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Allocation of Paraventricular and Supraoptic Neurons Requires Sim1 Function: A Role for a Sim1 Downstream Gene PlexinC1

Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21218

Address all correspondence and requests for reprints to: Chen-Ming Fan, Department of Embryology, Carnegie Institution of Washington, 3520 San Martin Drive, Baltimore, Maryland 21218. E-mail: fan{at}ciwemb.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
SIM1 is a transcription factor essential for the developmental expression of the endocrine hormone genes, e.g. vasopressin (Vp) and oxytocin (Ot), in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. Mice mutant for Sim1 lack structural PVN and SON, attributed in previous studies to the death of the PVN/SON progenitor cells. Here, we use a tau-LacZ knock-in allele at the Sim1 locus to trace Sim1 mutant cells and show that they are generated normally and survive to birth, contrasting to the previous proposal. Mutant cells adopt neuronal characteristics and maintain their PVN/SON identity as they continue to express PVN/SON progenitor markers. However, they occupy an ectopic position between the normal PVN and SON, indicating a defect in neuronal migration. To explore candidate molecular cues that contribute to PVN/SON neuronal migration, we focused on the Plexin family of genes. We found that PlexinA1 is expressed in regions surrounding the PVN and SON, whereas PlexinC1 is expressed within the PVN and SON. PlexinA1 expression becomes up-regulated in Sim1 mutant cells, whereas PlexinC1 expression is down-regulated. Finally, the PlexinC1 mutant has a selective defect in partitioning the VP and OT neurons coherently into the PVN and SON. Together, our results uncover a transcriptional regulation of neuronal migration cues initiated by Sim1 that contribute to the organization of the PVN and SON.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
THE PARAVENTRICULAR (PVN) and supraoptic (SON) nuclei of the anterior hypothalamus contain endocrine neurons that modulate pituitary secretion of various hormones (1, 2, 3). The PVN contains many types of neurons producing a diverse set of hormones, whereas the SON contains a limited set of hormone-producing neurons. Among them, oxytocin (OT)- and vasopressin (VP)-producing neurons are the only two common to both the PVN and SON. They are termed magnocellular neurons due to their large cell body. They project directly into the posterior pituitary where they release these two hormones into the blood stream. Endocrine neurons with small cell bodies are called parvocellular neurons, e.g. the TRH-producing neurons, and they project to the median eminence where they release hormones to be carried to the anterior pituitary to modulate secondary hormone secretion (reviewed in Refs. 1 and 4).

Anatomical studies and radioactive birth dating experiments of the rat hypothalamus have assigned the origin of the PVN and SON neurons to the anterior ventral lobes of the diencephalon (5). The ventricular region containing progenitors for the PVN and SON is easily identifiable due to its characteristic diamond shape (5). Most of the hypothalamic neurons organize into nuclei based on their birth dates, following an outside-in sequence. Formation of the PVN and SON is more complicated. The magnocellular neurons (i.e. VP and OT neurons) are born between embryonic d 10.5 and 12.5 (E10.5–E12.5) in the mouse (6, 7, 8). Thereafter, a portion of these neurons migrates to the lateral ventral surface just above the optic nerve to form the SON. The remaining magnocellular neurons stay near the ventricle to form the PVN together with later born parvocellular neurons. Thus, although born at the same location and time frame, VP and OT neurons are organized to form two distinct nuclei on opposite ends of the mediolateral axis. The molecular cues controlling their partitioning into distinct nuclei are under studied.

The genes encoding the transcription factors, Orthopedia (Otp) (9, 10), single-minded 1 (Sim1) (11), and aromatic-hydrocarbon receptor nuclear transporter 2 (Arnt2) (12, 13, 14) are essential for endocrine hormone gene expression of the PVN and SON neurons. Mice mutant for any of these three genes develops with neither a structural PVN or SON nor any associated hormone expression. The Otp gene, which encodes a homeodomain protein, also controls the proliferation, survival, and migration of the PVN/SON progenitors (9, 10). In two independently generated Otp mutants, the PVN/SON progenitor cells were marked by a knocked-in LacZ gene and followed for their destiny (9, 10). There was a drastic reduction in detectable LacZ-positive mutant cells, and these residual cells were found at neither the pia surface (the location of the SON) nor the periventricular area (the location of the PVN), but instead were found at a lateral position (9, 10). By contrast, the roles of Sim1 and Arnt2, both of which encode basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) domain proteins and act as dimer partners, in proliferation, migration, and neurogenesis of PVN/SON progenitors remain unclear because their original mutant alleles did not carry reporter genes for lineage tracing (11, 12, 13, 14).

To follow the axonal projections of Sim1-positive neurons from the mammillary body to the thalamus, a tau-LacZ knock-in allele at the Sim1 locus was created (Sim1-tLZ) (15). Using this allele, we have analyzed the properties of LacZ-positive Sim1 mutant cells at the PVN/SON progenitor region. In contrast to the previous proposal that the Sim1 mutant presumptive PVN/SON cells die after E14.5 (11), we found that they persist but occupy a position midway between the normal PVN and SON. This mislocalized pattern of Sim1 mutant cells is distinct from that reported for Otp mutant cells, indicating that Sim1 and Otp do not regulate the same set of migration cues to direct PVN/SON nuclear formation. To explore candidate genes acting downstream of Sim1 to control cell migration, we focused on the Plexin and Neuropilin families of genes, which are well known to mediate cell migration and axon guidance in the nervous system. We first documented the expression patterns of selective members of the Plexin and Neuropilin families (16) at the PVN/SON progenitor region. Among them, PlexinA1 and PlexinC1 expression is regulated by Sim1. We then showed that PlexinC1 mutants display a selective defect in the allocation of the magnocellular neurons.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Sim1 Mutant Cells Are Born Normally
The Sim1-tLZ allele was first described for tracing the mammillothalamic track initiated from Sim1-positive neurons in the mammillary body (15). Because the tau-LacZ cassette has a poly-A signal sequence and replaces the first exon of Sim1 that encodes the DNA binding and dimerization domains, it likely creates a null allele. Using X-gal staining to assess the LacZ reporter activity from the Sim1-tLZ allele in the heterozygote, we found that the stained domain is similar to the Sim1 expression domain assessed by in situ hybridization (ISH) in the E12.5 PVN/SON progenitor region (Fig. 1, compare A and B). Using adjacent sections of Sim1-tLZ heterozygous PVN/SON progenitor region at E12.5 to compare Sim1 (Fig. 1C) and LacZ (Fig. 1D) expression by ISH, we found that LacZ expression recapitulates endogenous Sim1 expression. We therefore used this allele to follow Sim1 mutant cells in the PVN/SON progenitor region. All data presented here used the Sim1-tLZ allele, and for simplicity it is referred to as Sim1^–.

View larger version (59K): [in this window] [in a new window]   Fig. 1. Sim1 Mutant PVN/SON Progenitors Have No Proliferation Defects A and B, Sim1 ISH (A) and X-gal staining (B) of Sim1-tLZ heterozygotes (Sim1+/–) at E12.5 PVN/SON progenitor region; coronal plane. C and D, Sim1 (C) and LacZ ISH (D) on adjacent sections of E12.5 Sim1+/– PVN/SON progenitor region. E–H, BrdU labeling and LacZ ISH of E10.5 (E and F) and E13.5 (G and H) PVN/SON regions of Sim1+/– (E and G) and Sim1–/– (F and H) brains. I, Tabulation of all BrdU (brown) and LacZ (blue) doubly positive cells from Sim1+/– (white bars) and Sim1–/– (black bars) PVN/SON regions at stages indicated at bottom; n = 3 for each stage, and both sides of each brain sample were counted; error bar = 1 SD. White dashed lines outline the positive signals for ISH and X-gal staining; blue arrows, BrdU and LacZ ISH overlapping regions. Scale bars,420 µm, A and B; 100 µm, C and D; 80 µm, E and F; and 110 µm, G and H.

Sim1 Mutant Cells Do Not Undergo Excessive Programmed Cell Death (PCD)
The gradual loss of Sim1 mutant transcripts as the developmental time course progresses has led to the proposal that Sim1 mutant cells fail to survive (11, 17). To test this, we performed terminal deoxy-UTP nick end labeling (TUNEL) assays to detect PCD between E10.5 and E16.5 at daily intervals. We used serial adjacent sections to first identify LacZ-positive domains in the heterozygous control and mutant (data not shown) and then used the immediate adjacent sections to perform the TUNEL assay. From E10.5 to E13.5, no apoptotic cells were found in the progenitor region of control or mutant embryos, although apoptotic cells were found in other regions on the same sections (data not shown). Between E14.5 and E16.5, there were observable apoptotic cells in the control PVN (Fig. 2, A, C, and E), but not in the lateral region or in the SON. By contrast, from E14.5 to E16.5 in the mutant, we did not find increased apoptotic cells in the presumptive PVN area (LacZ-positive domain) (Fig. 2, B, D, and F) compared with the control. When tabulated, there were less apoptotic cells in the LacZ-positive mutant cell domain (Fig. 2G). This result led us to conclude that Sim1 mutant cells are less likely to undergo PCD than wild-type cells during the time of PVN/SON progenitor generation and nucleus formation.

View larger version (42K): [in this window] [in a new window]   Fig. 2. The Sim1 Mutant PVN Region Does Not Have Increased PCD A–F, TUNEL assays to assess PCD in the PVN/SON area of Sim1+/– (A, C, and E) and Sim1–/– (B, D, and F) brains at stages indicated; white dashed lines outline the LacZ-positive regions in adjacent sections (data not shown); TG (white dotted lines), trigeminal ganglia used as a positive reference for PCD assay. G, Tabulation of all TUNEL-positive cells from Sim1+/– (white bars) PVN and Sim1–/– (black bars) presumptive PVN regions (determined by LacZ ISH on adjacent sections) at stages indicated; n = 3 for each stage, and both sides of each brain sample were counted; error bar = 1 SD. Scale bars, 200 µm.

At E10.5, the LacZ-marked heterozygous and mutant PVN/SON progenitor domains were small patches next to the ventricle with no discernible difference (Fig. 1, E and F). The difference between the shape of LacZ expression domains between heterozygotes and mutants was first noticeable but subtle at E12.5 (Fig. 3, compare A and B). At E13.5, when the separation of the SON first becomes detectable in the heterozygote, we noted that mutant cells were never found at the lateral pia surface (Fig. 3, C and D). At E14.5 when the normal PVN and SON neurons are clearly discernable as two groups (Fig. 3E), the mutant cells were less coherent and stayed as a scattered group of cells between the normal PVN and SON positions (Fig. 3F). After E14.5, mutant cells remained mislocalized (Fig. 3H). Even at postnatal day 0 (P0), no mutant cells were found at the normal PVN or SON position (Fig. 3J). Similar results have been reported recently (17). We noted that the nucleus of the lateral olfactory track (NLOT) does not form in the Sim1 mutant, a defect that appears to stem from a deficiency of NLOT progenitor cells as early as E12.5, before their migration.

View larger version (87K): [in this window] [in a new window]   Fig. 3. Sim1 Mutant Cells Have a Migration Defect A–J, LacZ ISH was performed to compare the distribution pattern of LacZ-positive cells in Sim1+/– (A, C, E, G, I) and Sim1–/– (B, D, F, H, J) brains at the PVN-SON level; embryonic stages are labeled at the left; asterisks in panels A and B indicate zona limitan (a reference site) expression not changed; brackets in C and D indicate LacZ-positive domain; brackets in F, H, and J indicate scattered mutant LacZ-positive populations compared with the normal PVN (blue arrows) and SON (white arrowheads) in the heterozygote (E, G, I). Black arrowheads indicate the presumed progenitors for NLOT and mature NLOT present in the heterozygote but not in the mutant.

Sim1 Mutant Cells Adopt Neuronal Characteristics
Because Sim1 mutant cells survive, it allows examination of their cellular properties. One possible explanation for the failure of the mutant cell to express endocrine hormone genes is that they fail to differentiate into neurons. To test this, we used the Tuj1 monoclonal antibody, which detects a neuronal specific isoform of ß-tubulin (18), together with an anti-LacZ antibody to assess the neuronal properties of Sim1 heterozygous (Fig. 4, A–C) and mutant cells (Fig. 4, D–F). As early as E12.5, mutant cells express the Tuj1 epitope, suggesting that they do adopt a neuronal fate. Of note, in the Sim1 heterozygote, Tuj1 level is relatively even in the LacZ domain, but in the mutant Tuj1 level is weaker at the ventral-medial subportion of the LacZ domain, suggesting that the lack of Sim1 function delays or compromises the neuronal differentiation of a subset of cells. Further analysis using the neuronal nuclei (NeuN) antibody, which detects the nuclei of neurons but not glia (19), revealed that the LacZ-positive mutant cells are NeuN-positive at P0 (Fig. 4, G–I), further indicating that Sim1 mutant cells do differentiate along the neurogenic pathway.

View larger version (73K): [in this window] [in a new window]   Fig. 4. Sim1 Mutant Presumptive PVN/SON Cells Do Become Neurons A, D, and G, Anti-LacZ IF (green). B and E, Tuj1 staining (red) of the same sections as in A and D. H, NeuN staining (red) on the same section as in G. C is an overlay image of A and B; F is an overlay of D and E; I is an overlay of G and H plus DAPI (blue) to identify nuclei. Stages and genotypes are labeled at the left. G–I is a region where the mutant cells are identified (based on LacZ staining) lateral to the normal PVN at P0. Scale bars, 80 µm, A–F; 7 µm, G–I.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Sim1 Mutant Cells Do Not Appear to Change Fate Expression of Otp (B, D, F, and H) and ARNT2 (J and L) are assessed by ISH and IF, compared with those of LacZ ISH (A, C, E, and G) and LacZ antibody staining (I and K). Scale bars, 100 µm, A–D; 130 µm, E–H; and 200 µm, I–L.

View larger version (135K): [in this window] [in a new window]   Fig. 6. Sim1 Mutant Hypothalamus Does Not Have a Global Change of Organization ISH was performed to compare LacZ (A–D), Dlx1 (E–H), and Six3 (I–L) expression in Sim1+/– and Sim1–/– at E12.5 and E15.5 PVN/SON areas on consecutive adjacent sections; coronal plane. Genotypes and embryonic stages are labeled on top, and probes used are labeled at left. Black dashed lines indicate the boundaries of Dlx1 and Six3 expression; circled areas indicate regions where Six3 expression domain expanded dorsally. Scale bars, 100 µm, A, B, E, F, I, and J; and 130 µm, C, D, G, H, K, and L.

Because the Sim1 mutant neuronal migration defect is largely restricted to LacZ-expressing cells, we narrowed our search to genes encoding receptors that are expressed in the PVN/SON progenitor because they are more likely to act cell-autonomously. We examined the Plexin and Neuropilin families of cell migration/axon guidance repellent receptors (reviewed in Refs. 16 and 22, 23, 24) for their expression patterns in the anterior hypothalamus. Listed in Table 1 are the gene members of each family that we examined for expression at E12.5, when we first detect a difference in PVN/SON progenitor cells in the wild-type vs. the Sim1 mutant. We also perform the same analysis at E14.5 or E15.5, when PVN and SON are clearly discernable. Using adjacent sections to detect Sim1 transcript for reference, we found four genes displaying patterns of expression in or around the developing PVN/SON. Below are descriptions of their expression pattern and regulation by Sim1:

2. Of the six Plexin genes examined (25, 26), we found that only PlexinA1 and PlexinC1 are expressed in the region of interest. PlexinA1 is detected generally in the anterior hypothalamus at E12.5 and is highly up-regulated in the LacZ- domain of the Sim1 heterozygote (Fig. 7, A and D). Curiously, its expression was down-regulated in the LacZ-positive domain as PVN/SON neuronal migration ensues (Fig. 7, B, C, E, and F). In the Sim1 mutant (Fig. 7, J–L), however, PlexinA1 is not down-regulated in the mutant cell population in adjacent sections (Fig. 7, N and O). These data suggest that down-regulation of PlexinA1 in Sim1-positive (inferred from LacZ expression) cells plays a role in their allocation to correct positions. PlexinC1 is also generally expressed in the anterior hypothalamus at a low level but up-regulated in the LacZ-positive domain of the Sim1 heterozygote at E12.5 (Fig. 7G). In contrast to PlexinA1, high levels of PlexinC1 expression persist to the mature PVN and SON up to E15.5 (Fig. 7, H and I). Because PlexinC1 expression is found in the entire SON and in scattered cells in the PVN, we reason that it is expressed in the magnocellular neurons. Importantly, PlexinC1 expression is not present at a high level in the Sim1 mutant cell domain marked by LacZ expression in the adjacent section (Fig. 7, P–R), suggesting that its expression is up-regulated by Sim1 and that it plays a positive role in allocating magnocellular neurons to the PVN and SON. Below we focus on the hypothalamic phenotype of the PlexinC1 mutant.

View larger version (83K): [in this window] [in a new window]   Fig. 7. PlexinA1 and PlexinC1 Expression in the PVN/SON Region Is Regulated by Sim1 ISH was performed to compare LacZ (A–C and J–L), PlexinA1 (D–F and M–O), and PlexinC1 (G–I and P–R) expression in Sim1+/– (A–I) and Sim1–/– (J–R) PVN/SON areas on consecutive adjacent sections of coronal plane. Genotypes are labeled at left and embryonic stages on top; blue arrows, PVN/SON progenitor and mature PVN; open blue arrowheads, SON; red arrowheads, strong PlexinA1 expression domains; red dashed lines, PlexinA1 down-regulated domain; blue brackets, mislocalized LacZ-positive mutant cells; open red arrows, PlexinA1 domain not as down-regulated in the mutant as in the heterozygotes; green arrows, strong PlexinC1 domain in PVN/SON progenitor and mature PVN; green arrowheads, strong PlexinC1 domain in the SON; open green arrows and arrowheads, a lack of strong PlexinC1 expression in the mutant LacZ-positive domains. Scale bars, 100 µm, A, D, G, J, M, and P; 110 µm, B, E, H, K, N, and Q; and 130 µm, C, F, I, L, O, and R.

View larger version (107K): [in this window] [in a new window]   Fig. 8. The PlexinC1 Mutant Has An Increased Number of OT and VP Neurons Scattered between the PVN and SON A and B, Ot; C and D, Vp; E and F, Sim1; ISH of PlexinC1+/– (A, C, and E) and PlexinC1–/– (B, D, and F) P2 brains at the PVN/SON level; coronal plane. G–J, VP IF of the adult brains (G and H) and pituitaries (I and J) of PlexcinC1+/– (G and I) and PlexinC1–/– (H and J) mice; coronal plane. K and L, Sim1; M and N, PlexinC1; ISH of PlexcinC1+/– (K and M) and PlexinC1–/– (L and N) E15.5 brains at SON and NLOT level; coronal plane. Black and white dashed circles indicate Sim1-expressing cells and VP and OT neurons scattered between the PVN and SON. Thin dashed lines in I and J outline the posterior pituitary. Black arrowheads indicate NLOT, whereas white arrowheads point to the position of SON in K–N. Scale bars, 150 µm, A–F and K–N; 180 µm, G–J.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Here we show that the presumptive PVN/SON progenitor cells of Sim1 mutant mice fail to localize to their normal positions. We have identified PlexinC1 as a downstream gene of Sim1 and partially responsible for the coherent localization of magnocellular neurons to the PVN and SON. Combining with previously published data implicating Netrin and DCC in this process (21), it appears that PVN/SON nuclei formation and maintenance require multiple steps mediated by distinctive cues.

Sim1 Mutant Cells Are Born and Differentiate into Neurons
Here we present data indicating that Sim1 is required neither for the generation of PVN/SON progenitors nor for their survival. A recent report using long-term labeling from E10.5 to E12.5 in the Sim1 mutant concluded that a reduction of BrdU-positive cells in the mutant compared with the wild type (17). The authors proposed that this was a result of excessive cell death between E10.5 and E12.5 in the mutant, but they did not evaluate PCD directly. Our PCD data in the Sim1 mutant hypothalamus argue against their proposal. The survival of Sim1 mutant cells is further supported by our data as well as their data that LacZ-positive mutant cells are abundantly detected at P0. We did note that Sim1 mutant cells migrate to different regions and are found in more coronal sections along the anterior-posterior axis. We suspect that Caqueret et al. (17) might have only counted BrdU-positive cells in a limited number of sections rather than the sum of total BrdU-LacZ doubly positive cells as reported here, hence the discrepancy.

Otp and Sim1 have been suggested to act in parallel for the terminal differentiation of neurons in the PVN and SON (9, 10). Like that reported for Otp mutants (9, 10), our data also indicate no increased PCD in Sim1 mutants. Different from the Sim1 mutant is that there is a severely reduced LacZ-expression (from the Otp locus) level and domain size in Otp mutants when compared with those in Otp heterozygotes at P0 (9, 10). One study concluded that there is a reduction in short-term BrdU-labeled cells in the Otp mutant (10), whereas the other concluded that mutant cells do die but not by PCD (9). Another possible explanation is that Otp autoregulates its own expression, which causes a failure to maintain high levels of knock-in LacZ gene expression in the mutant, hence an appearance of missing mutant cells. Whatever the mechanism may be for reduced LacZ-positive cells in Otp mutants, it appears that Sim1 and Otp do not act in a completely parallel manner.

Is There a Change of Fate for Sim1 Mutant Cells?
We have observed that the LacZ-positive Sim1 mutant presumptive PVN/SON cells adopt neuronal characteristics. In addition, Sim1 mutant cells maintain Sim1 promoter activity (reflected by LacZ expression), as well as Otp and Arnt2 expression (9, 17). Thus, the mutant cells appear to retain the identity of their original fate. Although we cannot exclude the possibility that these mutant cells have adopted another hypothalamic neuronal fate that is marked by the coexpression of all three genes, we propose that these mutant cells are more likely to be arrested at a stage before hormone gene expression.

Are SON and PVN Formed by a Passive or Active Program?
Using the LacZ reporter to trace Sim1 mutant presumptive PVN/SON cells, we show that the Sim1 mutant cells are mislocalized. Normally, within the PVN/SON progenitor region, the magnocellular neurons are born the earliest between E10.5 and E12.5 and are found at the pia surface as early as E13.5 (6, 7, 8). It is not clear whether their arrival at the SON is a passive event due to temporally controlled radial migration (1, 5). Our data would suggest that there is an active program involved in their migration, because Sim1 mutant cells are born and become neurons early but they neither colonize the pia surface nor congregate at the normal PVN position. These Sim1 mutant neurons scatter along the mediolateral axis, with some near the ventricle and some near the pia surface, distinct from the mostly lateral location documented for the Otp mutant cells (9, 10). This may reflect another difference in the function of Otp and Sim1.

The Roles of PlexinA1 and PlexinC1 in PVN/SON Neuronal Migration
Our data indicate that PlexinC1 is positively regulated by a Sim1-directed program in the progenitor and the mature PVN and SON. PlexinA1, on the other hand, is first expressed in the PVN/SON progenitor and becomes down-regulated in a subdomain of the future PVN and the future SON just when these neurons start to segregate (at E13.5). Because PlexinA1 down-regulation does not occur in the Sim1 mutant cells, we presume it to be negatively regulated by Sim1 in a temporally controlled manner. The regulation of PlexinC1 and PlexinA1 by Sim1 may be direct because SIM1 can act as an activator or a repressor in different contexts (28, 29, 30). They are presumably also regulated by Arnt2 because ARNT2 is a dimer partner of SIM1 (12, 13, 14). Alternatively, they may be differentially regulated by downstream effectors of SIM1/ARNT2. One primary suspect for such an effector is the transcription factor BRN2, which is essential for PVN/SON development (31, 32).

We further show that PlexinC1 mutant mice have a population of VP and OT neurons scattered between the PVN and SON. Because PlexinC1 is a receptor-like molecule, it is likely to be directly involved in receiving cues from a ligand to help allocate the magnocellular neurons to their final locations. We propose that this cue is a repellent expressed between the PVN and SON, thereby preventing magnocellular neurons from occupying this intermediate position. Curiously, except for a virally coded Semaphorin A39R (33, 34), there is yet no endogenous Semaphorin ligand definitively identified for PlexinC1 (27, 35). Our data indicate that such a ligand does exist, but whether it is a Semaphorin family member remains to be determined.

Because Sim1 controls multiple aspects of PVN/SON development and the Sim1 mutant is practically a Sim1;PlexinC1 double mutant for this neuronal lineage, it is expected that the migration defect of the Sim1 mutant will be more severe than that of the PlexinC1 mutant. The change of PlexinA1 expression in the Sim1 mutant may contribute to this more severe defect. The temporal switch of PlexinA1 expression from "on" at E12.5 to "off" at E13.5 in the PVN/SON progenitor region may indeed be important for controlling the timing of magnocellular neuron migration. Because it is normally down-regulated in the PVN and SON, a PlexinA1 mutant may not display a defect there. Aberrant up-regulation of PlexinA1 (as in the Sim1 mutant), on the other hand, may cause a migration defect.

Complexity of the Genetic Program Controlling PVN and SON Formation
Before this work, the only neuronal guidance molecule with an expression preference for the SON but not in the PVN is Netrin (21). Its receptor DCC is expressed in cells surrounding the SON (21). In either single mutant, the SON is poorly organized, and VP and OT neurons are scattered anteriorly along the optic track to reach the chiasm (21). Because the affected OT and VP neurons normally express the ligand (Netrin) instead of the receptor (DCC), there is no straightforward explanation for their mutant phenotype. In fact, the phenotype was suggested to be a secondary consequence of Netrin or DCC mutation (21). It is also not clear why they become dispersed along the anterior-posterior axis. Their mutant phenotype is distinct from that of the PlexinC1 mutant (OT and VP neurons dispersed along the medial-lateral axis), suggesting that for magnocellular neurons to reach and stay at their final location, multiple cues are required along different axes. Given the importance of the hypothalamic neuroendocrine system, it is surprising that so little is known about the molecules directing their migration. We hope that from this work forward, efforts will be directed toward the eventual discovery of the complete set of molecular cues orchestrating the neuronal migration of the hypothalamic neuroendocrine system.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Experimental Animals
The Sim1-tLZ knock-in mice (15) and the PlexinC1 knock-out mice (27) were maintained in the 129sv/C57B6 mixed background. Embryos and postnatal animals used in this study were progenies from sibling crosses between 18 and 23 generations. All animal experimentation described herein was conducted in accordance with accepted standards of humane animal care as outlined in ethical guidelines.

Sim1 and PlexinC1 Mutant Mice and Embryos
Heterozygous animals were bred to produce mutants. Tail or embryonic sac DNA was genotyped by PCR as described (15, 27). The vaginal plug date is designated to be E0.5, and the newborn is defined as P0.

ISH
Whole embryo heads (before E16.5) or dissected brains (after E16.5) were fixed in freshly made methancarn (60% methanol, 30% chloroform, and 10% acetic acid), dehydrated in methanol, embedded in paraffin, then sectioned at 10-µm thickness. The anatomical assignment and nomenclatures for the hypothalamus are based on the atlas by Altman and Bayer (36). ISH followed a standard protocol (37) using digoxygenin-labeled antisense RNA probes generated by T7, T3, or SP6 RNA polymerases using templates containing cDNA fragments of specified genes. The Sim1 probe was published (11). The LacZ probe was a gift from Dr. D. Epstein; Otp probe was a gift from Dr. A. Simeone; probes for the Neuropilin and Plexin families of genes were gifts from Drs. J. Rubenstein and A. Kolodkin or were generated by RT-PCR. All probes were sequenced for confirmation to assign the corresponding genes before use. Alkaline phosphatase conjugated antidigoxygenin antibodies (Roche, Nutley, NJ) were used to detect hybridized probes, followed by color reaction using the NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) (Roche) substrates to reveal signals. The images were taken using the Zeiss Axiocam digital camera (Carl Zeiss, Thornwood, NY) under either a dissection or a compound microscope.

BrdU Labeling and TUNEL Assays
For BrdU (Sigma, St. Louis, MO) labeling, 50 µg BrdU/g body weight was injected into the peritoneal cavity of pregnant females. One hour after injection, embryos were collected, fixed, and sectioned as described above. Sections were processed for ISH first, followed by BrdU immunohistochemistry staining according to the user’s manual of the kit (Zymed Laboratories Inc., South San Francisco, CA). For TUNEL assay, samples were similarly processed for detection using the Fluorescein In Situ Cell Death Detection Kit (Roche). Pictures were taken with a Leica confocal microscope (Leica, Solms, Germany). In both BrdU and TUNEL assays, three animals were used for quantification for each stage and genotype analyzed. Cell counts were averaged, and SE values were provided. Statistical analysis was performed with Student’s t test.

Immunofluorescence (IF)
IF was performed on paraffin sections except for adult brains. Adult mice were perfused with 4% paraformaldehyde in PBS, and brains were dissected out for further 4% paraformaldehyde fixation overnight and embedded in O.C.T. (Sakura Finetek, Torrance, CA), cryosectioned at 40 µm. Antibodies were diluted in PBS with 10% heat-inactivated sheep serum to specified final concentrations: rabbit anti-ß-galactosidase antibody (MP Biomedicals, Solon, OH), 1:100; rabbit anti-ARNT2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1:200; neuronal class III ß-tubulin (Tuj1) monoclonal antibody (Covance Research Products, Berkeley, CA), 1:500; NeuN monoclonal antibody (Chemicon International, Temecula, CA), 1:100; VP and OT monoclonal antibody (American Tissue Culture Collection, Rockville, MD), 1:10 of supernatant. All IFs were detected with Alex488- and/or Alex594- (Molecular Probes, Eugene, OR) conjugated fluorescent secondary antibody, and pictures were taken with a Leica confocal microscope.

	ACKNOWLEDGMENTS

 
We thank Drs. A. Kolodkin and J. Rubenstein for the plasmids to generate Plexin and Neuropilin probes. We thank Drs. Kolodkin and D. Ginty for sharing Np1, Np2, and PlexinC1 mutant brains for analysis. We also thank Fan laboratory members for critical readings of the manuscript.

	FOOTNOTES

 
This work was supported by National Institutes of Health Grant RO1 HD35596 (to C.-M.F.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 13, 2007

Abbreviations: Arnt2, Aromatic-hydrocarbon receptor nuclear transporter 2; BrdU, bromodeoxyuridine; E, embryonic day; IF, immunofluorescence; ISH, in situ hybridization; NeuN, neuronal nuclei; NLOT, nucleus of the lateral olfactory track; OT, oxytocin; Otp, Orthopedia; P, postnatal day; PCD, programmed cell death; PVN, paraventricular nucleus; Sim1, single-minded 1; SON, supraoptic nucleus; TUNEL, terminal deoxy-UTP nick end labeling; VP, vasopressin.

Received for publication January 18, 2007. Accepted for publication March 7, 2007.

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