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Pax6 regulates specification of ventral neurone subtypes in the hindbrain by establishing progenitor domains

Department of Developmental Neurobiology, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan

View larger version (65K): [in a new window]   Fig. 1. Mis-specification of ventral neurones in the Pax6 mutant hindbrain. (A,B) Summary of detailed time-course analyses on expression of ventral neurone markers in the wild-type (A) and Pax6 homozygous (B) embryos. Five types of neurones develop in the ventral hindbrain: from ventral to dorsal, branchiomotor (BM) and somatic (SM) motoneurones, and V2, V1 and V0 interneurones, with distinct marker expressions. Pax6 is already expressed in the hindbrain at E10.5 (not shown here). During normal development, SM neurones expressing Islet2 and HB9/MNR2 emerge by E11.5, and expression of HB9/MNR2 and Islet2 is maintained at E13.5. In the Pax6 mutant, a small number of cells expressing Islet2 and HB9/MNR2 are transiently seen during E12.75-13.0 at r7 level. In the wild type, En1 expression is first detected at E12.0. In the Pax6 mutant, En1 is transiently expressed in a small number of cells at r3-6 levels of E13.0-13.25 embryo. (C-J) Expression patterns of ventral interneurone markers. Adjacent sections of E13.0 the wild-type (C-F) and Pax6 mutant (G-J) hindbrain at r5 level hybridised with Pax6 (C,G), Chx10 (D,H), En1 (E,I) and Evx1 (F,J) antisense probes. (C,G) Pax6 is expressed in the ventricular zone of the ventrolateral domain in both the wild-type and mutant neural tubes. (D,H) In the Pax6 mutant, Chx10-positive cells are located in a slightly wider region than those seen in the wild type, spreading more dorsally (see bracket in H). (E,I) In the wild type, En1-positive cells are detected in the region dorsal to Chx10 expression (arrow). (I) In the Pax6 mutant, En1-positive cells are undetectable at E12.5 (not shown here) (Ericson et al., 1997), a small number of En1-positive cells are observed in a similar dorsoventral position (arrow). (F,J) In the wild type, Evx1-positive cells are detected in the dorsally adjacent region (arrowhead in F). In the mutant, Evx1 expression seems to expand ventrally at r5 (arrowheads in J). (K-R) Expression of motoneurone markers in the hindbrain. Adjacent sections of E12.75 wild-type (K-N) and Pax6 mutant (O-R) hindbrain at r7 level. Distribution of Pax6 (K,O), Islet1/2 (L,P) and HB9/MNR2 (N,R) proteins, and Islet2 transcripts (M,Q). (K,O) In the wild type, Pax6 is observed in the ventricular zone of the ventrolateral domain of the neural tube (K). In the Pax6 mutant, immunoreaction for Pax6 is not detected with the antibody that recognises the C-terminal region of normal Pax6 protein (O). (L-N) During normal development, SM neurones are positive for Islet1, Islet2 and HB9/MNR2, while BM neurones (bracket in L) are negative for Islet2 and HB9/MNR2. Some of BM neurones are located dorsolaterally, as they migrate dorsally at this stage (arrowheads). (P-R) In the Pax6 mutant, Islet2- and HB9/MNR2-positive cells are missing at E12.5 (not shown here) (Osumi et al., 1997), but a small number of Islet2-positive and MNR2/HB9-positive cells are observed at E12.75 (arrows in Q,R). Inset in (R) shows higher magnification of HB9/MNR2-positive cells indicated by white arrows.

View larger version (81K): [in a new window]   Fig. 2. Expression patterns of homeodomain (HD) protein genes in the hindbrain. (A-J) Adjacent sections of E12.5 wild-type (A-E) and Pax6 mutant (F-J) hindbrains at r7 level. Double immunostaining for Nkx2.2 and Pax6 (A,F), and in situ hybridisation for Nkx6.1 (B,G), Irx3 (C,H), Dbx2 (D,I), and Dbx1 (E,J). (A,F) In the wild type, expression of Nkx2.2 is observed in the ventricular zone, and the expression domain is adjacent ventrally to the floor plate and dorsal to Pax6 domain (white arrowhead in A). In the Pax6 homozygous embryo, Nkx2.2 domain expands dorsally (white arrowhead in F). In the wild type, Pax6 protein expression is at a very low level in most of the ventral area (white bracket in A) from which SM neurones develop. Immunoreactivity for Pax6 is not detected in the Pax6 mutant (F). (B,G) In the wild type, Nkx6.1 is localised in the ventral domain excluding the floor plate and adjacent to the Dbx2 domain (double black arrowhead). In the Pax6 mutant, the dorsal boundary of Nkx6.1 expression slightly became blurred (double green arrowhead). Nkx6.1 expression is also seen in BM neurones in the mantle zone of both the wild type and Pax6 mutant (arrowheads in B,G). (C,H) In the wild type, Irx3 is expressed in the regions that overlap with Pax6, except the SM neurone precursor domain (bracket in C). In the Pax6 mutant, Irx3 domain expands ventrally into Nkx2.2 domain (red arrowhead). In the wild type, Irx3 transcripts are also detected in SM neurones (arrows in C), while such population is not seen in the Pax6 mutant (H). (D,I) In the wild type, the expression domain of Dbx2 is ventral to the Nkx6.1 domain (black double arrowhead), and also covers a more dorsal region than Dbx1 domain. In the Pax6 mutant, The ventral boundary of Dbx2 expression also becomes blurred (green double arrowhead in I). (E,J) In the wild type, expression of Dbx1 is confined to a narrow region within the ventrolateral neural tube. In the Pax6 mutant, Dbx1-positive cells decrease in number and expand ventrally (red arrowhead in J). (K,L) Illustration of the expression of HD protein genes and subtypes of ventral neurones in the hindbrain of the wild type (K) and Pax6 mutant (L). In the Pax6 mutant rat, expression domains of HD protein genes are shifted, the boundaries become blurred, and ventral neurones are mis-specified. A small population of V1 interneurones and SM neurones seem to be generated from narrow V1 and SM progenitor domains, respectively. Note that mis-specification profiles are different depending on to anteroposterior levels.

View larger version (84K): [in a new window]   Fig. 3. Strategy of Pax6 misexpression in cultured rat embryos by electroporation. (A) Experimental schedules of electroporation (EP) into cultured wild-type and Pax6 homozygous mutant embryos starting from early E11.5 (22-somite stage) and E10.75 (16-somite stage). Electroporated embryos were cultured up to the stage corresponding to E12.5. (B) The electroporated area is monitored by GFP at harvest. White line indicates r7 level. (C-H) Exogenous Pax6 was transfected into r7 of the wild-type embryo (C-E) and Pax6 mutant (F-H) with GFP expression vector at E11.5, and these embryos were cultured up to E12.5 stage. After detection of Shh transcripts by in situ hybridisation (E,H), GFP protein is detected on the same sections by the antibody (C,F). GFP is transfected on the right side. (D,G) Adjacent sections of C and F, respectively. (D) Exogenous Pax6 protein is detected on the right, and endogenous Pax6 protein is detected on both sides. (G) In the Pax6 mutant, only exogenous Pax6 protein is detected on the right side, consistent with GFP expression. (E,H) When Pax6 is transfected in the region excluding the floor plate, endogenous expression of Shh is unaffected in the wild type and Pax6 mutant.

View larger version (64K): [in a new window]   Fig. 4. Alteration of HD protein gene expressions by Pax6 misexpression. (A-F) pCAX-mPax6 was introduced into the hindbrain of wild-type embryos at 22-somite stage, and the embryo was cultured for 26 hours. Serial sections obtained from the same electroporated embryo. Exogenous Pax6 is detected in the right side of the hindbrain at a high level, while endogenous Pax6 is seen in both sides (A). Note repression of Nkx2.2 expression (bracket in B) and ectopic expression of Irx3, Dbx2 and Dbx1 induced in the area where pCAX-mPax6 is transferred (arrowheads in D-F). Expression of Nkx6.1 is not changed by Pax6 overexpression (C). (G-L) pCAX-mPax6 was introduced into the hindbrain of Pax6 mutant embryos at 22-somite stage, and the embryo was cultured for 30 hours (corresponding to E12.5) as neuronal differentiation is slightly delayed in the mutant. Serial sections obtained from the same electroporated embryo. Exogenous Pax6 is detected in the right side at a high level (G). Note that expression of Nkx2.2 is downregulated (bracket in H), and ectopic expression of Dbx2 and Dbx1 is observed in the electroporated area (arrowheads in K,L). Expression of Nkx6.1 seems unchanged by Pax6 electroporation into the mutant hindbrain (I). Induction of Irx3 is not detected, as Irx3 expression shifts ventrally in the Pax6 mutant hindbrain (J).

View larger version (96K): [in a new window]   Fig. 5. Induction of En1 and Islet2 at r7 level in the Pax6 mutant by exogenous Pax6. (A-E) pCAX-mPax6 was introduced into the hindbrain of wild-type embryos at the 22-somite stage, and the embryos were cultured for 26 hours (corresponding to E12.5). (A) The same photograph as in Fig. 4A showing distribution of Pax6 protein. (B-E) These sections are adjacent to those shown as Fig. 4A-F, which were obtained from the same embryo. Ectopic expression of Chx10 and En1 is not observed (C,D), while Islet2 expression is relatively downregulated (B). Ectopic expression of Evx1 is observed (arrowheads in E) in the region where Dbx1 is ectopically expressed (compare with Fig. 4F). (F-J) pCAX-mPax6 was introduced into the hindbrain of Pax6 mutant rat embryos at 22-somite stage, and the embryos were cultured for 30 hours (corresponding to E12.5), as neuronal differentiation is slightly delayed in the mutant. (F) The same photograph as in Fig. 4G showing Pax6 expression. (G-J) These sections are adjacent to those shown as Fig. 4G-L, which were obtained from the same embryo. Although Islet2-positive cells are undetectable in both unelectroporated and electroporated sides (G), expression of En1 is recovered in the area where pCAX-mPax6 was electroporated (arrowheads in I). (H) The number of Chx10-positive cells is relatively decreased but sometimes observed at an ectopic position (arrowhead). (J) Evx1-positive cells are observed not only in the normal position (arrow) but also in ectopic positions (arrowheads), where Dbx1 is ectopically expressed (compare with Fig. 6L). (K-P) pCAX-mPax6 was introduced into the hindbrain of Pax6 mutant rat embryos at E10.75, and the embryos were cultured for 42 hours (corresponding to E12.5). (L,N,P) Higher magnifications of K,M,O, respectively. By electroporation into E10.75 Pax6 mutant hindbrain, a small number of SM neurones expressing Islet2 (M, N) and HB9/MNR2 (O,P) emerge in the area where Pax6 is transfected (K,L).

View larger version (68K): [in a new window]   Fig. 6. Expression patterns of Shh signalling molecules in the Pax6 mutant hindbrain. Adjacent sections of E12.5 wild-type (A-F) and Pax6 mutant (G-L) hindbrains at r7 level. In the Pax6 mutant hindbrain, Shh expression in the floor plate is no different from the wild-type (A,G). (B-F) In the wild type, Ptc1 is expressed in all ventral progenitor cells with a ventral-to-dorsal gradient, and is especially strong in the SM progenitor domain (bracket in C). Ventral limits of Gli1 and Gli2 expression domains are adjacent to the dorsal limit of Nkx2.2 domain (black arrowhead in B,D,E). The expression is missing in the BM progenitor cells expressing Nkx2.2, while it is detected at a high level in the SM progenitor cells (bracket in D,E). The dorsal limits of Gli1 and Gli2 expression domains correspond to that of Dbx1 domain (red arrowhead in D-F). (H-L) In the Pax6 mutant, strong Ptc1 expression in the ventral region was not seen. Gli1 and Gli2 was expressed in the region between dorsal limits of Nkx2.2 and Dbx1 (green and red arrowheads, respectively, in Fig. 6H,J-L).

View larger version (63K): [in a new window]   Fig. 7. Cell death and cell proliferation in the Pax6 mutant hindbrain. Sections at r7 level of E12.5 wild-type (A) and Pax6 mutant (C), and cultured wild-type (B) and Pax6 mutant (D) embryos. (E,F) Comparison of TUNEL- (E) and BrdU- (F) positive cells per section within the ventral region, including the Dbx1 domain using 10 sections from three embryos. (E) There is no significant difference in the number of apoptotic cells between the two groups. (B,D) Pulse labelling of BrdU was performed for embryos cultured for 20 minutes and BrdU labelled cells were detected with anti-BrdU antibody. The ratio of BrdU labelled cells is slightly increased in the Pax6 mutant (t-test; *P<0.001).

View larger version (13K): [in a new window]   Fig. 8. The proposed model of Pax6 function in neuronal specification. The model indicates that Pax6 functions in establishment of progenitor domains in a correct manner, thereby regulating specification of neuronal subtypes. Shh, Sonic hedgehog.