Stephanie I. Mok

Harvard College ‘09, moksi@mail.nih.gov

In neural development, Bhlhb5 and Prdm8 may have a cooperative role in the same regulatory processes underlying neuronal differentiation and maturation. Three potential models of interaction are proposed. The three models are: 1) Bhlhb5 and Prdm8 regulate the same target genes, but do not bind together in complex, 2) Bhlhb5 and Prdm8 interact in complex to regulate the same gene targets, and 3) Prdm8 and Bhlhb5 each regulate the transcription of unique proteins that are critical mediators of a common pathway. To address the hypothesis that Bhlhb5 and Prdm8 are crucial in the same neurons in development, the protein expression patterns of Bhlhb5 and Prdm8 were examined in the mouse brain and spinal cord via immunohistochemistry. Results show that Bhlhb5 and Prdm8 do not bind directly together in complex. Moreover, Prdm8-expressing neurons are significantly lost in the spinal cord of Bhlhb5 mutants, Bhlhb5 and Prdm8 are highly co-expressed in the brain and spinal cord, and Prdm8 mutants exhibit loss of the corticospinal tract. These results suggest that Bhlhb5 and Prdm8 are crucial regulators of a common pathway that underlies neuronal development in the mammalian central nervous system.

Introduction

The bHLH family of transcriptional regulators

One group of transcriptional regulators found to be critical in the early development of the central nervous system is a family of proteins known for their highly conserved Basic-Helix-Loop-Helix or BHLH structural domains. Many members of the BHLH family have been found to perform crucial regulatory roles in nervous system development in a broad array of cellular types (Massari and Murre, 2000). A particular BHLH gene of interest is Bhlhb5, which has been found to be transiently expressed in subpopulations of post-mitotic neurons throughout the central nervous system (Ross et al., unpublished data). These findings suggest that the Bhlhb5 transcription factor may have a regulatory role in cellular differentiation and maturation during neuronal development of key pathways necessary for proper movement and sensory mechanisms.

Bhlhb5 mutants have phenotypes indicating deficits in the nervous system

Investigators in the Greenberg Lab examined the potential role of Bhlhb5 in neural development through the generation of a Bhlhb5 knockout mouse (Bhlhb5 -/-). They found that Bhlhb5 mutants exhibited a series of physical and behavioral deficits (Figure 1: Supplementary Figures). Behavioral phenotypes observed include self injury due to excessive spot-grooming and the subsequent development of open sores, or skin lesions (Figure 1A). Moreover, in behavioral tests that evaluated sensitivity of Bhlhb5 mutants to chemical stimulation through exposure to capsaicin, a natural irritant found in chili peppers, Bhlhb5 mutants were found to demonstrate decreased response (measured by paw licking) to the harmful stimulant. Similarly, Bhlhb5 mutants also exhibited decreased sensitivity to thermal stimulation as indicated by the decreased rate of paw withdrawal when placed on a hot plate (Ross et al., unpublished data).
An unusual behavioral phenotype observed in the Bhlhb5 mutants was a “handstand” behavior in which the mice walked forward on their forelimbs, while retracting their hindlimbs to display a transient handstand posture (Figure 1C). Bhlhb5 mutants also displayed lack of motor coordination when compared to wildtype controls. This was indicated by a significant decrease in balancing skills when Bhlhb5 mutants were placed on a rotating rod and fell off at much higher rates than wildtype animals (Figure 1B).
While the nervous systems of Bhlhb5 mutants appeared normal at a gross level, closer inspection revealed several defects at the cellular level. For instance, a statistically significant loss of Bhlhb5-expressing neurons in the most superficial laminae of the dorsal horn of the spinal cord was observed in Bhlhb5 mutants when compared to that of wildtype controls (Figure 2). In addition, the corticospinal tract was missing in Bhlhb5 mutants (Ross et al., unpublished data) (Figure 3). The corticospinal tract (CST) consists of the bundle of neuronal fibers extending from the motor cortex of the brain into the spinal cord, and is therefore a key factor in regulating motor coordination in animals. The distinct absence of the CST in mutants thus indicates a potential role of Bhlhb5 in the formation of motor circuits in development. To identify the genes that are mis-expressed in Bhlhb5 mutants, Affymetrix array analysis examining RNA levels in Bhlhb5 knockout versus wildtype was performed. Prdm8 was the most dramatically misregulated gene (Sup. Figure 1) (Ross et al., unpublished data). In fact, Prdm8 was found to be up-regulated significantly in Bhlhb5 mutants (p<0.05) (Ross et al., unpublished data), thus suggesting that Bhlhb5 may directly repress the transcription of Prdm8 (Figure 4 in Supplementary Materials).

Figure 2. BHLHB5-expressing neurons are missng from a subpopulation of cells in the superficial layers of the spinal cord dorsal horn: (A) Control BHLHB5 cre/+ mice exhibit BHLHB5-expressing neurons marked by a reporter gene (eYFP) as shown in green. (B) Mutant BHLHBS cre/- mice exhibit fewer BHLHB5-expressing neurons as marked by a reporter gene (eYFP) as shown in green. The most superficial layers of the spinal cord are marked by layers A and B (outlined in white). (Ross et al., unpublished data)
Figure 3. Bhlhb5 mutants are missing the corticospinal tract. A) PKC-gamma immunostaining in BHLHB5 wt mice marks the CST (indicated by white arrow). B) PKC-gamma immunostaining in BHLHB5 mut mice exhibit no expression of PKC-gamma, thus demonstrating loss of the CST(indicated by white arrow). (Ross et al., unpublished data)
Figure 4. Proposed Models of Prdm8 and Bhlhb5 Regulation of Target Genes: (A) Bhlhb5 and Prdm8 bind to distinct regions of DNA, yet repress the same target genes. (B) Bhlhb5 and Prdm8 interact directly with each other in complex to co-repress the same target genes. (C) Bhlhb5 and PRdm8 each repress transcription of distinct target genes (X and Y, respectively), which are key proteins involved in a common pathway. In this model, both Bhlhb5 and Prdm8 act as indirect regulators of the same downstream processes, yet Bhlhb5 and Prdm8 do not interact with each other or target directly the same genes.

Similarity of phenotypes in Bhlhb5 and Prdm8 mutants

The Affymetrix RNA array results from the Bhlhb5 mutants suggested that Prdm8 may be an important transcriptional regulator in the same neuronal pathways as that of Bhlhb5. A putative transcription factor that is a member of the PR Domain family known for its histone methyl-transferase activity, Prdm8 has not been researched broadly and little is known about its role in the central nervous system.
However, Prdm8 analysis in the retina was being explored by investigators at the McInnes Lab; intriguingly, Prdm8 knockout mice exhibited similar behavioral phenotypes and deficits when compared to that of Bhlhb5 mutants (Sup. Figure 2). Like Bhlhb5 knockout mice, the Prdm8 mutants also displayed the unique “handstand” posture during movement. Furthermore, Prdm8 mutants also developed extensive skin lesions like the Bhlhb5 mutants. The combination of these observations in conjunction with the RNA Affymetrix array results indicating that Prdm8 is the most mis-regulated gene in Bhlhb5 mutants suggested that Prdm8 may be involved in a common pathway with Bhlhb5 in the development of the central nervous system in mice.
What is the mechanism through which Bhlhb5 and Prdm8 regulate transcriptional pathways such that removal of either factor induces the same phenotypic deficits in knockout mice? I propose three models of interaction to answer this question (Figure 4).
Three chief objectives were addressed: 1) Which model of interaction between Bhlhb5 and Prdm8 is best supported by the observed data? In Figure 8, several models of interaction that may characterize the true collaborative function of Bhlhb5 and Prdm8 in neuronal development are presented. Is there evidence to support the hypothesis that Bhlhb5 and Prdm8 regulate the same target genes, yet do not bind together directly in complex (Figure 4A)? Do they interact directly with each other to regulate the same gene targets (Figure 4B)? Or, do Prdm8 and Bhlhb5 each regulate distinct groups of genes that are critical mediators of a common pathway (Figure 4C)?
2) Is there evidence to suggest a mechanism of interaction between Bhlhb5 and Prdm8 in the regulation of processes important neuronal development? Do protein expression patterns of Bhlhb5 and Prdm8 in various regions of the brain and spinal cord indicate that these two factors may be crucial in the same neurons during differentiation and development?
3) To design a Prdm8 fusion protein construct for the development of an antibody that accurately recognizes Prdm8 protein in immunhistochemistry and immunoprecipitation experiments and allows for visualization of endogenous Prdm8 protein in fixed tissue and neural lysates.

Materials and Methods

Immunoprecipitation of Bhlhb5 or Prdm8 in transfected 293T cells

Supernatant of 293T cell lysate was incubated with primary Bhlhb5 (rat) antibody (or anti-Myc epitope (mouse)) antibody. Blocked Protein-A agarose beads were added to the cell lysate supernatant, pelleted from the cell lysate, and washed. Bhlhb5 protein (or Prdm8 protein) was then precipitated and bound to the beads through pull-down.

Design and generation of Prdm8-directed antibody

Multiple Prdm8 GST-fusion protein constructs were devised that consisted of all 6 combinations of 3 highly conserved domains in the full length Prdm8 sequence (labeled as Domain 1, Domain 2, and Domain 3). Primer-targeted PCR amplification was conducted, transforming constructs into competent cells, which were maxiprepped and sequenced. Constructs were then digested with restriction enzymes, ligated into pGEX vectors, and transformed the vectors into Top10 Competent Cells. The researcher then cultured in high volumes the correctly transformed GST-PRDM8 constructs, maxiprepped, and then transformed them into BL21 (E. coli) cells.

Induction and purification of Prdm8 GST-fusion protein constructs

Colonies of transformed BL21 cells were diluted, cultured in high volumes, and induced with IPTG. Cells were spun down, lysed, and incubated with Protein-G sepharose beads. Protein-coated beads were spun down, washed, and the protein eluted from the beads. A Bradford protein assay was conducted to quantify the eluted protein from the beads, and the purified protein was sent for injection into rabbits. To observe induction of each of the 6 PRDM8 GST-Fusion protein constructs in BL21 cells, cells were lysed in Western Lysis Buffer and protein sizes were examined via SDS-PAGE and coomassie staining (using a GelCode Blue Stain Reagent).

Immunoprecipitation of endogenous Bhlhb5 and Prdm8 protein in neurons

The supernatants of the neural lysate of Prdm8 mutant and wildtype mice were incubated with primary Bhlhb5 (rat) (or Prdm8 (rabbit)) antibody. Blocked Protein-A agarose beads were added to the cell lysate supernatant, and the beads were pelleted from the neural lysate and washed. Bhlhb5 protein (or Prdm8 protein) was precipitated bound to the beads through pull-down.

Western blot analysis of immunoprecipitation results

Lysates from the immunoprecipitation experiments were analyzed via SDS-PAGE and western blot using Prdm8 and Bhlhb5-specific antibodies. Prdm8 rabbit antibody was used to probe the upper half of the blot (Prdm8 protein band is approximately 72 kDa in size in neurons). For immunoprecipitation experiments conducted on 293T cell lysates, an anti-Myc (mouse) antibody to probe for the C-terminal epitope tag attached to Prmd8 transfected in cultured 293T cells was used. Bhlhb5 rabbit antibody was used to probe the lower half of the blot (Bhlhb5 protein is approximately 35 kDa in size).

Immunohistochemistry analysis

To analyze the cell-specific expression patterns of Prdm8 and Bhlhb5 in cortical and spinal cord tissue sections, immunohistochemistry analysis was performed on fixed tissue. Whole specimens were frozen in blocks of optimum cutting temperature (OCT) formula, and sagittal and coronal sections 20 microns thick were collected utilizing a cryostat, and the sections were transferred onto slides. Immunostaining analysis was conducted to visualize proteins of interest.

See Supplementary Materials for full Materials and Methods.

Results

No co-immunoprecipitation of Bhlhb5 or Prdm8 in 293T cells

To test the hypothesis of whether or not any evidence of protein-protein interaction between Bhlhb5 and Prdm8 could be observed, immunoprecipitation experiments were conducted. In this experiment, the researcher performed antibody-specific pull-down of a particular protein (Bhlhb5) via adhesion to beads and precipitation from the whole cell lysate. The precipitate was probed with antibodies specific for its partner protein (Prdm8) to observe for any co-immunoprecipitation. Through western blot analysis (Figure 5), no evidence was found of a direct interaction between Bhlhb5 and Prdm8. Antibody-specific immunoprecipitation of the Bhlhb5 protein was observed, and any associated Prdm8 pull down via probe identification of the Myc-epitope tagged to the C-terminal of the PRDM8 protein was analyzed. As seen in Figure 9, there is no observable protein band at approximately 98 kDa in the lane containing the precipitate pulled down via Bhlhb5 interaction. Conversely, immunoprecipitation of Prdm8 via its Myc-epitope tag exhibited no pull-down of Bhlhb5 in the precipitate (which would have been indicated by a band ~37 kDa in size).

Design and development of Prdm8 GST-fusion protein constructs

In order to conduct immunoprecipitation experiments on endogenous Bhlhb5 and Prdm8 protein from neurons, use of a Prdm8 antibody was required. Due to the lack of available Prdm8 antibody, however, a series of Prdm8 GST-Fusion protein constructs were designed and developed. Single constructs to be purified were selected and injected into rabbits for Prdm8 antibody production.
Sup. Figure 3 exhibits the design of the 6 different protein domains cloned into vectors with GST to create 6 individual GST-fusion protein constructs. Sup. Figure 4 displays the induction results for each of the Prdm8 GST-Fusion protein constructs in which the darkest band in each lane corresponds to the appropriate sized protein band for each of the Prdm8 fusion proteins. The middle region of the protein was found to be the only construct isolated in the soluble fraction during the protein purification process. This region (termed Domain 2) was selected as the Prdm8 antigen to be injected into rabbits for Prdm8 antibody production.

Designed antibody recognizes Prdm8 protein in neurons

To test for specificity and strength of the developed Prdm8 antibody to recognize endogenous Prdm8 in neural lysate, an experimental western blot on a wildtype whole mouse cortex and one mutant for Prdm8 was conducted (Sup. Figure 5). Prdm8 antibody derived from the serum of a rabbit injected with purified antigen (derived from the Prdm8 Domain 2 GST-Fusion protein construct) was used to probe for the presence of Prdm8 protein in the neural lysate. The Prdm8 antibody accurately identified the Prdm8 protein in the neurons as predicted. This finding was verified in the western blot through the observation of a ~72 kDa protein band, which corresponds to the approximate size of endogenous Prdm8 in neurons.

Figure 5. Western blot of immunoprecipitation conducted in 293T cells. 293T cells were transfected with both BhlHb5 and myc-tagged Prdm8. First lane displays IP for Bhlhb5, and no co-IP of Prdm8 is observed. Second lane displays IP for PRdm8 via its myc epitope tag, and no co-IP of Bhlhb5 is observed. Bottom half of blot(divded by dotted line) was blotted with antibody for Bhlhb5, upper half of blot probed with antibody for Myc-epitope tag of Prdm8.

No co-immunoprecipitation of Bhlhb5 or Prdm8 in neurons

Unlike the previous experiment performed in which 293T cells transfected with Bhlhb5 and Myc-Prdm8, this immunoprecipitaton experiment was performed on proteins expressed from endogenous genes in neural lysate drawn from the whole cortex of Prdm8 wildtype and mutant mice. The advantage of this protocol is that any associated co-factors necessary to facilitate proper Bhlhb5 and Prdm8 protein interaction would also be available in the neural lysate to maintain natural protein-protein interactions (such would not be possible in 293T cells).
In this experiment, antibody-specific pull-down of a particular protein (i.e. Bhlhb5) was conducted via adhesion to beads and precipitation from the whole cell lysate. The precipitate with the antibody specific for the partner protein (i.e. Prdm8) was then probed to look for any co-immunoprecipitation. As indicated in Figure 6, there is no protein band at approximately 72 kDa (MW of Prdm8 protein) in the lane that indicates the precipitate pulled down via Bhlhb5 interaction. Since Prdm8 is evident in the wildtype whole cell lysate when compared to the Prdm8 mutant control, endogenous Prdm8 is clearly present prior to immunoprecipitation. No Prdm8 was co-immunoprecipitated with Bhlhb5 in neurons. Similarly, immunoprecipiation was performed for Prdm8 and no associated pull-down of Bhlhb5 in the precipitate was found (Figure 7).

Figure 6. Western blot of BHLHB5 immunoprecipitation conducted in neural lysate. Neural lysate from whole cortex of both PRDm8 wt and Prdm8 mut P0 mice were utilized to IP for Bhlhb5 (both BHLhb5 wt). First and second lane display neural lysate (1% NP-40 lysis bufer) of Prdm8 mut and Prdm8 wt mice, respectively. Second lane clearly indicates the presence of PRDM8 protein in the neural lysate prior to the IP. Third and fourth lanes indicate IP for BHlhb5 in both Prdm8 mut and Prdm8 wt mice, respectively. No evidence of co-IP of Prdm8 is observed in the Prdm8 wt neurons.
Figure 7. Western blot of PRDM8 immunoprecipitation conducted in neural lysate. Neural lysate from whole cortex of both Prdm8 wt and Prdm8 mut P0 mice were utilized to IP for PRDM8. First and second lanes display neural lysate (SDS lysis buffer) of Prdm8 wt and Prdm8 mut mice (both Bhlhb5 wt), respectively. Presence of BHLHB5 protein in these lysates is observed by the dark band at the 35kDa mark in each lane. Third and fourth lanes indicate IP for PRDM8 in both Prdm8 wt and Prdm8 mut mice, respectively. No evidence of co-IP of BHLHB5 is observed in the Prdm8 wt neurons.

Prdm8-expressing cells lost in the dorsal horn of spinal cord of Bhlhb5 mutants

In previous research, Bhlhb5 mutants exhibited distinct phenotypes that included skin lesion development, handstand behavior, decrease in motor coordination, and decrease in sensitivity to thermal or chemical stimulation (Ross et al., unpublished data). When examined at the cellular level, Bhlhb5 mutants also exhibited a significant loss of Bhlhb5-expressing neurons in the superficial layers of the dorsal horn of the spinal cord (Ross et al., unpublished data). Recent findings that Prdm8 mutants indicate striking similarities comparable to those of Bhlhb5 mutants (i.e. skin lesion development and handstand behavior) (McInnes, unpublished data) suggested the theory that Bhlhb5 and Prdm8 may function using similar regulatory pathways of the central nervous system. As a result, it is possible that these two transcription factors are expressed in the same neurons during development. This reasoning led to the research hypothesis that: 1) Bhlhb5 mutants also exhibit significant loss of Prdm8-expressing neurons in the superficial layers of the dorsal horn of the spinal cord, 2) Prdm8 mutants exhibit the loss of Prdm8 and Bhlhb5 expressing neurons in the dorsal horn of the spinal cord.
To test the first hypothesis, immunohistochemistry was performed to compare Prdm8 expression in neurons in the dorsal horn of the spinal cord between wildtype controls and Bhlhb5 knockout mice at P0. A significant loss of Prdm8-expressing neurons in the dorsal horn of the spinal cords of Bhlhb5 mutants was detected(Figure 8A). The mean number of Prdm8-expressing neurons located in the dorsal horn of the spinal cord in Bhlhb5 mutants and controls were quantified to determine if a statistically significant difference existed. A two-tailed T-test with a confidence value of 95% (p-value = 0.0012) (Table 1) was utilized to perform the statistical analysis. A significant decrease in Prdm8-expressing neurons in Bhlhb5 mutant mice as compared to those of Bhlhb5 wildtype controls was observed. Moreover, the Prdm8-expressing neurons were lost to the outer-most laminae (superficial region) of the spinal cord dorsal horn.

Figure 8. BHLHB5 mutants exhibit significant loss of PRDM8-expressing neurons in dorsal horn of spinal cord. A) Immunostaining analysis exhibiting loss of PRDM8 (marked in red) in subpopulations of neurons located in the dorsal horn of the spinal cord (outlined in white). B) Graphical representation of the significant loss of PRDM8-expressing neurons in the dorsal horn of the spinal cord when comparing BHLHB5 mutants vs. wildtype mice.
Table 1. PRDM8 mutants exhibit significant loss of PRDM8-expressing neurons in dorsal horn of spinal cord. 2-Tailed statistical test (*2 sample unequal variance heteroskedastic) comparing the number of PRDM8-expressing neurons between Bhlhb5 mut and wt mice indicate a statistically significant difference (p-value<0.05, confidence value of 95%).

Co-expression of Bhlhb5 and Prdm8 in spinal cord dorsal horn

To test the second hypothesis, immunostaining was conducted for Bhlhb5 and Prdm8 in the spinal cord of Prdm8 wildtype controls and mutants at P0. Wildtype mice exhibited high levels of Bhlhb5 and Prdm8 co-expression in the dorsal horn region of the spinal cord (indicated by white dotted lines in Sup Figure 6A).
Co-expression of both transcription factors (indicated in the “merged” panels of Sup. Figures 6A and 6B, where neurons expressing both Bhlhb5 and Prdm8 appear purple or blue-green in color, respectively) was detected. Interestingly, in the analysis of immunostaining for Bhlhb5 and a GFP marker for Prdm8-expressing neurons in Prdm8 mutants, a qualitative loss was observed of Bhlhb5 and Prdm8 co-expressing neurons in the dorsal horn on the spinal cord (Sup. Figure 6B).

Co-expression of Bhlhb5 and Prdm8 in the frontal cortex

Due to the similarity in phenotypes observed in both Bhlhb5 and Prdm8 mutants (both behaviorally and at the cellular level in the spinal cord), there is another key hypothesis. The third hypothesis is that high levels of co-expression of both Bhlhb5 and Prdm8 would also be observed in neurons located in the cortex where important neuronal components of sensory and motor circuits are derived and send projections throughout the body. Immunostaining was conducted for Prdm8 and Bhlhb5 in sagittal sections of wildtype mice frontal cortex, which indicated high levels of co- expression for both proteins in populations of neurons at P0 (Figure 9). Figure 9A exhibits immunostaining for Bhlhb5 and Prdm8 in a wildtype control brain and Figure 9B exhibits immunostaining for Bhlhb5 and Prdm8 in a Prdm8 mutant cortex. Enlarged boxes of each panel in Figure 9 demonstrate the co-expression of both proteins in the same neurons in wildtype and Prdm8 mutants. In the analysis of the immunostaining, no qualitative difference between Bhlhb5 and Prdm8 expression patterns in the cortex was observed. Nevertheless, high levels of co-expression was localized near the outermost layers of the frontal cortex of both wildtype and Prdm8 mutants.

Figure 9. PRDM8 and BHLHB5 immunostaining in cortex. A) Immunostaining analysis of PRDM8 expression (red) and BHLHB5 expression (blue) in the frontal cortex of wildtype mice (P0). High levels of PRDM8 and BHLHB5 co-expression expression exhibited in the outer layers of the cortex (shown as purple neurons in enlarged box). B) Immunostaining analysis of PRDM8-expressing neurons via GFP knockin at Prdm8 locus (green) and BHLHB5 expression (blue) in the frontal cortex of PRDM8 mutant (-/-) mice (P0). Large population of neurons co-expressing PRDM8 and BHLHB5 also exhibited in the outer layers of the cortex (shown as blue-green neurons in enlarged box).

Prdm8 mutants and double heterozygotes are missing the CST

In previous research, the observation that both Bhlhb5 and Prdm8 mutants demonstrate similar behavioral phenotypes (i.e. development of skin lesions, handstand behavior) (Ross et al., unpublished data) led to the overarching theory that Bhlhb5 and Prdm8 may be two transcriptional regulators involved in common regulatory processes in neuronal development. This conceptual idea thus led to the reasoning that the phenotypes observed in Prdm8 mutants mirroring that of Bhlhb5 mutants would also extend to the cellular level throughout the central nervous system. Therefore, based on the finding that Bhlhb5 mutants exhibit distinct loss of the corticospinal tract (CST) (Figure 3), Prdm8 mutants might also exhibit loss of the CST.
To test this hypothesis, immunohistochemistry analysis of spinal cord sections of wildtype and Prdm8 mutant mice was performed at P0. In the immunostaining analysis, the presence of the CST was assessed through analyzing sectioned spinal cord tissue with a probe that recognizes a protein marker, Protein Kinase C-gamma (PKC-gamma), for the CST. PKC-gamma is highly expressed in the corticospinal tract (Moreno-Flores et al., 2006), which is composed of a bundle of fibers derived from neurons originating in the motor cortex of the brain (Purves, 2001). In the analysis of the immunostaining experiment, the CST was found to be lacking in Prdm8 mutants (Sup. Figure 7C).
The observation regarding the absence of the CST in Prdm8 mutants is identical to evidence demonstrating that Bhlhb5 mutants are also missing the corticospinal tract, established through research indicating that corticospinal axons may mis-project or stop growth prematurely and do not reach targets in the dorsal spinal cord (Ross et al., unpublished data). Therefore, the combination of past research exhibiting the absence of the corticospinal tract in Bhlhb5 mutants with this project’s findings that Prdm8 mutants may also lack the corticospinal tract further support the overarching hypothesis that Prdm8 and Bhlhb5 are both crucial factors in the development of circuit formation in the central nervous system. Intriguingly, it was also noted that double heterozygous mice for Prdm8 and Bhlhb5 (Prdm8-/+ Bhlhb5 -/+) also indicate loss of the CST through the absence of PKC-gamma protein expression in spinal cord sections at P0 (Sup. Figure 7B). However, this observation regarding the loss of the CST in heterozygous animals is not observed in animals heterozygous for only one gene.

Discussion

No evidence of Bhlhb5 or Prdm8 binding in 293T cells

Results from this experiment (Figure 9) did not present any evidence of co-IP. Conversely, when immunoprecipitation for Prdm8 was conducted, no presence of Bhlhb5 was observed in the precipitate. These results suggest that Bhlhb5 and Prdm8 do not interact, and therefore provide evidence in support of models in the Figures 4A and 4C. However, this experiment was conducted in transfected 293T cells, where many endogenous factors necessary for regulatory processes in neurons are absent. It is possible that other proteins present in neurons but absent in 293T cells are necessary co-factors to facilitate the complex formation of Bhlhb5 and Prdm8.
The immunoprecipitation experiment was replicated in neurons where pull-down of endogenous Prdm8 and Bhlhb5 in their natural substrate environments could be observed. To conduct this experiment in neurons, a direct Prdm8 antibody was required. The previous immunoprecipitation experiment conducted in 293T cells utilized a Myc-epitope tagged construct of Prdm8. Thus, immunoprecipitation and probing for Prdm8 in 293T cells could be performed via the use of an anti-Myc antibody (rather than a direct antibody for Prdm8). A chief obstacle in this study that arose was the lack of available Prdm8 antibody to use in immunoprecipitation and immunohistochemistry experiments on neurons and fixepd tissue.

Design and development of the Prdm8 antibody

To resolve the issue of the absence of a Prdm8 antibody, a series of Prdm8 GST-Fusion protein constructs was designed and developed (Supp. Figures 3 and 4), from which a purified Prdm8 antigen could be selected and injected into rabbits for production of an antibody.

No evidence of endogenous Bhlhb5 and Prdm8 binding in neurons

Immunoprecipitation for Bhlhb5 in Prdm8 wildtype and mutant neurons from the whole cortex of mice demonstrated clear pull-down of the Bhlhb5 protein, yet no evidence of any Prdm8 was found in the precipitate (Figure 6). Similarly, no co-IP of Bhlhb5 was observed when pull-down for Prdm8 was conducted (Figure 7). Again, these results suggested that Bhlhb5 and Prdm8 do not interact, and therefore provided evidence in support of models in the Figures 4A and 4C.
Nevertheless, it is also possible that conditions of the immunoprecipitation experiment itself may have contributed to the dissociation of proteins in complex from one another. Considering the multiple steps involved in immunoprecipitation (i.e. washing of the beads, incubation of the antibody, and incubation with beads), it is very plausible that the nature of the protein-protein interaction between Bhlhb5 and Prdm8 may be very sensitive to the concentration of the lysis buffer ingredients, and that such may have affected the natural affinity between Bhlhb5 and Prdm8. Future experiments to clarify the interactive relationship between Bhlhb5 and Prdm8 should therefore call for a series of tests examining the effects of different lysis buffer ingredients, concentrations, and wash conditions on the immunoprecipitation and co-immunoprecipitation of proteins.

Significant loss of Prdm8-expressing neurons in Bhlhb5 mutant spinal cord

To further explore the relationship between Bhlhb5 and Prdm8 in neural development, the expression patterns of each protein in both Bhlh5 and Prdm8 mutants were characterized. Immunohistochemistry analysis was conducted on Bhlhb5 mutant mice, and a significant loss of Prdm8 was observed in the dorsal horn of the spinal cord at P0. The researcher confirmed this observation through immunostaining analysis (Figure 8A) and statistical quantification (Table 1, Figure 8B). These results, in conjunction with past research that Bhlhb5 is significantly lost from populations of neurons in the superficial layers of the dorsal horn of the spinal cord (Ross et al., unpublished data), combine to support the primary hypothesis: that Prdm8 and Bhlhb5 may be important regulatory factors in the same pathways in central nervous system development. With evidence from past research that Bhlhb5-expressing neurons are lost in the dorsal horn of the spinal cord, this finding that Prdm8-expressing neurons are also lost in the same region of the spinal cord suggested that Prdm8 and Bhlhb5 may be highly expressed in the same neurons.

High levels of co-expression of Prdm8 and Bhlhb5 in dorsal horn of spinal cord

Due to the finding that a significant loss of Prdm8-expressing neurons was observed in the same regions of the dorsal spinal cord where a significant loss of Bhlhb5-expressing neurons are absent in Bhlhb5 mutants, it was asked whether or not Bhlhb5 and Prdm8 co-expression would also be observed in the dorsal horn of the spinal cord. Sup. Figure 6A depicts immunostaining results from a wildtype mouse spinal cord. A high degree of co-localization of Prdm8 and Bhlhb5 protein expression in the dorsal horn of the spinal cord was observed. Furthermore, when the wildtype immunostaining results were compared to those from Prdm8 mutant spinal cord, a qualitative loss was determined of neurons co-expressing both Prdm8 and Bhlhb5 in the dorsal horn of the spinal cord (Sup. Figure 6B). The finding that Prdm8 and Bhlhb5 are both highly expressed in the same neurons thus provides further evidence in support of the hypothesis that Bhlhb5 and Prdm8 are involved in the same pathways in nervous system development.
Due to the lack of available Prdm8 mutant mice, there was not a sufficient sample size to conduct a quantitative statistical analysis to determine the significance of this loss of neurons co-expressing Prdm8 and Bhlhb5 in the dorsal horn of the spinal cord. Future experiments, however, should call for a quantification of the number of co-expressing neurons in wildtype versus Prdm8 and Bhlhb5 mutant mice, and statistical calculation to test for significance in difference between the two groups.

High levels of co-expression of Prdm8 and Bhlhb5 in frontal cortex

Due to the high levels of observed Bhlhb5 and Prdm8 co-expression in the spinal cord, in addition to the underlying theory that these two genes are crucial in the development of the central nervous system, Bhlhb5 and Prdm8 expression patterns in the mouse brain were analyzed. Immunostaining analysis was conducted for Bhlhb5 and Prdm8 on sagittal sections of the mouse cortex. Expression was observed for each of these proteins in cortical neurons, and for any distinguishing features between Bhlhb5 and Prdm8 expression patterns in wildtype versus Prdm8 mutant P0 mice. From the results (Figure 9), high levels of Prdm8 and Bhlhb5 co-expression in the cortex was found. The qualitative analysis further determined that the areas of greatest co-expression were the outermost layers of the frontal cortex. Qualitative examination of Bhlhb5 and Prdm8 expression in the frontal cortex between wildtype and Prdm8 mutant mice did not reveal any outstanding disparities in expression patterns. Nevertheless, further analysis following the availability of more Prdm8 mutant mice would be necessary to examine critically the differences in Prdm8 and Bhlhb5 expression patterns in the cortex of mutant mice.

Prdm8 mutants and Prdm8-Bhlhb5 heterozygotes exhibit loss of CST

From the finding that Bhlhb5 mutants exhibit distinct loss of the corticospinal tract (CST) (Figure 3), it was hypothesized that Prdm8 mutants would also exhibit loss of the CST. Immunostaining analysis was conducted utilizing a CST marker (PKC-g) (Sup. Figure 7C), which indicates the distinct loss of the CST in Prdm8 mutants. This result supportes the hypothesis that Prdm8 and Bhlhb5 mutants are both missing the CST and further provides evidence in support of the theory that Prdm8 and Bhlhb5 are crucial in the development of neurons involved in the same pathways of the central nervous system.
More intriguingly, however, the loss of the CST in double heterozygous mice (Bhlhb5 -/+ Prdm8 -/+) (Sup. Figure 7B) was noted. This finding is particularly striking, since no loss of the CST was observed in mice heterozygous for only one gene (i.e. Bhlhb5 -/+ Prdm8 +/+ or Bhlhb5 +/+ Prdm8-/+). Therefore, the finding that a heterozygous copy of both Prdm8 and Bhlhb5 could result in the absence of the CST, a phenotype found only in Bhlhb5 or Prdm8 full knockouts, suggestes that Bhlhb5 and Prdm8 may have an additive impact where the absence of a single copy of both Prdm8 and Bhlhb5 in mice may be equivalent in phenotype to missing both copies of either gene. The discovery of the absence of the CST in double heterozygous mice is exceptionally intriguing and prompts the hypothesis that such mice will also exhibit other identical phenotypes at the cellular (i.e. significant loss of Bhlhb5 or Prdm8-expressing neurons) and behavioral (i.e. skin lesion, handstand) levels. This hypothesis thus calls for future experiments to explore the phenotypes of double heterozygous mice through a series of behavioral assays (i.e. rotarod balance experiments, observation of self-injurious behavior, thermal and chemical stimulation assays) and examination of cellular expression patterns (i.e. analysis of Bhlhb5 and Prdm8-expressing neurons in the spinal cord and brain) and comparing such results to those of Prdm8 or Bhlhb5 single gene knockout mice.

Future Directions

Although results gathered in this study does not suggest direct interaction between Bhlhb5 and Prdm8, it is still not clear what the precise nature of their regulatory relationship may be. Three potential models of interaction were proposed, of which one (Figure 4B) was not supported by the immunoprecipitation results. However, two other models (Figures 4A and 4C) are possible. Therefore, a future direction of this study would be to distinguish between the two mechanisms or identify other alternative models that may best describe the relationship between Bhlhb5 and Prdm8.

Conclusions

The major finding in this study was that no indication of direct interaction between Bhlhb5 and Prdm8 proteins was observed. The models in Figures 8A and 8C are potential mechanisms of interaction between Bhlhb5 and Prdm8 in transcriptional regulation. In these models, Bhlhb5 and Prdm8 do not bind to each other in complex, targeting either the same genes (Figure 4A) or distinct genes in the same pathway (Figure 4C). Nevertheless, due to the nature of the experimental conditions and the possible sensitivity of the proteins’ affinities for each other, it is necessary that further experiments be conducted before the model in which Prdm8 and Bhlhb5 interact in complex (Figure 4B) may be rejected.
Despite the lack of evidence identifying the precise model of interaction between Bhlhb5 and Prdm8, immunostaining results provide strong evidence in support of the overarching theory that Bhlhb5 and Prdm8 function as important regulatory factors in the same pathway of neuronal development in the central nervous system. This conclusion was drawn from four critical findings: 1) Prdm8-expressing neurons are significantly lost in the dorsal horn of the spinal cord in Bhlhb5 mutants, 2) high levels of co-expression of Prdm8 and Bhlhb5 are observed in the dorsal horn of the spinal cord, 3) dramatic loss of neurons co-expressing Prdm8 and Bhlhb5 is observed in the dorsal horn of the spinal cord, and 4) loss of the CST is observed in Prdm8 mutants and double heterozygous mice (Bhlhb5 het, Prdm8 het). The combination of these four findings demonstrate that Prdm8 mutants, like Bhlhb5 mutants, express similar Prdm8 and Bhlhb5 expression patterns in the brain and spinal cord. This study additionally suggests that the missing subpopulation of neurons in the dorsal horn of the spinal cord co-express both Bhlhb5 and Prdm8, thus providing evidence in support of the hypothesis that Bhlhb5 and Prdm8 are crucial factors in a common pathway underlying neural differentiation and development.
Technical difficulties encountered included the lack of available mice of desired Prdm8 and Bhlhb5 genotypes. With a greater number of Prdm8 mutants, double heterozygotes, and double mutants, more immunostaining experiments could be conducted to discern for any nuances in phenotype that distinguish Bhlhb5 mutants from Prdm8 mutants. Furthermore, the availability of more mice of desired mutant genotypes would allow for statistical analysis to be performed so that the significance of Bhlhb5 and Prdm8 co-expression may be calculated.
In sum, the overarching motivation that propelled this research was the pursuit of a better understanding of the processes underlying central nervous system development in mammals. The striking phenotypes exhibited by both Bhlhb5 and Prdm8 mutant mice was the first signal that a cooperative relationship may exist between the two proteins, and that their function was critical in neural development. These results provide support for the view that a novel relationship between Bhlhb5 and Prdm8 exist in the same neural pathways; they also help illustrate a process within the formation of the central nervous system that have significance for the future understanding of not only how the mammalian nervous system develops, but how it matures and ages with time.

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