We designed two primer sets specifically for Ptprg transcripts: one to amplify long transcripts and another to amplify short transcripts. The first (“long”) γEx1_Fwd and γEx30_Rev primer pair amplifies all possible Ptprg transcripts that initiate with Exon 1 and terminate with Exon 30, which includes Ptprg-V1 (NM_008981) and the hypothetical Ptprg-X1 assembly (XM_006517956). The second (“short”) γEx14a_Fwd and γEx30_Rev′ primer pair amplifies the Ptprg-V2 transcript (NM_001347593), which initiates in Exon 14a and terminates in Exon 30, 146 nt 3′ to the termination of the previous reverse primer (i.e., γEx30_Rev). We clone the purified PCR bands into the pCR-Blunt plasmid using the Zero Blunt PCR Cloning Kit as described above.

Determining the presence of different Ptprz1 transcripts is more challenging than is for Ptprg (which has one validated ¹ short transcript, one validated long transcript, and one hypothetical long transcript; see previous paragraph) for three reasons: (a) Ptprz1 has as many as nine assemblies, four validated and five hypothetical; (b) Ptprz1 has multiple “short” (<5.4 kb), one “intermediate” (5.4–8.0 kb), and multiple “long” (>8.0 kb) Ptprz1 transcripts; and (c) the differences that arise from alternate utilization of three of the cassettes (exon 16, which may be spliced in or out; exon 21“a” or 21“b” ² ) are so small that the alternative splicing yields multiple full-length transcripts that are difficult to distinguish in a single PCR on the basis of size alone. Therefore, we designed sets of nested primer pairs (Table 2) to amplify cDNA fragments from at most two possible transcripts per PCR and then verified the identity of these transcripts through cloning the cDNA and sequencing. In both Table 2 and the lists below, “V” designates previously verified murine Ptprz1 variants, whereas “X” designates variants previously designated as hypothetical. As described above, we cloned PCR bands for all short, intermediate, and long Ptprz1 transcripts into pCR-Blunt for sequencing.

V1: To amplify the lone intermediate-length Ptprz1-V1 transcript, we used, in the round-one PCR, ζEx1_Fwd together with the ζEx12_Rev primer. We then used the purified amplicon from round one as template in a second, nested PCR using the ζEx1_Fwd′ and ζEx12_Rev′ primer pair.

Other variants: To amplify all possible “short” and “long” Ptprz1 full-transcripts that commence with exon 1 and end with exon 30, in round-one PCR, we used the primers ζEx1_Fwd and ζEx30_Rev. For the second round, we used the amplicons from round one as templates, together with the ζEx1_Fwd′ and ζEx30_Rev′ primer pair.

To identify the presence of all other alternative Ptprz1 cassettes from transcripts that commence with exon 1 and end with exon 30, we used first-round PCR amplicons amplified from PCRs primed with ζEx1_Fwd and ζEx30_Rev as the cDNA template in second-round nested PCRs. These second-round PCRs were primed with new sets of “diagnostic” nested primers (also summarized in Table 1) that produce shorter, variant-specific amplicons. We outline the rationale for the designs of these novel second-round primers in the following list:

V3/X3: We designed the ζEx12a-13_Fwd and ζEx20-21a_Rev primer pair that will amplify an 820-bp fragment of Prprz1-V3 and/or a 799-bp fragment of Prprz1-X3 cDNA if the transcripts are present in TRNA.

V4/5: The ζ12b-13_Fwd and ζ20-21a_Rev primer pair amplifies an 826-bp fragment of Prprz1-V4 and/or 805-bp fragment of Prprz1-V5 if these transcripts are present in TRNA.

X1/X2: We designed the ζEx12a-13_Fwd and ζEx20-21b_Rev primer pair to amplify an 827-bp fragment of Prprz1-X1 and/or 806-bp fragment of Prprz1-X2 if mRNAs are present in the TRNA sample.

X4/5: The ζ12b-13_Fwd and ζEx20-21b primer pair amplify an 832-bp fragment of Ptprz1-X4 and/or 811 bp fragment of Ptprz1-X5 if the transcripts are present in the TRNA sample.

From mixed neuron–astrocyte HC cultures, we obtained 10 colonies, 40% of which contained the complete 4,329-bp open reading frame (ORF) for Ptprg-V1 (Figure 1B). We submitted three of these four clones to GenBank, which assigned the accession numbers [OR710276], [OR710277], and [OR710278]. From pup and adult HC tissue, we obtained 10 colonies each, 30% of which contain the same complete 4,329-bp ORF for Ptprg-V1. For both pup and adult, we submitted two samples for pup and three for the adults of these three clones to GenBank. GenBank assigned accession numbers [OR710279] and [OR710280] for the pup, and [OR710281], [OR710282], and [OR710283] for the adults.

In addition to the 40% or 30% of the PCR cloning colonies that yielded Ptprg-V1 cDNA (see V1 transcripts section), we found that the remaining colonies represent the hypothetical murine assembly [XM_006517956]. Specifically, 60% of the colonies from mixed neuron–astrocyte HC cultures (five submitted, yielding GenBank accession numbers [OR900076]–[OR900080]) and 70% of the PCR cloning colonies from pup ([OR900081]–[OR900088]) or adult HC tissue ([OR900089]–[OR900093]; Figure 1B). This novel transcript lacks exon 14 (representing 29 amino acids), which encodes the intracellular region immediately after the TM domain in Ptprg-V1.

Interestingly, in a single pup P0–P2 culture clone (i.e., [OR900082]), sequencing on both cDNA strands revealed the existence of a single G→A nucleotide substitution, 584 nt into the Ptprg ORF. In [OR900082], this substitution changes the ⁵⁸³AGA⁵⁸⁵ ORF codon to ⁵⁸³AAA⁵⁸⁵, resulting in an R195K amino acid change in the translated RPTPγ protein. This conservative substitution, located on the external surface of the CALD, is the single amino acid linking the S186-N194 α-helix and the I197-S207 β-strand (Bouyain and Watkins, 2010). As a point of reference, R195K is located within the CALD, 20 downstream from residue Q175, which is at the position equivalent to one of the three Zn²⁺-coordinating histidines in active CAs.

Note that we did not amplify or clone cDNA corresponding to mouse Ptprg-V2 from any of our three mouse preparations (Figure 1B).

Figure 2A displays a schematic representation of the topology and structural domains of the three translated RPTPγ variants. RPTPγ-V1 [NP_033007] and RPTPγ-V“3” (formerly hypothetical [XP_006518019]) both possess extracellular CALD and FNIII domains, followed by a single TM domain, and the D1 and D2 phosphatase domains. They differ only by the omission of 29 juxta-membrane amino acids that, in RPTPγ-V“3”, are encoded by exon 14 (Figure 1A). In Figure 2A, we represent this omission by the dashed line. RPTPγ-V2 [NP_001334522]—undetected in the present study—lacks the CALD, FNIII, and transmembrane domains and therefore is an exclusively intracellular variant.

Figure 2B displays the amino acid sequence alignment for the three RPTPγ variants. The RPTPγ-V“3” protein corresponds to the protein previously only reported by NCBI RefSeq as a hypothetical mRNA XM_006517956 and the hypothetical translation product [XP_006518019].

About 14 days after initiating cultures from WT P0–P2 mouse pups, we stained the primary neuron–astrocyte HC cultures with DAPI (Figure 3A, blue in leftmost column), a MAP2 mouse monoclonal antibody (green) to identify the neurons, and a RPTPγ chicken IgY antibody (red; see Mafficini et al., 2007). The rightmost column shows the merged images. We determine that RPTPγ (Figure 3A, red) is localized throughout the neuronal soma and major neuronal processes. When we stained parallel cultures, not with an MAP2 antibody but with a GFAP mouse monoclonal antibody to identify the astrocytes (Figure 3B, green), RPTPγ-stained projections (red) from adjacent neurons are clearly visible surrounding the astrocytes. However, the staining never colocalizes within the GFAP-positive cells (i.e., astrocytes).

Because we derived the mixed neuron–astrocyte HC cultures from P0–P2 pups, we also studied RPTPγ expression in native P0–P2 HC tissue. We examined stained sections from four regions of P0–P2 pup hippocampus: cornu ammonis 1 (CA1), CA2, CA3, and dentate gyrus (DG). In each row of Figure 4, the leftmost panel is at relatively low magnification, whereas the panels to the right are high-magnification images from selected regions of interest (ROIs). We observed that MAP2-stained neurons (Figure 4A, green) in all four HC regions also stained for RPTPγ (red). Throughout the CA regions, the brightest RPTPγ staining was in the SP and became more diffuse in the adjacent stratum oriens (SO) and stratum radiatum (SR). Within the DG, RPTPγ staining was concentrated in the stratum granulosum (SG) but extended more diffusely into the neuropil of the adjacent molecular layer (ML).

In parallel sections, we stained astrocytes with anti-GFAP (Figure 4B, green) and observed no colocalization of RPTPγ (red) throughout the CA1, CA2, CA3, or DG with GFAP-positive cells (i.e., astrocytes) or processes. Positive RPTPγ staining was only diffusely visible in the surrounding neuropil.

Finally, following our approach for cultures and pups, we examined RPTPγ expression in native adult (8- to 12-week-old animals) HC tissue. We found expression patterns for RPTPγ (Figure 5A) that are nearly identical to what we observed in the P0–P2 pup tissue. In particular, RPTPγ colocalizes strongly with MAP2-positive cells (i.e., neurons) in the CA1–3 SP HC regions. In the DG, RPTPγ staining localizes to the soma of the granule cell within the SG.

In adult mouse HC tissue stained for GFAP (Figure 5B, green), but not for MAP2, we consistently observed GFAP-positive cells (i.e., astrocytes) to be devoid of RPTPγ staining (red). Rarely, and especially at low magnification, we observe yellow pixels in a merged image (i.e., GFAP from astrocytes plus RPTPγ). The yellow could reflect (a) a neuron and an astrocyte in the same optical section or (b) the rare astrocyte that expresses RPTPγ (see Discussion). Nevertheless, the dominant pattern is that GFAP-positive cells are RPTPγ negative. GFAP-positive astrocyte soma and, in some cases, the early branches of their processes are most clearly visible in the CA1–3 SR in the high-magnification ROIs of Figure 5B, where GFAP and RPTPγ do not appear to colocalize within individual cells or cell processes.

To validate the RPTPγ chicken IgY antibody, we immunostained adult Ptprg ^−/− HC sections following the same protocols as used in Figures 4A, B, 5A, B. After counterstaining with either MAP2 (Figure 5C) or GFAP (Figure 5D), we did not observe any non-specific signal from the anti-RPTPγ chicken IgY.

The mouse Ptprz1 gene (Gene ID: 19283) contains 30 exons on chromosome 6. When the present investigation commenced, the NCBI RefSeq database contained four validated murine splice variants (i.e., V1, V3, V4, and V5) that had also been described by Fujikawa et al. (2017) and five hypothetical transcript assemblies (i.e., X1 through X5).

Here, we define exon 12 as the full-length exon. We propose the names 12“a” and 12“b” for the shorter alternative exons that arise from splicing at one of two splice sites within exon 12, such that 12“a” is entirely within 12 and 12“b” is entirely within 12“a”.

Ptprz1-V1 (Exons 1–12). Variant 1 (Ptprz1-V1, [NM_011219]) is encoded by exons 1–12 (Figure 6A; Table 3). V1 is the only variant to utilize the 5479-nt exon 12 that, due to the presence of the ⁵⁴⁵⁶AAUAAA⁵⁴⁶¹ cleavage and polyadenylation specificity factor binding site, will be cleaved and polyadenylated 18-nt downstream, preventing its splicing to any additional exons. Utilization of exon 12 in Ptprz1-V1 results in the ORF continuing only as far as an in-frame amber (TAG) stop codon, located at a position analogous to 3-nt downstream from the exon 12/13 splice site in V3. The remainder of the Ptprz1-V1 mRNA, downstream of the amber (TAG) stop codon, consists of a 1,938-nt 3′-UTR. At the protein level, V1 encodes an RPTPζ variant that has 1,211 amino acids between the end of the FNIII domain and the termination of the protein, which lacks transmembrane and phosphatase domains (Figure 6B). Thus, V1 is secreted (S) into the extracellular fluid. Fujikawa et al. (2017) originally designated V1 as PTPRZ-S to reflect the ultimate fate of the protein.

Exon 1–30 variants (Ptprz1-V3 to Ptprz1-V5 and Ptprz1-X1 to Ptprz1-X5). The eight other validated or hypothetical Ptprz1 transcripts all commence with exon 1 and end with exon 30 (Figure 6A). None of these eight variants includes exon 12. Instead, the source of variation comes from (a) utilization of one of the two other exon-12 sites that splice to exon 13, yielding shorter exon 12 variants (i.e., 12“a” or 12“b”); (b) inclusion vs omission of exon 16; and (c) utilization of one of two possible exon-21 alternatives (i.e., 21“a” or 21“b”; Table 3). Below, we outline the alternative cassette usage by each of the eight exon 1–30 variants.

Ptprz1-V3 [NM_001081306]—Exon 12“a”, exon 16, exon 21“a”. (a) If the splice site 3,547-nt into exon 12 is utilized, the result is alternative exon 12“a”, and this splices to exon 13 (Figure 6A; Table 3). At the protein level, V3 has 1,232 amino acids between the end of the FNIII domain and the start of the TM domain (Figure 6B; Table 3). (b) The middle portion of the Ptprz1-V3 transcript, beginning at exon 13, includes exon 16. (c) The last portion of the Ptprz1-V3 transcript, beginning at exon 17, includes exon 21“a” rather than 21“b” (Figure 6A; Table 3). The full Ptprz1-V3 transcript, which Fujikawa et al. (2017) named Ptprz-A, comprises 8,018 nt and encodes a 2,305-amino acid protein.

Ptprz1-V4 [NM_001311064]—Exon 12“b”, exon 16, exon 21“a”. If the splice site at the position 1,000-nt into exon 12 is utilized, the result is exon 12“b”. This is the only difference between the variant 4 transcript and that of V3 mentioned above (Figure 6A; Table 3). At the protein level, the amino acid linker between the end of the FNIII domain and the start of the TM domain of RPTPζ-V4 is only 383 amino acids long (Figure 6B; Table 3). The full Ptprz1-V4 transcript, which Fujikawa et al. (2017) named Ptprz-B, comprises 5,537 nt and encodes a 1,463-amino acid protein.

Ptprz1-V5 [NM_001361349]—Exon 12“a”, Δexon 16, exon 21“a”. The only difference between the V5 transcript and that of V4 is the omission of exon 16 (Figure 6A; Table 3). Exon 16 encodes 7 amino acids (TLKEFYQ) within the helix-turn-helix “wedge” segment of the D1 phosphatase domain, which may be important in the allosteric modulation of phosphatase activity or in interactions with adjacent phosphatase domains (Bilwes et al., 1996; Figure 6B). The full Ptprz1-V5 transcript, which Fujikawa et al. (2017) named Ptprz1-BΔex16, comprises 5,471 nt and encodes a 1,456-amino acid protein.

Ptprz1-X3 [XM_006505014]—Exon 12“a”, Δexon 16, exon 21“a”. The NCBI RefSeq database also predicted the hypothetical transcript variant Ptprz1-X3, which is identical to V3 except for the lack of exon 16. Although listed by NCBI as hypothetical, Fujikawa et al. (2017) in their Figure 1B had included this transcript as Ptprz1-AΔex16, which they detected in the mouse brain (Figure 6A; Table 3). The full Ptprz1-X3 transcript comprises 8,018 nt and encodes a 2,305-amino acid protein.

Finally, at the time that the present investigation commenced, the NCBI RefSeq nucleotide database cataloged four other hypothetical Ptprz1 transcript variants that, if they were assembled as mature mRNAs and translated, would yield transmembrane RPTPζ variants with all the essential components: a signal peptide, CALD, FNIII, TM domain, and D1 and D2. We discuss these hypothetical constructs next.

Ptprz1-X1 [XM_006505012]—Exon 12“a”, exon 16, exon 21“b”. The only difference between the X1 transcript and that of V3 is the use of exon 21“b” rather than 21“a” (Figure 6A; Table 3). If Ptprz1-X1 were expressed and translated, the inclusion of exons 12“a” and 16 indicates that it would possess both the longer 1,232-amino acid extracellular FNIII-TM linker and the larger “wedge” motif in the intracellular D1 phosphatase domain (Figure 6B; Table 3). The utilization of exon 21“b” would encode (compared to exon 21“a”) six additional residues' N-terminal to the catalytic site of D1. The full Ptprz1-X1 transcript would comprise 8,285 nt and encode a 2,318-amino acid protein.

Ptprz1-X2 [XM_006505013]—Exon 12“a”, Δexon 16, exon 21“b”. The only difference between the X2 transcript and that of X1 (immediately above) is the omission of exon 16. (Figure 6A; Table 3). The only difference between the X2 transcript and that of X3 is the use of exon 21“b” rather than 21“a” (Figure 6A; Table 3). The absence of exon 16 from this variant predicts that the D1 phosphatase domain would exhibit the smaller “wedge” motif, and the presence of 21“b” would produce the 6-amino acid insert N-terminal to the catalytic domain (Figure 6A; Table 3). The full Ptprz1-X2 transcript would comprise 8,264 nt and encode a 2,311-amino acid protein.

Ptprz1-X4 [XM_006505015]—Exon 12b, exon 16, exon 21“b”. The only difference between the X4 transcript and that of V4 is the use of exon 21“b” rather than 21“a” (Figure 6A; Table 3). The inclusion of exon 12“b” would produce the shorter extracellular FNIII-TM linker; exon 16 would encode the larger “wedge” motif in D1; and exon 21“b” would produce the 6-amino acid insert near the catalytic domain (Figure 6B; Table 3). The full Ptprz1-X4 transcript would comprise 5,510 nt and encode a 1,469-amino acid protein.

Ptprz1-X5 [XM_006505017]—Exon 12“b”, Δexon 16, exon 21“b”. The only difference between the X5 transcript and that of V5 is the use of exon 21“b” rather than 21“a” (Figure 6A; Table 3). The only difference between the X5 transcript and that of X4 is the absence of exon 16. Thus, the protein would have the shorter extracellular FNIII-TM linker (because of exon 12“b”), smaller “wedge” motif in the intracellular D1 (lack of exon 16), and six extra residues near the catalytic site (exon 21“b”; Figure 6B; Table 3).

Ptprz1-X1, Ptprz1-X2, Ptprz1-X4, and Ptprz1-X5 contain an alternatively spliced form of exon 21 that we propose to name exon 21“b”. Exon 21“b” is the full-length exon 21 variant, as it does not use the splice site located 18 nt into the exon. We named it 21b because at the beginning of the present investigation, all RPTPζ variants with verified expression used the shorter form of exon 21, which we propose to name exon 21“a”. That is, exon 21“b” had only been present in hypothetical assemblies. RPTPζ products of exon 21“b” containing transcripts will have an extra LSELFQ motif in the D1 phosphatase domain adjacent to the N-terminal side of the catalytic site (Figure 6B).

To determine which of the above described Ptprz1 transcripts express in mixed neuron–astrocyte HC cultures, P0–P2 pup HC tissue, or adult HC tissue, we prepared cDNA from these three sources and then used GSPs (Table 2) in a series of PCRs designed to amplify sets of cDNA fragments that (with sequencing verification) are diagnostic for each of the Ptprz1 variants.

Ptprz1-V1 (Exons 1–12). Figure 6C shows that by using the ζEx1_Fwd′ and ζEx12_Rev′ GSPs, we amplified 5,565-bp cDNA fragments from P0–P2 pups and adults, but not from mixed neuron–astrocyte cultures. Cloning these amplicons into the pCR-Blunt plasmid yielded two colonies from P0–P2 pup and two additional colonies from adult HC samples.

Sequencing of the first cloned P0–P2 pup HC amplicon [PP524764] revealed a Ptprz1-V1 transcript with two minor variations compared to the consensus [NM_011219] RefSeq entry: (a) omission of nucleotides ¹⁸⁶CTCTCT¹⁹¹ within the exon-1/5′-UTR and (b) a non-synonymous T→C point mutation at exon 1/nucleotide 319 of the consensus sequence that would result in a L4P amino acid substitution in the RPTPζ-V1 signal peptide.

Sequencing of the second cloned P0–P2 pup HC amplicon [PP524765] reveals that this Ptprz1-V1 clone contains three differences compared to the consensus [NM_011219] RefSeq entry: (a) omission of nucleotides ¹⁸⁵CTCTCT¹⁹¹ from the exon-1/5′-UTR; (b) a non-synonymous G→A point mutation in exon 3 that would result in an E91K mutation in the translated protein; and (c) retention of a 1,017-nt intron between exon 9 and exon 10, which would result in translation of a truncated 375-amino acid RPTPζ variant that would contain only the first 66% of FNIII. The final four C-terminal residues after L371 would be -VTIR*.

Sequencing of the first cloned adult HC amplicon [PP524766] revealed that it is identical to consensus [NM_011219] RefSeq entry with the exception of a synonymous T→C point mutation in exon 12 at position T³⁷²⁰ of the consensus sequence (thus the codon for residue Threonine-1138 would change from ACT to ACC).

Sequencing of the second cloned adult HC amplicon [PP524767] revealed that compared to [NM_011219], this clone lacks nucleotides ¹⁹⁰CT^{19 1} from the exon-1/5′-UTR. We found no other changes within the ORFs of either of the adult clones, which we predict would result in the expression of an RPTPζ-V1 protein with an amino acid sequence identical to that of the RefSeq consensus [NP_035349].

Exon 1–30 variants (Ptprz1-V3 to Ptprz1-V5 and Ptprz1-X1 to Ptprz1-X5). For our first-round PCRs, our GSPs were ζEx1_Fwd and ζEx30_Rev, which should amplify all transcripts except Ptprz1-V1 (which contains exon 12). Using these products as templates, we performed second-round nested PCRs using the GSP combinations outlined in Table 2. We then determined which of the remaining possible validated or hypothetical Ptprz1 variants are present in each of the three HC preparations.

Ptprz1-V3 vs. Ptprz1-X3 (Exon 12“a”, ±exon 16, exon 21“a”). PCRs primed with ζEx12a-13_Fwd and ζEx20-21a_Rev (Table 2) amplified ∼800-bp cDNA fragments from mixed HC neuron–astrocyte cultures, P0–P2 pups, and adults (Figure 6D, “V3 or X3” lanes). Cloning and sequencing of the ∼800-bp cDNA fragments determined that 69% (9/13) of the cloned amplicons from the cultures were 820-bp fragments from Ptprz1-V3 transcripts (i.e., containing exons 12“a”, 16, and 21“a”; [PP524793]–[PP524801]. The remaining 31% (four colonies) were 799-bp fragments of Ptprz1-X3 transcripts (exons 12“a”, Δ16, 21“a”; [PP524840]–[PP524843].

Regarding the pups, 60% (3/5) of the clones contain the 820-bp fragment specific for Ptprz1-V3 [PP524802]–[PP524804], and 40% (2/5) contain the 799-bp fragment specific for Ptprz1-X3 [PP524844]–[PP524845].

Regarding the adults, 67% (4/6) of the clones contain the 820-bp fragment specific for Ptprz1-V3 [PP524805]–[PP524808] and 33% (2/6) contain the 799-bp fragment specific for Ptprz1-X3 [PP524846]–[PP524847].

Ptprz1-V4 vs. Ptprz1-V5 (Exon 12“b”, ±exon 16, exon 21“a”). PCRs primed with ζEx12b-13_Fwd and ζEx20-21a_Rev (Table 2) amplified ∼800-bp cDNA fragments from mixed HC neuron–astrocyte culture cDNA, P0–P2 pups, and adults (Figure 6D, “V4 or V5” lanes). Cloning and sequencing the ∼800-bp cDNA fragments established that 80% (12/15) of the cloned amplicons from the cultures were 826-bp fragments from Ptprz1-V4 transcripts (exons 12“b”, 16, and 21“a”; [PP524809]–[PP524820]). The remaining 20% (three colonies) were 805-bp fragments from Ptprz1-V5 transcripts (exons 12“b”, Δ16, and 21“a”; [PP524829]–[PP524831]).

Regarding the pups, 50% (5/10) of the clones contain the 826-bp fragment specific for Ptprz1-V4 [PP524821]–[PP524825] and 50% (5/10) contain the 805-bp fragment specific for Ptprz1-V5 [PP524832]–[PP524836].

Regarding the adults, 50% (3/6) of the clones contain the 826-bp fragments specific for Ptprz1-V4 [PP524826]–[PP524828] and 50% (3/6) contain the 805-bp fragment specific for Ptprz1-V5 [PP524837]–[PP524839].

Ptprz1-X1 vs. Ptprz1-X2 (Exon 12“a”, ±exon 16, exon 21“b”). PCRs primed with ζEx12a-13_Fwd and ζEx20-21b_Rev (Table 2) amplified ∼800-bp cDNA fragments from mixed HC neuron–astrocyte cultures, P0–P2 pups, and adults (Figure 6D, “X1 or X2” lanes). Cloning and sequencing of the ∼800-bp cDNA fragments determined that 50% (6/12) of the cloned amplicons from the cultures were 827-bp fragments from Ptprz1-X1 transcripts (exons 12“a”, 16, and 21“b”; [PP524784]–[PP524789]). One of the above six cloned amplicons [PP524784] lacks the thymine-5529 nucleotide of the [XM_006505012] consensus sequence. This deletion would cause a frame shift and premature stop in the RPTPζ ORF, 10 amino acids downstream from W1559 (at the end of the transmembrane domain). The remaining predicted residues would be RKCFQTAHF^I* [where ^ designates the location of the frameshift; I (i.e., isoleucine) is an abnormal residue; and * is the C-terminus]. The truncated protein would comprise only 1,669 amino acids. The other five of six 827-bp Ptprz1-X1 transcript fragments ([PP524785]–[PP524789]) are identical to the [XM_006505012] consensus sequence. The remaining 50% of the cloned amplicons (six colonies) were 806-bp fragments from Ptprz1-X2 transcripts (exons 12“a”, Δ16, 21“b”; [PP524768]–[PP524773]).

Regarding the pups, 33% (3/9) of the clones contain the 827-bp fragment specific for Ptprz1-X1 [PP524790]–[PP524792] and 67% (6/9) contain the 806-bp fragment specific for Ptprz1-X2 [PP524774]–[PP524779].

Regarding the adults, 100% (4/4) of the clones contain the 806-bp fragment specific for Ptprz1-X2 [PP524780]–[PP524783].

Ptprz1-X4 vs. Ptprz1-X5 (Exon 12“b”, ±exon 16, exon 21“b”). Finally, PCRs primed with ζEx12b-13_Fwd and ζEx20-21b_Rev (Table 2) amplified ∼800-bp cDNA fragments from mixed HC neuron–astrocyte cultures, P0–P2 pups, and adults (Figure 6D, “X4 or X5” lanes). Cloning and sequencing of the ∼800-bp cDNA fragments determined that 100% (three colonies) of the cloned amplicons from the cultures were 833-bp fragments from Ptprz1-X4 transcripts (exons 12“b”, 16, and 21“b”; [PP524848]–[PP524850]).

Regarding the pups, 9% (2/22) of the clones contain the 833-bp fragment specific for Ptprz1-X4 [PP524851]–[PP524852] and 91% (20/22) contain the 812-bp fragment specific for Ptprz1-X5 (exons 12“b”, Δ16, and 21“b”; [PP524857]–[PP524876]).

Regarding the adults, 20% (4/20) of the clones contain the 833-bp fragment specific for Ptprz1-X4 [PP524853]–[PP524856] and 80% (16/21) contain the 812-bp fragment specific for Ptprz1-X5 [PP524877]–[PP524892].

About 14 days after initiating cultures from WT P0–P2 mouse pups, we identified cells with DAPI and neurons with the MAP2 mouse monoclonal antibody (Figure 7A, green). We observed RPTPζ expression throughout the soma and cellular projections in these MAP2-positive cells (i.e., neurons) by counterstaining using a novel RPTPγ rabbit polyclonal primary antibody (red) that we developed against an extracellular RPTPζ epitope between the FNIII domain and transmembrane domain. In parallel cultures labeled by an anti-GFAP to identify the astrocytes (Figure 7B, green), we observed that RPTPζ (red) co-stained cells shaped like neurons but never did the RPTPζ signal colocalize within the GFAP-positive cells (i.e., astrocytes).

To validate the specificity of our new RPTPζ antibody, we performed immunocytochemical analyses on mixed neuron–astrocyte HC cultures similar to those above but derived from P0–P2 Ptprz1 ^−/− pups. By employing the same staining conditions that we used to visualize RPTPζ expression in WT neurons (Figure 7A) but not RPTPζ astrocytes (Figure 7B), here in the cultures from Ptprz1 ^−/− tissue (Figure 7C), we did not observe significant cross-reaction of our novel RPTPζ antibody with other proteins expressed in these mixed neuron–astrocyte HC cultures.

In the next step, we examined RPTPζ expressed in four regions of P0–P2 pup HC tissue (CA1, CA2, CA3, and DG). In all four HC regions, the MAP2-positive cells (i.e., neurons, green) co-stained for RPTPζ (red), as indicated in yellow in merged images (Figure 8A). In the CA1–CA3 regions, RPTPζ staining is the strongest in the neuronal somata of the SP and also in some somata throughout the neuropil of the SO and SR. Likewise in the DG, we observe RPTPζ staining in the neuronal somata, especially in the SG and, to a lesser extent, ML (Figure 8A).

In parallel sections, we stained astrocytes with the GFAP antibody (Figure 8B, green) and observed no RPTPζ colocalization (red) with GFAP-positive (i.e., astrocytic) somata or projections throughout the CA1, CA2, CA3, or DG. RPTPζ-positive staining is very diffuse in the surrounding neuropil and likely represents adjacent neuronal projections.

When we stained HC sections obtained from P0–P2 Ptprz1 ^−/− pups following the same protocols that we used to stain sections from WT pups, we observed no significant cross-reaction of our novel RPTPζ antibody with other proteins expressed in either neurons (Figure 8C) or astrocytes (Figure 8D).

In all four regions of adult HC tissue (Figures 9A, B), RPTPζ appears in MAP2-positive cells (i.e., neurons) but not in GFAP-positive cells (i.e., astrocytes), generally following patterns reminiscent of those noted above for tissue from P0–P2 pups.

We detected no significant cross-reaction of the RPTPζ antibody with other proteins expressed in HC sections from adult Ptprz1 ^−/− mice (Figures 9C, D).

Comparable to Fujikawa et al. (2017), we detected the expression of Ptprz1-V1, Ptprz1-V3, Ptprz1-V4, and Ptprz1-V5 transcripts (Figure 6), in their nomenclature, respectively, Ptprz-S, Ptprz-A, Ptprz-B, Ptprz-BΔex16 (Table 3), in almost all of our mouse HC preparations. The notable exception was that Ptprz1-V1 is absent from our mixed neuron–astrocyte HC cultures. However, this absence of V1 from the culture aligns with early reports that mature CNS cells secrete RPTPζ-V1/RPTPζ-S/phosphacan (Canoll et al., 1996). Thus, it is possible that our HC cultures are not sufficiently mature to express this variant, even 14–20 days post isolation of the cells from the P0–P2 brain.

Computational analyses of the Mus musculus chromosome 6 sequence (NC_000072) can predict hypothetical Ptprz1 variants. During the course of the present investigation, NCBI performed several updates to the RefSeq database. In the process, NCBI validated the hypothetical assemblies Ptprz1-X3 and Ptprz1-X4, originally predicted from genomic source sequences [AC133599] and [AC134445]—both RPCI-24 BAC library constructed from male C57BL/6J mouse spleen and/or brain genomic DNA, as expressed variants Ptprz1-V6 and Ptprz1-V7, respectively (Table 3).

The NCBI probably did not validate the Ptprz1-X3 assembly earlier because, although Fujikawa et al. (2017) had previously published their Ptprz1-AΔex16 (Table 3) transcript from the mouse brain, the cDNA sequences were not in either the NCBI or Ensembl databases. Moreover, the approach of Fujikawa et al., who amplified between exons 12“a” and 18, would not have allowed them to distinguish between Ptprz1-X3 and Ptprz1-X2. The validation occurred after the submission of transcriptomic evidence for Ptprz1-X3 expression from the sequence-read archive (SRA) runs SRR14777531.436575 and SRR14777534.444365, which are a part of the PRJNA667257 and PRJNA547800 BioProjects. PRJNA547800, “Targeted long read sequencing of neuronal cell surface receptor–encoding genes” specifically sources transcript material from the C57BL/6J mouse retina and cerebral cortex. Although NCBI validated Ptprz1-X3 as Ptprz1-V6 based on, presumably, a single sequence read of one cDNA strand, we now provide independent evidence for Ptprz1-V6 mRNA expression in eight clones (four from cultures, two from pups, and two from adults), in which we sequenced both cDNA strands.

NCBI validated Ptprz1-X4 as Ptprz1-V7 (Table 3) on the basis of its presence in SRR9219380.17411 and SRR9219381.21863, sourced from the C57BL/6J retina and cerebral cortex. Although NCBI validated Ptprz1-X4 as Ptprz1-V7 based on, presumably, two single-sequence reads of one cDNA strand each, in the present investigation, we now provide independent evidence of Ptprz1-V7 mRNA expression in nine clones (three from cultures, two from pups, and four from adults), in which we sequenced both cDNA strands.