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Molecular Endocrinology, doi:10.1210/me.2003-0372
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Molecular Endocrinology 18 (5): 1251-1262
Copyright © 2004 by The Endocrine Society

Sim2 Contributes to Neuroendocrine Hormone Gene Expression in the Anterior Hypothalamus

Eleni Goshu, Hui Jin, John Lovejoy, Jean-François Marion, Jacques L. Michaud and Chen-Ming Fan

Department of Embryology (E.G., H.J., J.L., C.-M.F.), Carnegie Institution of Washington, Baltimore, Maryland 21210; and Research Center (J.-F.M., J.L.M.), Hôpital Sainte-Justine, Montréal, Québec, Canada H3T 1C5

Address all correspondence and requests for reprints to: Chen-Ming Fan, Department of Embryology, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, Maryland. E-mail: fan{at}ciwemb.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Paraventricular (PVN) and supraoptic nuclei of the hypothalamus maintain homeostasis by modulating pituitary hormonal output. PVN and supraoptic nuclei contain five major cell types: oxytocin-, vasopressin-, CRH-, somatostatin-, and TRH-secreting neurons. Sim1, Arnt2, and Otp genes are essential for terminal differentiation of these neurons. One of their common downstream genes, Brn2, is necessary for oxytocin, vasopressin, and CRH cell differentiation. Here we show that Sim2, a paralog of Sim1, contributes to the expression of Trh and Ss genes in the dorsal preoptic area, anterior-periventricular nucleus, and PVN. Sim2 expression overlaps with Trh- and Ss-expressing cells, and Sim2 mutants contain reduced numbers of Trh and Ss cells. Genetically, Sim1 acts upstream of Sim2 and partially compensates for the loss of Sim2. Comparative expression studies at the anterior hypothalamus at early stages reveal that there are separate pools of Trh cells with distinctive molecular codes defined by Sim1 and Sim2 expression. Together with previous reports, our results demonstrate that Sim1 and Otp utilize two common downstream genes, Brn2 and Sim2, to mediate distinctive sets of neuroendocrine hormone gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE NEUROENDOCRINE HYPOTHALAMUS integrates central and peripheral signals to modulate secretion by the pituitary of peptidic hormones involved in homeostasis. Many of the neuropeptides secreted by the hypothalamus have been identified (reviewed in Ref.1). They are expressed in two types of neurons: the magnocellular and the parvocellular neurons (reviewed in Refs.2, 3, 4). The magnocellular neurons project to the posterior pituitary where they release vasopressin (VP) and oxytocin (OT) into the general circulation. They are located within the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. The parvocellular neurons project to the medial eminence where they release hormones that are carried to the anterior pituitary by the portal vasculature. Three distinctive parvocellular cell types have been fully characterized: CRH and TRH cells in the PVN, and the somatostatin (SS) cells in the anterior periventricular (aPV) nucleus, which is contiguous with the PVN. These hypophysiotropic peptides modulate the secretion of pituitary hormones including ACTH, TSH, and GH.

Expression studies have determined that VP, OT, CRH, TRH, and SS-positive neurons of the aPV/PVN/SON appear at different times between embryonic d 12.5 (E12.5)–E18.5 in the mouse (4, 5, 6, 7, 8, 9, 10). Transcription factors involved in directing hormone gene expression of these neurons during development have just begun to be uncovered. Recent molecular studies in the mouse indicated that as early as E10.5, transcription factors important for terminal differentiation of these cell types are specifically expressed in an area previously proposed for neuroendocrine progenitors (11). They include Otp (encoding a homeodomain protein), Sim1, and Arnt2 (both encoding basic-helix-loop-helix-per-arnt-sim proteins) (12, 13, 14, 15, 16, 17). Inactivation of any of these genes (12, 13, 14, 15, 16, 17) results in the elimination of PVN and SON structures and neuroendocrine hormone expression. One of their common downstream genes, Brn2 (encoding a POU-homeodomain protein), appears necessary only for differentiation of a subset of cell types, i.e. the CRH, OT, and VP neurons (18, 19). Consistently, Brn2 is mainly expressed at the mid-to-posterior PVN and SON, where the CRH, VP, and OT cells reside (12). In contrast, genes that act downstream of Otp, Sim1, and Arnt2 to direct Ss and Trh expression in the aPV and anterior PVN, respectively, remain elusive.

Sim2 is a paralog of Sim1 (20, 21, 22, 23, 24, 25). It is expressed in the PVN but not in the SON (15, 26). Here we describe the molecular analysis of the Sim2 mutant and demonstrate a role of Sim2 for proficient Trh and Ss expression in the preoptic area, aPV, and PVN.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sim2 Is Expressed in the Preoptic Area, aPV, and PVN
Sim2 was documented to be expressed in the PVN (15, 26), but a more detailed description is lacking. We therefore performed in situ hybridization (ISH) on postnatal d 0 (P0) brains by coronal and sagittal sections using a Sim2 antisense probe with a focus on the anterior hypothalamus (Fig. 1Go, A–D'). Along the rostro-caudal axis, Sim2 expression starts at the posterior preoptic area, primarily occupying the dorsal area (referred as the dP for dorsal preoptic area). Sim2 expression continues into the anterior and mid-PVN, including the aPV. Toward the posterior PVN, Sim2 expression diminishes. We next compared the expression of Sim2 to that of Sim1 and Brn2, the former being expressed throughout the PVN with the latter primarily detected in the mid- to posterior PVN (12). Using coronal (see below) and midsagittal sections (Fig. 1Go, E–G), we found that Sim2 expression was indeed more anteriorly localized than Brn2 expression and that the Brn2 and Sim2 expression domains together appeared to overlap with the Sim1 domain. This result suggests a function for Sim2 in the anterior Sim1 domain.



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  		Fig. 1. Comparison of Sim1, Sim2, and Brn2 Gene Expression in the Newborn Hypothalamus

A–D, Coronal sections of P0 brains around the anterior hypothalamus area, after an anterior (A) to posterior (D) sequence. Sections were subjected to ISH using 35S-UTP-labeled probes. A'–D' are overlaid images of the dark field (to visualize the silver granule deposits resulting from the hybridized radioactive probe) photos of Sim2 expression taken with a red filter (hence the pink granules) and the bright-field photos of A–D taken under a blue filter (see Materials and Methods). The dorsal preoptic area (dP), anterior PVN (aPVN), aPV, mid-PVN (mPVN), posterior PVN (pPVN), optic nerve (opn), and optic chiasma (oc) are as labeled. White dashed lines mark the histological area of the labeled nuclei on the left side of the brain. An asterisk indicates the fornix, and white dots outline the suprachiasmatic nucleus (SCN). E is the histological image of E', a midsagittal section of a P0 brain hybridized to Sim2. F is an immediate adjacent section of E hybridized to Brn2. G is a midsagittal section of a similar plane from a sibling’s brain hybridized to Sim1. E', F, and G are overlaid images of dark-field/red filter and bright-field/blue filter photos as in A'–D'. In E–G, the anterior is at left and the posterior is at right. The anatomical assignments are based on histology (42 ) and other brain reference structures in nearby sections (data not shown). The lateral ventricle (black arrow), third ventricle (open arrow), anterior commissure (ac), oc (bracketed), and SCN (white dots) are as labeled. The ac helps define the anterior limit of the hypothalamus, whereas the SCN approximates the anterior and posterior limits of the PVN.

 
Sim2 Expression Domain Correlates with that of Ss- and Trh-Expressing Cells
To correlate Sim2 expression to hormone-expressing neurons, we performed ISH on P0 hypothalamus using adjacent coronal sections hybridized to the Sim2 probe and the probe of each of the major hormone markers, Trh, Ss, Crh, Vp, and Ot. In the dP, Trh cells are found in the Sim2 domain (Fig. 2Go, A and A'). Within the anterior (Fig. 2Go, C and C') to mid-PVN (data not shown), Sim2 and Trh expression is also found. At the aPV (Fig. 2Go, B and B'), Ss cells are found in a tight cluster in the Sim2 expression area. There are other dorsolaterally located Ss cells but only the aPV population overlaps with Sim2 expression. Sim2 expression at the midposterior PVN is medial and does not correlate with the expression of Crh (Fig. 2Go, D and D'), Vp, or Ot (data not shown). At the most posterior PVN, Crh (data not shown), Vp, and Ot neurons are present in abundance (Fig. 2Go, E' and F'), whereas Sim2 expression is not detectable (Fig. 2Go, E and F).



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  		Fig. 2. Sim2 Expression in the Newborn Brain Is Similar to that of the Trh and Ss in the dP, aPV, and Anterior to Mid-PVN

Sim2 expression was examined by coronal sections and compared with Trh, Ss, Crh, Vp, and Ot expression as pairs by ISH on adjacent sections throughout the P0 hypothalamus. Selected sections for each comparison are shown. The corresponding anterior-posterior levels of the sections are labeled at left. Images are composites of blue filter/bright-field and red filter/dark-field photos as in Fig. 1Go. A–F are Sim2 expression (white arrowheads). A'–F' are adjacent sections of A–F hybridized to Trh, Ss, Trh, Crh, Vp, and Ot probes (as labeled), respectively. Their expression at the region of interest is indicated by white arrows. At the preoptic level, Trh cells are in a punctate pattern in which the periventricular cells (A') are within the Sim2 domain (A). At the aPV, Sim2 expression (B) occupies a slightly larger domain than the Ss expression domain (B'). Of note, the dorsal Sim2 domain above the aPV (B) is a Trh-positive domain representing the most anterior PVN (data not shown). At the mPVN, Sim2 (C) and Trh (C') show scattered expression patterns similar to each other. At the aPVN, there are few Crh cells (data not shown) and at the mid-to-posterior PVN level (m-pPVN) where Sim2 signal starts to weaken (D), Crh cells are in abun dance (D'). At this level, there are some Trh-positive cells at the position similar to that Sim2 (data not shown). Little or no Sim2 expression is found at the most posterior PVN (E and F), where Vp (E') and Ot (F') are abundant. Open arrowheads indicate the lateral Trh cells in the dP (A') and in the ventral lateral hypothalamic area (C'), the lateral and dorsal Ss cells (B'), and the Vp (E') and Ot (F') cells in the SCN.

 
The Sim2 Mutant Has Reduced TRH and SS Neurons
We have previously reported that the PVN of the Sim2 mutant is histologically indistinguishable from that of its wild-type (wt) siblings (26). The above results predict that if Sim2 plays a role in the PVN, it may only contribute to anterior cell types such as the TRH and SS neurons, and the cellular deficit in the Sim2 mutant PVN may not be easily detectable.

To determine whether Sim2 has a role in the development of TRH and SS neurons, we analyzed the mutant at the molecular level. The above five major cell types were surveyed and compared in the wt and mutant P0 brains (Fig. 3Go). For SS and VP neurons, immunohistochemistry (IC) was used to detect their protein expression. To detect Trh, Crh, and Ot gene expression, ISH was used. Sim2 mutants contain Trh cells in the dP and PVN, as well as SS cells in the aPV (Fig. 3Go, A'–C'). However, the numbers of Trh (combined cell counts in the dP and PVN) and SS (in the aPV) neurons were reduced to 57% and 17%, respectively (Fig. 3Go, A'–C'; Table 1Go). By contrast, Crh, Ot, and VP cells were normal as judged by signal levels [Crh: 3697 ± 485(wt) vs. 3778 ± 513(–/–) signal pixels, n = 3, P > 0.25, Fig. 3Go, D and D'; Ot: 9688 ± 1043(wt) vs. 9373 ± 689(–/–) signal pixels, n = 3, P > 0.25, not shown) and cell number (VP: 516 ± 32(wt) vs. 508 ± 43(–/–) cells, n = 3, P > 0.25, Fig. 3Go, E and E'). Ot expression level and VP cell number in the Sim2 mutant SON are also normal (data not shown). These results reveal that Sim2 is not required for Crh-, VP-, or Ot-expressing cells. Instead, Sim2 is required for the development of normal numbers of Trh- and SSexpressing cells in the anterior hypothalamus.



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  		Fig. 3. Sim2 Mutant Has Reduced Trh and SS Cells

P0 wt (A–F) and Sim2 mutant (Sim2–/–) (A'–F') brains were examined for reduction of Trh and SS cells. IC was used to assess SS and VP protein expression. Trh cells were distributed as scattered single cells and discernable for cell count. Crh and Ot (data not shown) ISH signals were quantified by densitometry of the silver granules (see Materials and Methods). Coronal sections of the dP, aPV, and PVN levels and the markers used are labeled at left. At the Sim2 mutant dP, periventricular Trh cells are reduced (white arrowhead in A'); at the aPVN, they are also affected but to a lesser degree (white arrowhead in C'). The aPV SS cell number is drastically reduced in the mutant (white arrowhead in B'); open arrowheads indicate the dorso-lateral SS populations; although they may appear increased in number in B', examination of three sample sets reveal that they are not affected. Crh (D') and VP (E') neurons are not affected in the mutant PVN and SON (data not shown). F and G, ISH using 35S-UTP-labeled Sim2 probe simultaneously with DIG-labeled Ss and Trh probes, respectively. For visualization of the silver granules in the bright field, no counter stain was used. F, Sim2 expression (high-density areas of silver granules) and Trh (brown) expression at the aPV; G, Sim2 and Ss (brown) at the aPVN. The insets in F and G are higher magnifications to show colocalization at the single-cell level. Open arrowheads indicate examples of Trh- or Ss-positive cells that also express Sim2. White arrowheads indicate some cells that are Sim2 positive but do not express Trh or Ss. Black arrowheads indicate Trh-positive cells that do not contain Sim2 signal higher than background. White dashed lines indicate the position of the ventricle.

 

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  		Table 1. TRH and SS Cell Numbers Are Affected in Various Sim1 and Sim2 Mutant Allelic Combinations

 
To assess whether Trh and Ss cells coexpress Sim2, we performed ISH on sections simultaneously hybridized to 35S-UTP-labeled Sim2 probe and digoxigenin (DIG)-labeled Trh or Ss probes. In the dP (data not shown) and PVN (Fig. 3FGo), we found both Sim2positive and Sim2-negative Trh-expressing cells. By contrast, almost all Ss-expressing cells in the aPV also express Sim2 (Fig. 3GGo). Control ISH using sense probes for these genes detected no specific signals (supplemental Fig. 1Go; published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).

Developmental Defects of TRH and SS Cell Types
To address whether the reduction of Trh- and Ss-expressing cells in the Sim2 mutant occurs early, we examined the early time points when their expression is detected. For Trh cells, E12.5 and E14.5 embryos were examined. At E12.5, Trh cells are found around the optic recess [equivalent to E14.5 rat (8)]. In the adjacent sections, we found that Sim2 occupied a lateral region contiguous from the preoptic to the postoptic (prospective PVN) level (Fig. 4Go, A–C), where Trh expression was also found (Fig. 4Go, A'–C'). In the Sim2 mutant, a reduction of Trh-positive cells was observed around this region (Fig. 4Go, B'' and C''). At the preoptic and optic levels of E14.5 brains, more Trh cells were found within the Sim2 domain, which expanded more medially than at E12.5 (Fig. 4Go, D, D', E, and E'). A reduction of Trh cells in the corresponding regions was also found in the Sim2 mutant. At the E14.5 prospective PVN, three populations of Trh-positive cells could be distinguished (Fig. 4Go, F and F'): the medial population without Sim2 expression, the intermediate population within the Sim2 domain, and the most lateral population also without Sim2 expression. All three populations of Trh cells were affected in the Sim2 mutant (Fig. 4FGo'').



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  		Fig. 4. The Reduction of Trh- and Ss-Positive Cells in the Sim2 Mutant Occurs at the Time of Their Initial Expression

Trh and Sim2 expression were compared in the wt and Sim2 mutant (Sim2-/-) (labeled at bottom) hypothalamus at E12.5 and E14.5 (labeled at top) by coronal sections. Sim2 expression in the mutant is omitted because the mutant transcript is not detectable (26 ). Sim2 expression at E12.5 preoptic (pre-op, possibly corresponding to the future dP, panel A), optic (op, panel B), and postoptic (post-op, corresponding to the prospective PVN, panel C) levels occupies slightly larger domains than Trh cells (A'–C', adjacent sections to A–C, respectively). In Sim2–/–, Trh cells are reduced at all levels (A''–C''). B/B'/B'' are at the midoptic recess level, and A/A'/A'' and C/C'/C'' are 80–90 µm anterior and posterior to B/B'/B'', respectively. Sim2 expression at E14.5 preoptic (D), optic (E), and postoptic (F) levels is compared with the Trh cells in adjacent sections (D'–F'). Trh cells are reduced at all levels in the mutant (D''–F''). At the prospective PVN (F'), only the intermediate zone of Trh cells resides in the Sim2 domain. Open arrowheads indicate the expression sites of Trh and Sim2. Asterisks indicate regions where Trh cells are reduced in the mutant. These sections are 150 µm apart with the reference midpoint of E/E'/E'' at the optic recess (or). Other abbreviations: rpe, retinal pigmented epithelium; zli, zona limitans; t, tongue. Sim2 (G) and Ss (G') expression were compared by adjacent coronal sections of wt E16.5 prospective aPV (white arrowheads, above the anterior joining of the optic nerve, opn). The dorso-lateral Ss populations do not express Sim2. At the periventricular area, Sim2 expression occupies a larger domain than the Ss cells. The dorsal Sim2 domain (asterisk in G) likely represents the Trh population in the most anterior PVN. Only very few Ss cells are found in the prospective aPV in the Sim2 mutant (G'').

 
Ss expression was detected at E19 aPV in the rat (6). We found that the Ss transcripts were not detected at E15.5 and appeared at E16.5 in the prospective aPV (Fig. 4GGo'). Sim2 expression in the adjacent sections was much weaker than Ss expression and occupied a larger domain than Ss expression, similar to their expression at P0 (Fig. 4GGo). The dorsal domain of Sim2 was its anterior PVN expression. In the Sim2 mutant, only a few Ss cells were found in the aPV (Fig. 4GGo''). Thus, Sim2 function is required at early periods of Trh and Ss expression.

The Genetic Interaction between Sim1 and Sim2 in Trh and Ss Expression
The presence of residual Trh and Ss cells in the dP, aPV, and PVN of the Sim2 mutants suggests the presence of a compensatory or an independent pathway for their expression. Because Sim1 is a paralog of Sim2 and is expressed in the same area, it is a likely candidate for such a role. To test this, we examined Trh and SS cell numbers in compound mutants of Sim1 and Sim2. The results are summarized in Table 1Go. Sim1+/– does not have reduced SS and Trh cells. Yet, Sim1–/– completely loses Trh and SS cells in the PVN and aPV (12) as well as Trh cells in the preoptic area (Table 1Go). In contrast, Sim2+/– has reduced Trh and SS cells, 78% and 47%, respectively, of the wt. This intermediate phenotype (compared with Sim2+/+ and Sim2–/–) indicates a dosage-dependent contribution of Sim2. In the Sim2+/– background, the numbers of SS and Trh cells are not affected by the presence of one or two copies of Sim1. However, in the Sim2–/– background, further loss of a Sim1 copy worsens the phenotype for both Trh and SS cells. These data suggest that Sim2 and Sim1 genetically interact but not by simple compensatory or parallel pathways.

Sim2 Is a Downstream Gene of Sim1
One possible explanation for the above result is that Sim1 acts upstream of Sim2, and Sim1 can also partially compensate for the loss of Sim2. To address this, we examined Sim2 expression in the Sim1 mutant and vice versa. As shown in Fig. 5Go, A–B', throughout the E12.5 preoptic, optic, and postoptic levels, Sim2 is expressed within the Sim1 domain. In the Sim1 mutant, however, Sim2 expression in the prospective dP/PVN is no longer detected (Fig. 5Go, C–C'), although the expression of Sim2 in the zona limitans (zli) and mamillary body (data not shown) is normal. By contrast, in the E12.5 Sim2 mutant, Sim1 expression in the prospective dP/PVN (Fig. 5Go, D–D'') is normal, consistent with Sim1 acting upstream of Sim2.



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  		Fig. 5. Sim2 Is a Downstream Gene of Sim1

Sim1 (A–A') and Sim2 (B–B'') expression were compared in the E12.5 wt brain by coronal adjacent sections at the preoptic (pre-op, A and B), optic (op, A' and B') and postoptic (post-op, A'' and B'') levels. Open arrowheads indicate the normally coexpressed regions of Sim1 and Sim2. C–C'', Sim2 expression in these regions is missing (white arrowheads) in the Sim1 mutant (Sim1–/–), although its zli expression is normal. D–D'', Sim1 expression in the Sim2 mutant (Sim2–/–) appears normal. v, Lateral ventricle; t, tongue; rpe, retinal pigmented epithelium; zli, zona limitans; or, optic recess.

 
Sim2 expression in these regions was also not found in the Sim1 mutant at E14.5 and P0 (data not shown). On the other hand, Sim1 expression in the dP, aPV, and PVN was normal in levels and domain size at E14.5 (data not shown) and P0 in the Sim2 mutant (compare Fig. 6Go, A–C to A'–C'), suggesting that Sim2 mutant cells were alive, continued to express Sim1 and occupied the same position as the wt cells. The persistent Sim1 expression may help to initiate, albeit partially, some Trh- and SS-expressing cells in the Sim2 mutant. These data demonstrate a regulatory hierarchy between these two paralogs.



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  		Fig. 6. Sim1 Expression in Sim2 Mutant Newborn Brain Is Not Reduced

Expression of Sim1 in the wt (A–C) and Sim2 mutant (Sim2–/–, A'–C') was assessed by coronal sections of P0 brains. Sim1 expression does not appear to be altered in the Sim2 mutant. The corresponding levels of the sections are labeled at left. Sim1 expression at the dP is indicated by white arrowheads, at the aPVN by white arrows, at the aPV by white open arrowheads, and at the SON by black-lined white arrowheads. These sections are approximately 180–200 µm apart following the anterior to posterior direction. The suprachiasmatic nucleus (SCN) is as labeled.

 
Sim2 Expression in the Anterior Domain of Sim1 Is Distinct from that of Brn2
Brn2 expression occupies the medial portion of the Sim1 domain at the prospective PVN at E12.5 (14). We show here that Sim2 expression occupies a lateral Sim1 domain at the same stage (Fig. 5Go). A direct comparison of their expression patterns should shed some light on gene expression pattern and anterior hypothalamus development. Adjacent sections of E12.5 brains were subjected to ISH using Sim1, Brn2, and Sim2 probes. As described above, Sim1 and Sim2 expression in the hypothalamus region is present throughout the posterior preoptic, optic, and postoptic levels (Fig. 7Go, A, A', B , B', C, and C'), whereas within the Sim1 domain, Brn2 expression is found mainly at the postoptic level (Fig. 7Go, A''–C''). Only at the postoptic level where the prospective PVN is located, are both Sim2- and Brn2-expressing cells found. Within this neuroepithelial patch, Sim2 occupies the lateral subdomain whereas Brn2 occupies the medial subdomain. Importantly, their combined expression overlaps with the Sim1 expression domain, and their expression in the presumptive PVN is lost in the Sim1 mutant (Ref.12 ; Fig. 5Go, C–C'). These data, together with the genetic evidence, support that Sim1 controls Brn2 and Sim2 expression in subdomains of the PVN progenitor region to dictate distinctive neuroendocrine hormone gene expression.



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  		Fig. 7. Sim2 and Brn2 Define Specific Progenitor Domains within the Prospective dP and PVN/SON as Early as E12.5

Sim1 (A–C), Sim2 (A'–C'), and Brn2 (A''–C'') were hybridized to adjacent sections of E12.5 (A–A'', B–B'', C–C'') brains. Brn2 is expressed in the ventricular and periventricular but not in the differentiated field of the hypothalamic regions before the optic recess (or) (A'' and B''). At the E12.5 prospective PVN/SON domain, Sim2 (C') is expressed in the lateral part of the Sim1 domain (C). Brn2 (C'') is expressed in the medial domain. Of note, only the dorsal half of this Brn2 domain is lost in Sim1 and Arnt2 mutants (12 14 ). The white lines mark the diencephalic sulcus. pre-op, Preoptic level; op, optic level; post-op, postoptic level; zli, zona limitans; v, lateral ventricle.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We show here that Sim2 plays a role in the expression of two neuroendocrine hormone genes in the dP, aPV, and PVN: the Trh and Ss genes. Together with previous work, our results provide a framework for understanding the molecular genetic pathway that orchestrates diversified neuroendocrine hormone gene expression in these regions.

A Commonality between the dP, aPV, and PVN
The Trh and Ss cells are contained within the Sim1/Sim2 expression domain from the time of their emergence. Around this area, Sim1 and Sim2 are expressed in a contiguous domain from the preoptic to the postoptic level (the prospective PVN) from E12.5 to birth. These data suggest that these regions are specified around the same time by a similar mechanism. We therefore propose that the dP, aPV, and PVN may originate from a large contiguous area of similar molecular identity as early as E12.5.

The Molecular Genetic Pathway for dP, aPV, PVN, and SON Development
The proposed functions and genetic hierarchy for Sim1, Arnt2, Otp, Brn2, and Sim2 genes are outlined in Fig. 8Go. In Otp, Arnt2, and Sim1 mutants, the prospective PVN/SON cells are born and specified, but the cells do not terminally differentiate to express neuroendocrine hormone genes. Otp also contributes to progenitor cell proliferation, survival, and migration (13, 15). Otp, Sim1, and Arnt2 utilize a common downstream gene, Brn2, to mediate the survival and terminal differentiation steps of CRH, VP, and OT neurons (i.e. their hormone gene expression) in the PVN and SON (18, 19).



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  		Fig. 8. Genetic Pathway for the Genesis of dP, aPV, PVN, and SON Neuronal Cell Types

This proposed genetic pathway is based on the phenotypic analyses of each mouse mutant. The functions and hierarchy of Otp, Sim1, Arnt2, and Brn2 in the PVN and SON were published: only Otp was shown to participate in cell proliferation and migration, whereas all four genes were suggested to participate in the differentiation program leading to hormone gene expression. The roles of Sim1 and Sim2 in the dP, and Sim2 in the aPV and anterior-mid-PVN are shown in this study. We propose that Sim2 functions only at the hormone gene expression step but not at the steps of proliferation or migration. Here, the role of Otp in regulating Sim2 in the dP and the partnership between Sim2 and Arnt2 in these regions are proposed but not established.

 
We show here that Sim2 acts downstream of Sim1 in the dP, aPV, and anterior PVN for proficient Trh and Ss expression. Sim2 expression was also reported to be lost in the Otp mutant P0 brain at the presumptive PVN (15). We therefore propose that Sim2 expression in the dP also requires Otp, which is expressed in the dP as well (Refs.13 and 15 , and data not shown). Accordingly, the Otp mutant may have a defect in Trh expression in the dP. Because Sim2 mutant transcript is undetectable, the mutant cells cannot be traced in our mouse model. However, the absence of proliferation and cell death defects in the progenitor region (supplemental Fig. 2Go; published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), along with the normal Sim1 (which labels the progenitors) expression domain in the Sim2 mutant, suggests that Sim2 function is required only for hormone gene expression and not for proliferation, survival, or migration.

Spatially Distinct Expression of Sim2 and Brn2
At an early stage of PVN cell differentiation (E12.5), Brn2 and Sim2 are expressed in distinct domains within the Sim1 domains. In the anterior regions, only Sim2 expression is found. At the postoptic prospective PVN region, Brn2 is expressed in the medial cells whereas Sim2 is expressed in the lateral cells. At P0, Brn2 expression is located at the midposterior PVN and SON and colocalizes with CRH, VP, and OT cells (12, 19). Sim2 expression is more anteriorly localized and tracks with the TRH and SS cells. Their intermingled expression at the P0 mid-PVN most likely results from cell migration later during the course of PVN formation. Together with the fact that the two mutations affect distinct cell types, these data support the hypothesis that cell type diversification is coded by region-specific transcription factors. In this model, activation of Sim1/Arnt2/Otp provides a general code for the common progenitor region of dP, aPV, and PVN/SON cells. Within this region, subregional expression of Sim2 and Brn2 dictates the local cell types generated (Fig. 8Go). Because Brn2 and Sim2 cells at the prospective PVN likely originate from the same ventricular precursors, their segregated expression domains may result from signaling molecules present in local environments. As Brn2 and Sim2 each contributes to more than one cell type, they likely activate another tier of downstream transcription factors to refine individual cell type-specific hormone gene expression.

The Upstream and Partially Compensatory Role of Sim1 for Sim2
In the absence of Sim1 function, there is no Sim2, Trh, and Ss expression, reflecting a general and upstream role of Sim1. In the absence of Sim2 function, Trh and Ss cells are reduced even with normal Sim1 expression, reflecting a semidominant and downstream role of Sim2. Furthermore, without Sim2 function, Sim1 dosage has an influence on Trh and Ss cell numbers, demonstrating that Sim1 has some capacity to compensate for Sim2. Two other lines of evidence support this proposal. First, overexpression of Sim1 and Sim2 under the control of a Wnt1 enhancer activates Shh expression in the mouse midbrain (27), demonstrating that Sim1 and Sim2 can act similarly in a given embryonic context. Second, both SIM1and SIM2 can bind to the same DNA sequence (28, 29) and act similarly in cell-based transcription assays (22, 29, 30). Because their transcriptional activities are harbored within their C termini, the lack of extensive homology between their C termini may explain why SIM1 cannot completely compensate for SIM2.

Multiple Populations of TRH Neurons
The first population of hypothalamic Trh cells appears at the lateral hypothalamus posterior to the PVN in the rat (8) at a time equivalent to E11.5 mouse. The second Trh population around the optic area appears shortly after and is localized to the lateral portion of the Sim1/Sim2 domain (Fig. 4Go, A–C and A'–C'). At E14.5 PVN, the entire Trh population is localized within the Sim1 domain. Only the intermediate Trh pool falls within the Sim2 domain. Yet, all Trh cells at this level are affected in the Sim2 mutant, strongly suggesting a noncell autonomous influence of Sim2 in Trh gene expression. Taken together with the reduced Trh cell numbers in different Sim1 and Sim2 mutant allelic combinations, these data suggest that Sim1 alone can initiate the expression of Trh gene and also activate Sim2 for more proficient Trh expression, either directly or indirectly. The lack of complete colocalization of Sim2 and Trh expression at E14.5 and P0 is consistent with both direct and indirect actions of Sim2 on Trh expression. We also found Sim2-positive cells that do not express Trh or Ss. They may either express Trh or Ss later or belong to other types of hormone-expressing neurons.

The Dimerization Partner of SIM2
SIM1 and SIM2 require a heterodimer partner to function (reviewed in Refs.31 and 32). Both of them can dimerize with ARNT2 (Refs.14 , 28 , and 30 , and our unpublished data). Because Sim1 and Arnt2 mutant mice have identical defects in the hypothalamus (12, 14, 16, 17), they are presumed to be partners. SIM2 also likely uses ARNT2 as its partner to promote Trh and Ss expression. Whether SIM1, SIM2, and ARNT2 directly participate in Trh and Ss gene activation remains to be determined. Sim2 mutants have additional defects in the palate and ribs (26, 33). There, Sim2 likely forms a partnership with Arnt because Arnt2 and Bmal1 are not expressed in these tissues (34, 35). Lastly, the human SIM2 gene is located on chromosome 21 (20), triplication of which causes Down syndrome (DS). Transgenic mouse models overexpressing Sim2 also display some of the DS phenotypes (36, 37). We also show here that the requirement of Sim2 for Trh- and Ss-expressing cells is dosage sensitive. It is therefore possible that the thyroid dysfunction (38) and impaired GH secretion (39) found in the DS patients are caused by dysregulated expression of Trh and Ss due to increased Sim2 dosage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Experimental Animals
Mice genetically modified at the Sim1 (12) and Sim2 (26) loci of 129sv/C57B6 mixed background were used. Embryos and newborns used in this study were progenies from sibling crosses between 12 and 17 generations. All animal experimentation described herein was conducted in accord with accepted standards of humane animal care as outlined in Ethical Guidelines.

Sim1 and Sim2 Mutant Mice
Mutant alleles of Sim1 (12) and Sim2 (26, 33) were previously described. Heterozygous animals of both alleles were bred to produce compound mutants. Tail or embryonic sac DNA was genotyped by Southern analysis as described (12, 26). The embryonic stages are defined by the vaginal plug date to be E0.5. The newborn is defined as P0.

In Situ Hybridization (ISH)
Whole embryos (before E16.5) or dissected brains (after E16.5) were fixed in Carnoy’s and dehydrated in ethanol. They were then embedded in paraffin and sectioned at 10 µm. Adjacent sections were used to compare gene expression whenever possible. 35S-UTP-labeled antisense RNA probes of Sim1, Brn2, Trh (12), Sim2 (26), and Crh (40) were prepared as described. Ss, Vp, and Ot probes were generated by T7 polymerase using templates containing corresponding cDNA fragments amplified from hypothalamus total RNA using RT-PCR; the primers are: Ss: 5'-AGCGGCTGAAGGAGACGCTACC and 5'-CATAATCTCACCATAATTTTAT; Vp: 5'-CGCTCGAGATGCTCGCCAGGATGCTCAA and 5'-CGGATATCCAGCTGTACCAGCCTTAGCA; Ot: 5'-CGCTCGAGTTGCTGCCTGCTTGGCTTAC and 5'-CGGATATCAGGTATTCCCAGAAAGTGGG. Sense probes of the above genes gave no specific signals (data not shown). The slides were processed, exposed to K5 emulsion (Eastman Kodak Co., Rochester, NY) for 7 d (except for Sim2, 20 d), developed, counterstained with hematoxylin, and mounted in Permount (VWR International, West Chester, PA) (23). The images were taken using a Dage digital camera. Bright-field (with blue filter) and dark-field (with red filter) photos of the same section were taken to reveal the histology and the silver granules of the hybridization signal, respectively. Each ISH image is presented as superimposed images of the dark- and bright-field photos. For colocalization, DIG-labeled Trh or Ss probes were used together with the 35S-UTP-labeled Sim2 probe on fresh frozen P0 brain sections (at 12 µm). Trh and Ss expression was revealed by anti-DIG-horseradish peroxidase (HRP) followed by 3,3'-diaminobenzidine reaction for 30 min (41). Slides were then processed as described above for radioactive ISH to reveal Sim2 signal without counterstaining. Sense probes for these genes were tested as negative controls and gave no specific signals (supplemental Fig. 1Go).

Immunocytochemistry (IC)
Brains were fixed in Carnoy’s, embedded in paraffin, and sectioned at 10 µm. IC was performed according to the manufacturers’ protocol: anti-SS (Peninsula Laboratories, Inc., Belmont, CA) was used at 1:100 dilution and anti-VP (Chemicon, Temecula, CA) was used at 1:1000 dilution, followed by the Vector Stain HRP/3,3'-diaminobenzidine kit for color development. Sections were lightly counterstained with hematoxylin, and images were taken under bright-field microscopy. Controls with secondary HRP-conjugated antibody alone followed by color reaction gave no specific signals (supplemental Fig. 1Go).

Cell Counts
Coronal sections at 10 um thickness of P0 brain samples were processed by IC to detect SS and VP, and by ISH to detect Crh, Ot, and Trh as described above. Sections containing hormone-specific positive neurons in the hypothalamic regions of interest were included for quantification: SS in the aPV; TRH in the dP and PVN; and CRH, OT, and dVP in the PVN. SS cells on both sides of the aPV (four to five sections per brain) were counted. VP cells on the left side of the PVN were counted (16–18 sections per brain). TRH, CRH, and OT proteins are not expressed at high enough levels for IC detection before the establishment of hypothalamic/pituitary connection. Therefore, ISH was used. Trh cells viewed under high magnification appeared as distinct single cells for counting. Only the left side of the dP and PVN Trh cells was counted (22–25 sections per brain). For Crh and Ot, quantitative densitometry of the silver granules was used to measure expression levels by pixel numbers using the ImageQuant program (Molecular Dynamics, Sunnyvale, CA). Only the left side of the PVN was quantified for their expression levels (16–20 sections per brain). For each genotype analyzed, three animals were used for quantification of each cell type. Cell counts and expression levels were averaged and subjected to two-tailed t test. SEs and P values are provided in the text and Table 1Go.


   ACKNOWLEDGMENTS
 
We thank Dr. Sandy Peterson for her advice on performing ISH with two probes for colocalization.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant RO1 HD35596 (to C.M.F.) and Canadian Institutes for Health Research Grant MOP-15458 (to J.L.M.).

Abbreviations: aPV, Anterior periventricular nucleus; DIG, digoxigenin; dP, dorsal preoptic area; DS, Down syndrome; HRP, horseradish peroxidase; IC, immunocytochemistry; ISH, in situ hybridization; OT, oxytocin; PVN, paraventricular nucleus; SON, supraoptic nucleus; SS, somatostatin; VP, vasopressin; wt, wild-type.

Received for publication September 24, 2003. Accepted for publication February 9, 2004.


   REFERENCES
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 DISCUSSION
 MATERIALS AND METHODS
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