. D)Pax6 and phospho-histone H3 staining of the ventricular region. Pax6 (α-Pax6, panels 1&3) and phospho-histone H3 (α-pH3, panels 2&4) immuno-staining of ventricular regions of P16 WT (+/+, panels 1&2) and Nfix-/- (-/- panels 3&4) brains. Arrows in panels 2&4 denote some pH3-positive cells. Note that most of the Pax6 positive ventricular cells in the -/- brains do not react with α-pH3. Bar in panel 1 = 100 μm for D1-4 and 500 μm for both panels of C. E) Doublecortin (DCX) immuno-staining in p69 WT (+/+) and Nfix-/- brains. Panels 2&4 are higher magnification images of the boxed regions of panels 1&2, respectively. Arrows show the locations of some DCX-positive cells. Bars in panels 2&4 = 50 μm and in panel 3 = 2 mm.

Mentions: We had shown previously that Nfia, Nfib and Nfix are all expressed in both prenatal and adult brains [8], and that loss of either Nfia or Nfib results in defects in brain development [12,13,16]. In addition, a previous description of an Nfix-/- mouse indicated some neuroanatomical defects [18]. We therefore examined the morphology of the brains of Nfix-/- animals. Our initial studies were performed on serial coronal sections of P22 brains of Nfix+/+ and Nfix-/- mice not maintained on supplemented diets. Gross examination of P22 Nfix-/- brains showed an apparent posterior extension of the cortical region that extended over the cerebellum (Fig. 3A, double arrows in -/- versus +/+). This was the first indication of abnormalities in the brains related to cortical development. In Fig. 3A the olfactory bulbs (OB) of the -/- animals appear somewhat smaller, but most or all of this size difference is due to accidental severing of portions of the bulb during brain removal. No major change in olfactory bulb size was seen in sections through the OB of 3 animals (not shown) but it is possible that subtle changes exist in some of the OB cell layers. We plan on examining OB structure in more detail in future studies. While WT and Nfix-/+ brains (not shown) exhibited the normal compact structure of the lateral ventricles (Fig. 3B, +/+ arrow), the ventricles of Nfix-/- mice were expanded and filled with cells of an unknown origin (Fig. 3B. -/- arrow). These cells stained darkly with Cresyl violet, were tightly packed, and serial sections showed that they filled the most anterior regions of the lateral ventricles at P22. Additional studies showed that these cells were also present in the ventricles of dough-supplemented animals (not shown) and all subsequent analyses were performed on diet-supplemented animals. Analysis of brains at different ages indicated that these aberrant cells were present as early as P0 and they remained until at least P69 (not shown). Since the cells filled regions of the lateral ventricles, we performed immuno-staining with markers previously shown to stain ventricular and subventricular cells. Pax6 is a transcription factor that is expressed at high levels in prenatal ventricular zone cells [19] and has previously been used as a marker for both prenatal and postnatal neural stem cells [20]. At P12–P16 (N = 3), Pax6 is highly expressed in both the normal ventricular zone cells in +/+ mice (Fig. 3C, +/+) and in the aberrant cells in the lateral ventricles of Nfix-/- mice (Fig. 3C, arrow -/-). The expression of this marker of neural progenitor cells suggests that these cells could represent an aberrant type of neural stem cell in the Nfix-/- animals. To determine whether any of these Pax6 positive cells were undergoing active proliferation we stained adjacent sections of brains with antibodies directed against Pax6 and phospho-histone H3 (pH3), a known marker of mitosis during cell proliferation. While 30–50% of the aberrant cells stained with α-Pax6, only a few cells in the ventricular zone of both +/+ and Nfix-/- mice stained with α-pH3 (Fig. 3D, arrows in panel 4). Thus at most only a small fraction of these aberrant cells are actively proliferating.


The transcription factor Nfix is essential for normal brain development.

Campbell CE, Piper M, Plachez C, Yeh YT, Baizer JS, Osinski JM, Litwack ED, Richards LJ, Gronostajski RM - BMC Dev. Biol. (2008)

Aberrant cortex depth and ventricular zone cells in Nfix-/- mice. Brains from WT (+/+) and Nfix-/- animals were harvested, fixed in 4% PFA and imaged before (A) or after (B-E) sectioning, staining and mounting. A) Gross structure of P22 brains of WT (+/+) and Nfix-/- animals. Note difference in cortex anterior-posterior length (double-headed arrows). B) Cresyl violet-stained coronal sections of P22 WT (+/+) and Nfix-/- brains. Arrows denote normal ventricular region in Nfix+/+ and aberrant ventricular zone cells in Nfix-/- brains. C) Immunostaining of Pax6 in coronal sections of P16 WT (+/+) and Nfix-/- brains. Arrow shows Pax6 expression in aberrant ventricular zone cells of an Nfix-/- brain and in the ventricular zone of a +/+ littermate brain. D)Pax6 and phospho-histone H3 staining of the ventricular region. Pax6 (α-Pax6, panels 1&3) and phospho-histone H3 (α-pH3, panels 2&4) immuno-staining of ventricular regions of P16 WT (+/+, panels 1&2) and Nfix-/- (-/- panels 3&4) brains. Arrows in panels 2&4 denote some pH3-positive cells. Note that most of the Pax6 positive ventricular cells in the -/- brains do not react with α-pH3. Bar in panel 1 = 100 μm for D1-4 and 500 μm for both panels of C. E) Doublecortin (DCX) immuno-staining in p69 WT (+/+) and Nfix-/- brains. Panels 2&4 are higher magnification images of the boxed regions of panels 1&2, respectively. Arrows show the locations of some DCX-positive cells. Bars in panels 2&4 = 50 μm and in panel 3 = 2 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2414869&req=5

Figure 3: Aberrant cortex depth and ventricular zone cells in Nfix-/- mice. Brains from WT (+/+) and Nfix-/- animals were harvested, fixed in 4% PFA and imaged before (A) or after (B-E) sectioning, staining and mounting. A) Gross structure of P22 brains of WT (+/+) and Nfix-/- animals. Note difference in cortex anterior-posterior length (double-headed arrows). B) Cresyl violet-stained coronal sections of P22 WT (+/+) and Nfix-/- brains. Arrows denote normal ventricular region in Nfix+/+ and aberrant ventricular zone cells in Nfix-/- brains. C) Immunostaining of Pax6 in coronal sections of P16 WT (+/+) and Nfix-/- brains. Arrow shows Pax6 expression in aberrant ventricular zone cells of an Nfix-/- brain and in the ventricular zone of a +/+ littermate brain. D)Pax6 and phospho-histone H3 staining of the ventricular region. Pax6 (α-Pax6, panels 1&3) and phospho-histone H3 (α-pH3, panels 2&4) immuno-staining of ventricular regions of P16 WT (+/+, panels 1&2) and Nfix-/- (-/- panels 3&4) brains. Arrows in panels 2&4 denote some pH3-positive cells. Note that most of the Pax6 positive ventricular cells in the -/- brains do not react with α-pH3. Bar in panel 1 = 100 μm for D1-4 and 500 μm for both panels of C. E) Doublecortin (DCX) immuno-staining in p69 WT (+/+) and Nfix-/- brains. Panels 2&4 are higher magnification images of the boxed regions of panels 1&2, respectively. Arrows show the locations of some DCX-positive cells. Bars in panels 2&4 = 50 μm and in panel 3 = 2 mm.
Mentions: We had shown previously that Nfia, Nfib and Nfix are all expressed in both prenatal and adult brains [8], and that loss of either Nfia or Nfib results in defects in brain development [12,13,16]. In addition, a previous description of an Nfix-/- mouse indicated some neuroanatomical defects [18]. We therefore examined the morphology of the brains of Nfix-/- animals. Our initial studies were performed on serial coronal sections of P22 brains of Nfix+/+ and Nfix-/- mice not maintained on supplemented diets. Gross examination of P22 Nfix-/- brains showed an apparent posterior extension of the cortical region that extended over the cerebellum (Fig. 3A, double arrows in -/- versus +/+). This was the first indication of abnormalities in the brains related to cortical development. In Fig. 3A the olfactory bulbs (OB) of the -/- animals appear somewhat smaller, but most or all of this size difference is due to accidental severing of portions of the bulb during brain removal. No major change in olfactory bulb size was seen in sections through the OB of 3 animals (not shown) but it is possible that subtle changes exist in some of the OB cell layers. We plan on examining OB structure in more detail in future studies. While WT and Nfix-/+ brains (not shown) exhibited the normal compact structure of the lateral ventricles (Fig. 3B, +/+ arrow), the ventricles of Nfix-/- mice were expanded and filled with cells of an unknown origin (Fig. 3B. -/- arrow). These cells stained darkly with Cresyl violet, were tightly packed, and serial sections showed that they filled the most anterior regions of the lateral ventricles at P22. Additional studies showed that these cells were also present in the ventricles of dough-supplemented animals (not shown) and all subsequent analyses were performed on diet-supplemented animals. Analysis of brains at different ages indicated that these aberrant cells were present as early as P0 and they remained until at least P69 (not shown). Since the cells filled regions of the lateral ventricles, we performed immuno-staining with markers previously shown to stain ventricular and subventricular cells. Pax6 is a transcription factor that is expressed at high levels in prenatal ventricular zone cells [19] and has previously been used as a marker for both prenatal and postnatal neural stem cells [20]. At P12–P16 (N = 3), Pax6 is highly expressed in both the normal ventricular zone cells in +/+ mice (Fig. 3C, +/+) and in the aberrant cells in the lateral ventricles of Nfix-/- mice (Fig. 3C, arrow -/-). The expression of this marker of neural progenitor cells suggests that these cells could represent an aberrant type of neural stem cell in the Nfix-/- animals. To determine whether any of these Pax6 positive cells were undergoing active proliferation we stained adjacent sections of brains with antibodies directed against Pax6 and phospho-histone H3 (pH3), a known marker of mitosis during cell proliferation. While 30–50% of the aberrant cells stained with α-Pax6, only a few cells in the ventricular zone of both +/+ and Nfix-/- mice stained with α-pH3 (Fig. 3D, arrows in panel 4). Thus at most only a small fraction of these aberrant cells are actively proliferating.

Bottom Line: On standard lab chow Nfix-/- animals show a decreased growth rate from ~P8 to P14, lose weight from ~P14 to P22 and die at ~P22.A fraction of the animals survive to adulthood and are fertile.The weight loss correlates with delayed eye and ear canal opening and suggests a delay in the development of several epithelial structures in Nfix-/- animals.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept. of Biochemistry and New York State Center of Excellence in Bioinformatics and Life Sciences, State University of New York at Buffalo, 3435 Main St,, Buffalo, NY 14214, USA. cc59@buffalo.edu

ABSTRACT

Background: The Nuclear Factor I (NFI) multi-gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects; Nfib-deficient mice have defects in lung maturation and show callosal agenesis and forebrain defects resembling those seen in Nfia-deficient animals, while Nfic-deficient mice have defects in tooth root formation. Recently the Nfix gene has been disrupted and these studies indicated that there were largely uncharacterized defects in brain and skeletal development in Nfix-deficient mice.

Results: Here we show that disruption of Nfix by Cre-recombinase mediated excision of the 2nd exon results in defects in brain development that differ from those seen in Nfia and Nfib KO mice. In particular, complete callosal agenesis is not seen in Nfix-/- mice but rather there appears to be an overabundance of aberrant Pax6- and doublecortin-positive cells in the lateral ventricles of Nfix-/- mice, increased brain weight, expansion of the cingulate cortex and entire brain along the dorsal ventral axis, and aberrant formation of the hippocampus. On standard lab chow Nfix-/- animals show a decreased growth rate from ~P8 to P14, lose weight from ~P14 to P22 and die at ~P22. If their food is supplemented with a soft dough chow from P10, Nfix-/- animals show a lag in weight gain from P8 to P20 but then increase their growth rate. A fraction of the animals survive to adulthood and are fertile. The weight loss correlates with delayed eye and ear canal opening and suggests a delay in the development of several epithelial structures in Nfix-/- animals.

Conclusion: These data show that Nfix is essential for normal brain development and may be required for neural stem cell homeostasis. The delays seen in eye and ear opening and the brain morphology defects appear independent of the nutritional deprivation, as rescue of perinatal lethality with soft dough does not eliminate these defects.

Show MeSH
Related in: MedlinePlus