Cortical and Commissural Defects Upon HCF‐1 Loss in Nkx2.1‐Derived Embryonic Neurons and Glia

Abstract Formation of the cerebral cortex and commissures involves a complex developmental process defined by multiple molecular mechanisms governing proliferation of neuronal and glial precursors, neuronal and glial migration, and patterning events. Failure in any of these processes can lead to malformations. Here, we study the role of HCF‐1 in these processes. HCF‐1 is a conserved metazoan transcriptional co‐regulator long implicated in cell proliferation and more recently in human metabolic disorders and mental retardation. Loss of HCF‐1 in a subset of ventral telencephalic Nkx2.1‐positive progenitors leads to reduced numbers of GABAergic interneurons and glia, owing not to decreased proliferation but rather to increased apoptosis before cell migration. The loss of these cells leads to development of severe commissural and cortical defects in early postnatal mouse brains. These defects include mild and severe structural defects of the corpus callosum and anterior commissure, respectively, and increased folding of the cortex resembling polymicrogyria. Hence, in addition to its well‐established role in cell proliferation, HCF‐1 is important for organ development, here the brain.

In this study, we investigated the functions of Hcfc1 in the mouse brain. Conditional loss of HCF-1 in ventral telencephalic Nkx2.1 + progenitors did not appear to affect their proliferation, and yet fewer Nkx2.1derived GABAergic interneurons and glia arose upon loss of HCF-1, owing to increased apoptosis. Reduced migration of GABAergic interneurons and glia was accompanied with corpus callosum defects and abnormal formation of the anterior commissure as well as severe cortical defects that resembled polymicrogyria.

Mice
All experimental studies have been performed in compliance with the EU and national legislation rules, as advised by the Lemanic Animal Facility Network (Resal), concerning ethical considerations of transportation, housing, strain maintenance, breeding, and experimental use of animals. Mice were housed four to five per cage at 23°C with ad libitum food and water access. For staging of embryos, midday of the day of vaginal plug formation was considered as embryonic day 0.5 (E0.5). WT mice maintained in a C57Bl/6 genetic background were used. We used heterozygous GAD67 _ GFP knock-in mice, described in this work as GAD67-GFP mice (Tamamaki et al., 2003). GAD67 _ GFP embryos could be recognized by their GFP fluorescence. PCR genotyping of these lines was performed as described previously (Niquille et al., 2009). We used Hcfc1 lox/lox (Minocha et al., 2016b), Nkx2.1-Cre (Xu et al., 2008), and GLAST-Cre:ERT2 (The Jackson Laboratory, Bar Harbor, Maine, USA, Tg(Slc1a3-cre/ERT)1Nat/J)) (Minocha et al., 2015b) transgenic mice described previously. The reporter Rosa26R-GFP mouse line was used to reliably express GFP under the control of the Rosa26 promoter upon Cre-mediated recombination. The control Nkx2.1-Cre -/Rosa26-GFP + and Hcfc1 lox/Y /Rosa26-GFP + did not show any GFP labeling. The control GLAST-Cre:ERT2 + /Rosa26-GFP + brains did not show any GFP labeling without tamoxifen treatment. For the induction of CreERT, tamoxifen (20 mg ml −1 , Sigma, St Louis, MO) was dissolved at 37°C in 5 ml corn oil (Sigma) pre-heated at 42°C for 30 min. A single dose of 4 mg (250-300 μl) was administered to pregnant females.

Tissue Immunohistochemistry and Histology
Embryos were collected after Caesarean section and quickly killed by decapitation. Their brains were dissected out and fixed by immersion overnight in a solution of 4% paraformaldehyde in 0.1 M of phosphate buffer (pH 7.4) at 4°C. Postnatal mice were profoundly anesthetized and perfused with the same fixative and their brains post-fixed for 4 h. Brains were cryoprotected in a solution of 30% sucrose in 0.1 M phosphate buffer (pH 7.4), frozen and cut in 50-μm-thick coronal sections for fluorescence immunostaining. For diaminobenzidine (DAB) immunostaining, the brain tissues were paraffin-embedded and sectioned into 8 μm thick sections using a MICROM HM325 microtome. For each immunostaining, we made use of several mice (between three and six) for both control and mutant strains analyzed. The method for paraffin-and cryosections staining was as follows: a DAB and fluorescence immunostaining: The paraffin-embedded sections were first (i) deparaffinized in xylene, (ii) rehydrated through graded alcohol washes, and (

Nissl staining
Standard Nissl staining was performed on both deparaffinized and rehydrated paraffin sections and cryo-sections as described (Paul et al., 2008).

Hematoxylin and eosin staining
Standard hematoxylin and eosin staining were performed on deparaffinized and rehydrated lung sections (Fischer et al., 2008).

TUNEL Assay
Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) was performed on brain sections with the in situ cell death detection kit (Roche Applied Science, cat. # 11684795910), according to the manufacturer's directions.

Immunoblotting
For immunoblotting, approximately 100 mg of brain tissue was homogenized in RIPA buffer (50 mM Tris-HCl ph 7.4, 150 mM NaCl, 1 mM EDTA, 0.2% sodium deoxycholate, 1 mM DTT, 1 mM PMSF, and 1% Triton X) containing protease inhibitors (Roche). Samples (10-20 μg) were boiled for 5 min before PAGE and transfer to nitrocellulose membrane. Membranes were blocked for 60 min with 5 ml of LI-COR blocking buffer, incubated with primary antibody in 50% LI-COR blocking buffer and 50% PBST (PBS containing 0.1% Tween 20) overnight at 4°C, washed three times and incubated with secondary antibody (dilution 1:10,000) for 30 min at RT. The membranes were washed three times and scanned with an Odyssey infrared imager (LI-COR).

Quantitation and Statistical Analyses
For each analysis, all cells in a representative field of either 8 µm-thick paraffin sections or 50 µm cryosections were counted. Mutant embryos/pups were always compared with controls originating from the same litter. In each case, entire fields acquired at same magnification were quantitated. For all analyses, values from at least three independent experiments were first tested for normality and the variance of independent populations were tested for equality. Student's t-test was performed using the R package (www.rproje ct.org). To show the degree of significance for quantitation included in the study, we added the number of asterisks based on the following standard P-value criteria: ***P < 0.001; **P < 0.01; *P ≤ 0.05.

Atlas and Nomenclature
The neuroanatomical nomenclature is based on the "Atlas of the prenatal mouse brain" (Schambra et al., 1991).

Broad Hcfc1 Expression in Mouse Brain
Before investigating the effects of loss of Hcfc1 function in brain cells, we assayed its expression in the developing mouse brain. The human HCFC1 and mouse Hcfc1 genes are highly expressed in actively dividing tissue culture cells, and in embryonic and placental tissues and in adult tissues (Wilson et al., 1995;Frattini et al., 1996;Kristie, 1997;Huang et al., 2012;Minocha et al., 2016b). Previously, using a well-characterized antibody generating little to no non-specific reactivity (H12), we have shown that HCF-1 is ubiquitous and predominantly nuclear in E6.5-to-E12.5 embryos, postnatal day 0 (P0) brains, and 10-week-old young adult brains (Minocha et al., 2016b). Here, we further investigated in detail the cellular and subcellular localization of HCF-1 in the early mouse brain by immunostaining wild-type C57BL/6 postnatal day 0 (P0) brains. We focused on the cortex (Ctx), corpus callosum (CC), and anterior commissure (AC)brain regions affected by the Nkx2.1-Cre-engineered Figure 1 Hcfc1 is broadly expressed in the mouse postnatal brain. (A-C) Immunofluorescence analysis of cryosections from wildtype C57BL6 brains at P0 stained with DAPI (blue) and antibody against HCF-1 (green). Staining with only anti-HCF-1 (A1, B1, and C1) and colocalization between anti-HCF-1 and DAPI staining (A2, B2, and C2) is shown in cortex (Ctx; A), corpus callosum (CC; B), and anterior commissure (AC; C) region. (D) Immunofluorescence analysis of cryo-sections from wildtype C57BL6 brains at P0 stained with antibodies against HCF-1 (green) and astroglial marker, glial fibrillary acidic protein (GFAP; red). The inset shows a GFAP-positive glia at higher magnification. (E) Immunofluorescence analysis of cryo-sections from wildtype C57BL6 brains at P0 stained with antibodies against HCF-1 (green) and neuronal marker, NeuN (red). (F) Immunofluorescence analysis of cryo-sections from wildtype C57BL6 brains at P0 stained with antibodies against HCF-1 (green) and oligodendrocyte marker, Olig2 (red). Scale bars are indicated in the figure.
Developmental Neurobiology loss of HCF-1 described here. In these three regions, HCF-1 was found to be ubiquitous ( Fig. 1A-C) and predominantly nuclear in the GFAP-positive astroglia ( Fig. 1D), NeuN-positive neurons (Fig. 1E), and Olig2-positive oligodendrocytes (Fig. 1F), with astroglial-cell processes showing additional relatively faint staining (Fig. 1D, see arrow). Immunoblotting analysis of postnatal and adult mouse brains ranging from P1 to 1.2 years old demonstrated continued expression of Hcfc1 into adulthood (Supp. Fig. 1A1) (Minocha et al., 2016b), but with a progressive reduction in relative HCF-1 protein levels with age (Supp. Fig. 1A2). Such a broad and long-term expression profile of Hcfc1 suggests that HCF-1 plays roles in both young and adult mouse brains.

Induced Loss of HCF-1 in Nkx2.1-Positive Cells in the Ventral Telencephalon
We wished to probe such roles for HCF-1 in brain development and decided to use Nkx2.1-Cre mediated Hcfc1-gene inactivation for this purpose. In the embryonic brain, the Nkx2.1 homeobox gene is expressed in a region of the forebrain that develops into the ventral telencephalon which includes the medial ganglionic eminence (MGE), the anterior entopeduncular area (AEP), the anterior preoptic area (POA), the septum (SEP) and parts of the amygdala (Lazzaro et al., 1991;Kimura et al., 1996;Sussel et al., 1999;Puelles et al., 2000;Flames et al., 2007). In the MGE, Nkx2.1 is expressed in the rapidly dividing ventricular (VZ) and sub-ventricular zones (SVZ) as well as the mantle zone comprised of migratory cells (Sussel et al., 1999), and is important for the generation of both neuronal (GABAergic interneurons) and glial (astroglia and oligodendrocytes) cell types (Sussel et al., 1999;Anderson et al., 2001;Corbin et al., 2001;Marin and Rubenstein, 2001;Kessaris et al., 2006;Minocha et al., 2015a;2015b). These cells ensure proper cortical development and function, and commissure formation (Wonders and Anderson, 2006;Lindwall et al., 2007;Minocha et al., 2015b). Importantly, as shown in Figure 1, Hcfc1 is highly expressed in these cell types.
We induced conditional deletion of the Hcfc1 gene in these cell types by crossing males heterozygous for an Nkx2.1-Cre transgene (referred to as Nkx2.1-Cre + ) (Xu et al., 2008) with Hcfc1 lox/lox females (Minocha et al., 2016b). The X-linked Hcfc1 lox allele contains two loxP sites, one in intron 1 and another in intron 3 that undergo recombination in the presence of Cre recombinase, deleting exons 2 and 3 to generate the conditional knockout (cKO) allele encoding a highly truncated 66 amino acid long N-terminal HCF-1 protein (Minocha et al., 2016b). Thus, hemizygous Hcfc1 lox/Y males carrying the Nkx2.1-Cre + allele were expected to generate a complete embryonic Nkx2.1specific knock out. The resulting male progeny of the aforementioned cross generated the control strain Nkx2.1-Cre + ; Hcfc1 +/Y and the Nkx2.1-Cre-induced knockout strain Nkx2.1-Cre + ; Hcfc1 lox/Y . The Nkx2.1-Cre + ; Hcfc1 lox/Y postnatal mice appeared to be ill, probably owing to improper lung formation (compare Supp. Fig. 2A to B and C; see [Lazzaro et al., 1991]), and often suffered from maternal cannibalism or died during early postnatal ages around P5.
Developmental Neurobiology
Developmental Neurobiology with active or recent proliferation. Furthermore, Ki67-positive cells appeared to be evenly distributed in the MGE of Nkx2.1-Cre + ; Rosa26-GFP + ; Hcfc1 lox/Y embryos (Fig. 2H). Similar results were also observed at E14.5 (data not shown). Thus, although HCF-1 has been implicated in cell proliferation in vitro (Goto et al., 1997) and in liver regeneration (Minocha et al., 2016b), these results indicate that HCF-1 is not required for proliferation of MGE neuronal and glial progenitor cells in the developing embryonic mouse brain.
Next, we assayed for the possibility of increased cell death in the ventral telencephalon of Nkx2.1-Cre + ; Rosa26-GFP + ; Hcfc1 lox/Y vs. control embryos. Indeed, TUNEL assays revealed a significantly increased number of apoptotic cells, specifically in the SVZ and the mantle zone, both at E12.5 (compare Fig. 5A and C to B and D, respectively) and E14.5 (compare Fig. 5E and G to F and H, respectively) (see quantification in Fig. 4C). Thus, the reduced number of GFP-positiveand hence HCF-1-negative cells -in the developing cortex (see Figs. 2 and 4A) is likely due to an increased incidence of cell death as opposed to a direct effect on cell proliferation.
Developmental Neurobiology telencephalic region apparently fails to reach their target regions.
Similar results were obtained upon using a temporally activatable Cre-recombinase transgene called GLAST-CreERT2, which is primarily expressed in astroglia (Mori et al., 2006). We compared the effects of tamoxifen treatment of GLAST-CreERT2 + ; Hcfc1 lox/Y and GLAST-CreERT2 -; Hcfc1 lox/Y mice at E16.5 on astroglia development. We observed a reduction in the number of GFAP-positive astroglia in both the corpus callosum (compare Supp. Fig. 5A to B) and anterior commissure region (compare Supp. Fig. 5C to D). Quantification revealed a loss of approximately 50% GFAP-positive glia within the corpus callosum and 40% GFAP-positive glia within the anterior commissure region (Supp. Fig. 5E). Thus, these results also suggest a role of HCF-1 in brain astroglia development.

Loss of HCF-1 Causes Corpus Callosum and Anterior Commissure Defects
Here, we investigated the effects of the loss of Nkx2.1-lineage cells upon the absence of HCF-1 on brain development. In the analysis of the disappearance of Nkx2.1-lineage cells in Nkx2.1-Cre + ; Rosa-GFP + ; Hcfc1 lox/Y brains shown in Figure 6, we noted a number of morphological defects, particularly commissural and cortical. We first investigated the commissural and then the cortical defects, and used in each case glial-and neuronal-specific immunofluorescence markers to follow the contribution of glia and neurons to the defective structures observed. To avoid a conflicting GFP fluorescence signal from the Rosa-GFP transgene, it was excluded from the mouse strains used in the experiments described below.
Relative to the commissures, we observed mild and strong defects in the corpus callosum and anterior commissure regions, respectively. The corpus callosum was clearly thinner but otherwise appeared normal in Nkx2.1-Cre + ; Hcfc1 lox/Y knockout brains (compare Fig. 7A to B; Fig. 7E). In contrast, the anterior commissure exhibited gross distortions, which is consistent with the finding that selective ablation of Nkx2.1-lineage post-mitotic cells leads to deflection of anterior commissure axons from their normal trajectory and improper formation of the anterior commissure (Minocha et al., 2015b).
Hence, loss of HCF-1 in Nkx2.1-lineage cells affects their survival and as a result midline commissures, particularly the anterior commissure, are malformed in mutant brains.

Early Loss of HCF-1 Causes Cortical Defects
Nkx2.1 plays a key role in the specification and production of GABAergic interneurons and glia from the ventral telencephalon that migrate into the striatum and cerebral cortex (Sussel et al., 1999;Anderson et al., 2001;Corbin et al., 2001;Marin and Rubenstein, 2001; Developmental Neurobiology Kessaris et al., 2006;Minocha et al., 2015a;2015b). To elucidate the effect on cortical development of loss of migratory neurons and glia upon Hcfc1-gene disruption in Nkx2.1-Cre + ; Hcfc1 lox/Y knockout brains, we analyzed P0-to-P1 mice.
Although less frequent than the commissure defects, cortical defects were still common. Such defects are clearly visible in both hemispheres in the Nkx2.1-Cre + ; Hcfc1 lox/Y knockout brain serial sectioning shown in Supplemental Figure 6 (black arrows). Indeed, we did not observe a preference for defects in one or the other hemisphere. Eleven of 19 analyzed Nkx2.1-Cre + ; Hcfc1 lox/Y knockout brains exhibited severe cortical defects in at least one hemisphere (see Fig. 9B and D compared to A and C) whereas the rest exhibited milder cortical defects (see Fig. 9F compared to E) and yet the cortices were thinner when compared to control embryos (Fig. 9K). In comparison to the unaltered six-layered cortex of control Nkx2.1-Cre -; Hcfc1 lox/Y brains ( Fig. 9G and I), the laminar organization of the Nkx2.1-Cre + ; Hcfc1 lox/Y knockout brain cortices ( Fig.  9H and J) was disturbed, perhaps missing layers.
Developmental Neurobiology Calbindin (Fig. 10K,L), and Ctip2 (Fig. 10M,N) for layer V; and Parvalbumin ( Fig. 10E,F) and Tbr1 ( Fig. 10O,P) for layer VI. From the cortical layer analysis and NISSL staining (Fig. 9), it appeared that layer I though deformed is present, whereas layers II, III, and V though present are relatively thinner in Nkx2.1-Cre + ; Hcfc1 lox/Y knockout brains when compared to control brains. Interestingly, it appeared that the layer IV and layer VI are either much thinner or absent.
Although Nkx2.1-Cre + ; Hcfc1 lox/Y knockout brains exhibit these malformations, the cells within the cortex are essentially all HCF-1 positive and are proliferating as found in normal brains (Supp. Fig. 7). Thus, it is likely that it is the absent Nkx2.1-lineage cells that are causing the cortical aberrations. These aberrations resemble those that occur in asymmetric polymicrogyria, which are typically characterized by the presence of irregular cortical folds and a reduced number of cortical layers (Barkovich, 2010;Guerrini and Parrini, 2010). Such malformations could be one way by which human patients carrying HCFC1 mutations display intellectual disability.

DISCUSSION
We have shown that conditional loss of HCF-1 in Nkx2.1-derived neurons and glia leads to commissural defects affecting primarily the AC as well as asymmetric polymicrogyria-like cortical defects. These defects appear to arise because of a reduced number of embryonic Nkx2.1-derived neurons and glia owing to their increased cell death. This reduced presence of Nkx2.1-derived cells leads to formation of severely malformed AC whose axons deviate from the normal path, being deflected both dorsally and ventrally. Also, the laminar organization of the cortex is locally disturbed generating irregular folds where cortical layers are reduced in number and thickness. Hcfc1 lox/Y (n = 21) brains at P0. The difference between cortical thickness in control Nkx2.1-Cre -; Hcfc1 lox/Y and knockout Nkx2.1-Cre + ; Hcfc1 lox/Y brains was highly significant (P-value 6.65 × 10 −7 ).

The Activity of HCF-1 Differs Depending on Cell Context
HCF-1 is a transcriptional co-regulator and is important for several aspects of the cell cycle in tissue culture cells and during liver regeneration (Goto et al., 1997;Reilly and Herr, 2002;Julien and Herr, 2003;Minocha et al., 2016a;2016b). Interestingly, loss of HCF-1 does not seem to affect the proliferation capacity of Nkx2.1-positive precursor cells in the ventral telencephalic region. These results are consistent with a previous report where knockdown of HCF-1 was shown to display increased proliferation of neural precursors in an in vitro neurosphere assay (Jolly et al., 2015). Our results show that, instead of inhibiting cell proliferation, loss of HCF-1 leads to increased cell death of Nkx2.1-derived post-mitotic cells (GABAergic interneurons and glia) in the ventral telencephalic region. These results demonstrate that the role of HCF-1, likely as a transcriptional regulator of many genes, can differ depending on the cell context. This finding of differing roles of HCF-1 in different cell contexts complements that in the context of resting adult mouse hepatocytes where loss of HCF-1 leads to hepatocyte malfunction (Minocha et al., 2018). HCF-1 appears to have evolved to play a multitude of cell-specific roles in the regulation of gene expression, probably principally gene transcription but also through protein stabilization as in the case of PGC1α in hepatocytes (Ruan et al., 2012;Minocha et al., 2018). In this manner, it serves as a broad and versatile potentiator of cell function.

Effects of HCF-1 Loss in Nkx2.1-lineage Cells on Embryonic Brain Development
Nkx2.1-derived cells (GABAergic interneurons and glia) have the striking capacity to migrate to numerous areas of the brain including the commissures and cortex after their initial formation primarily in the MGE and POA. Here, owing to their death, Nkx2.1-derived cells cannot migrate apparently causing Nkx2.1-Cre + ; Hcfc1 lox/Y knockout mice to display the numerous diverse brain defects observed.
These results are consistent with previous reports where it has been shown that polymicrogyria primarily develops due to either reduced proliferation of neural precursors, disturbed neuronal migration or aberrant Developmental Neurobiology cortical organization (Guerrini and Filippi, 2005;Guerrini and Parrini, 2010). A role of GABAergic interneurons in cortical development has also been shown in Arx-deficient mice that recapitulate features of cortical malformation called as X-linked Lissencephaly with absent corpus callosum and Ambiguous Genitalia (XLAG) in humans (Bonneau et al., 2002;Kitamura et al., 2002). XLAG is characterized by agenesis of CC, poorly laminated cortex, microcephaly, and epilepsy (Bonneau et al., 2002). Together, these results all point to the importance of GABAergic interneurons in cortical development.
Cases of polymicrogyria are known to occur sporadically, though several families have also been observed with loci mapping to the X chromosome, including Xq28 (Geerdink et al., 2002;Villard et al., 2002;Barkovich, 2010). In humans, HCFC1 resides on Xq28 (Frattini et al., 1994;Wilson et al., 1995) and has been strongly implicated in development of intellectual disability (Huang et al., 2012;Yu et al., 2013;Jolly et al., 2015). Intellectual disability is often a clinical manifestation of cortical malformations such as polymicrogyria. HCF-1 is essential for survival of Nkx2.1-lineage cells, GABAergic interneurons and glia, whose absence in turn causes cortical defects strongly resembling polymicrogyria. We suggest that these cortical malformations observed upon loss of HCF-1 in subpopulations of GABAergic interneurons and glia may well also be present in human intellectual disability patients carrying HCFC1 mutations.

Uncovering a New Role of Nkx2.1-Lineage Cells in Polymicrogyria
The effect of Nkx2.1-Cre-induced loss of HCF-1 on anterior commissure formation closely mimics those observed upon ablation of Nkx2.1-derived cells, both neurons and glia, with the help of diphtheria toxin (Minocha et al., 2015b). But, interestingly, ablation of Nkx2.1-derived cells did not show any cortical aberrations (Minocha et al., 2015b), such as those visible in brains lacking HCF-1 in Nkx2.1-derived cells. These previous studies involving ablation of Nkx2.1-derived cells failed to observe an effect on cortical development apparently due to the delayed accumulation of diphtheria toxin where precursors are not affected and also because embryos died before birth probably owing to an effect on lung development. In our study, as we were able to analyze the brains of neonatal pups lacking HCF-1 in Nkx2.1-derived cells, we could observe the cortical aberrations. Hence, our study uncovers a new role of Nkx2.1-derived cells during cortical development.
We thank Danièle Pinatel for technical assistance; Catherine Moret for help with staining and tissue sectioning; Cécile Lebrand and Jean-Pierre Hornung for cortical marker antibodies, Nkx2.1-Cre tg and GAD67-GFP tg mice, and valuable advice; and the Cellular Imaging Facility of the University of Lausanne Faculty of Biology and Medicine for microscope access. We are indebted to Cécile Lebrand for critical reading of the manuscript.

AUTHOR CONTRIBUTIONS
The experiments were conceived and designed by S.M. and W.H. The experiments were performed by S.M. S.M. and W.H. analyzed the data and prepared the manuscript. Both authors participated in the discussion of the data and in production of the final version of the manuscript.