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Endocrinology, doi:10.1210/en.2003-0996
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Endocrinology Vol. 145, No. 1 360-366
Copyright © 2004 by The Endocrine Society

Differential Colocalization of Islet-1 and Estrogen Receptor {alpha} in the Murine Preoptic Area and Hypothalamus during Development

Aline M. Davis, Marianne L. Seney, Heather J. Walker and Stuart A. Tobet

Department of Biomedical Sciences (M.L.S., H.J.W., S.A.T.), Colorado State University, Fort Collins, Colorado 80523; and Biology Department (A.M.D.), Framingham State College, Framingham, Massachusetts 01701

Address all correspondence and requests for reprints to: Stuart Tobet, Ph.D., Colorado State University, College of Veterinary Medicine, and Biomedical Sciences, Department of Biomedical Sciences, 1680 Campus Delivery, Fort Collins, Colorado 80523-1680. E-mail: stuart.tobet{at}colostate.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen receptor (ER) expression and regulation is vital to the correct functioning of the neuroendocrine brain. Islet-1 (Isl-1) is a LIM homeodomain-containing transcription factor that has been implicated in neuronal differentiation, is located in the hypothalamus, and can alter ER function in vitro. We have determined that Isl-1 is localized in several regions of the hypothalamus, including the ER rich areas of the ventromedial nucleus (VMH), the preoptic area, and the anterior hypothalamus. Using double-label immunocytochemistry, we examined the overlap between immunoreactive ER{alpha} and Isl-1 in these different hypothalamic brain regions. In the developing brain, almost 100% of VMH cells that contain immunoreactive ER{alpha} also contain Isl-1. However, in older animals, the percentage of double-label cells decreased below 70%. This change is due to a decrease in the number of cells containing Isl-1, because there was no difference in the number of ER{alpha}-containing cells. By contrast, in more anterior regions of the hypothalamus, cells containing both Isl-1 and ER{alpha} were less common, with the two populations adjacent to each other, rather than overlapping. These data suggest that, although Isl-1 and ER{alpha} can interact, they are not always found in the same cells and that regulation of ER{alpha} function is not under the same control in the VMH, preoptic area, and the anterior hypothalamus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GENERAL MECHANISMS AND key molecular players that control the organization and differentiation of the developing vertebrate brain are continually emerging (1). Transcription factors are vital components of the machinery necessary for the correct timing and location of gene expression during development. Islet-1 (Isl-1) is a LIM homeodomain-containing protein that acts as a transcription factor and mediates intranuclear protein-protein interactions (2). LIM homeodomain proteins play key roles in determining neuronal identity during development (3). Although first characterized by its ability to bind to the promoter region of the insulin gene (4), the main focus of studies regarding Isl-1 has been in motor neuron differentiation (5, 6). Isl-1 protein expression in the hypothalamus has been reported previously (7, 8), but its exact distribution within specific nuclei and changes in expression with age have not been examined.

The localization of estrogen receptor (ER) {alpha} protein in the preoptic area (POA) and anterior hypothalamic area has been well characterized in many species (9, 10, 11, 12, 13), including mice (14). These brain areas also have many regions that are sexually dimorphic in area or volume, such as the sexually dimorphic nucleus (SDN)-POA and the anteroventral periventricular preoptic area (AVPv) (15, 16). Many of the cells in these sexually dimorphic regions also express ER{alpha}, and its activation is a mechanism by which many of these sex differences develop (17, 18). The POA has also been shown to play a role in the regulation of male reproductive behavior in many rodent species (19, 20). Isl-1 is expressed in this anterior brain region (7), but expression during development and possible interactions with ERs have not been examined.

The ventromedial nucleus of the hypothalamus (VMH) regulates several physiological and behavioral mechanisms including feeding, cardiovascular function, and reproduction. It has also been shown to have distinct cellular arrangements (chemoarchitecture) in different regions of the nucleus (21, 22, 23). It is likely that these specific cellular arrangements are, at least in part, responsible for organizing the varied behaviors and processes that are regulated by this nucleus. The murine VMH becomes visible as a nucleus by Nissl stain around embryonic d 17 (E17); however, by this stage of development, some of the key cell populations are already organized (24). Previous investigations have revealed that there are a variety of different transcription factors present in the developing rodent VMH (25, 26, 27, 28, 29), yet a detailed examination of Isl-1 relative to the nucleus has not been completed.

Acting as a nuclear transcription factor, ER{alpha} is known for regulating the expression of several genes, including the progesterone receptor (30). Recently, protein-protein interactions have been found between Isl-1 and ER{alpha} in vivo and in vitro in adult rats, and it was concluded that these two proteins could interact directly, resulting in an alteration of ER{alpha} function (31). However, the degree of cellular colocalization of these transcription factors in vivo during development has not been reported.

To address whether Isl-1 plays a role in murine hypothalamic development, we examined the location of Isl-1 immunopositive cells in the developing hypothalamus. We found sites for possible interactions of Isl-1 and ER{alpha} in vivo by using fluorescent double-label immunocytochemistry. We determined that Isl-1 was expressed in cells distributed throughout hypothalamic nuclei in the murine brain, but the degree of colocalization between Isl-1 and ER{alpha} differed in varying nuclei of the hypothalamus and with developmental age.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Embryonic mice were generated from timed mating of C57Bl/6J mice housed at the Shriver Center of the University of Massachusetts Medical School. Males were placed with females overnight, and the presence of a vaginal plug the next morning was considered E0. On E17, dams were deeply anesthetized using a combination of ketamine and xylazine (80 mg/kg and 8 mg/kg, respectively), and the embryos were removed individually by cesarean section and placed on ice. Embryos were transcardially perfused with 2 ml of 4% paraformaldehyde using a 27-gauge needle on a handheld syringe. Pups collected on the day of birth, or postnatal d 0 (P0), were removed from their mother, anesthetized using ice cryoanesthesia, and perfused in a similar manner to the embryos but with 5 ml of fixative. After perfusion, the gonads of the E17 and P0 animals were examined to determine sex. All other animals used had been separated from the dam after about P20 and housed in same-sex groups of two to four. The weanling animals (24–32 d of age) and the adult animals (P47) were anesthetized similar to the dams used to collect the embryos and perfused using constant pressure (80–90 mm Hg). Weanling and adult animals were transcardially perfused with 4% paraformaldehyde, preceded by 0.05 M PBS (pH 7.4) containing heparin (2000 U/10 ml PBS). For the embryos and pups, the heads were removed and the brains exposed by a midsagittal cut through the skin and skull. Heads were then placed overnight in fixative at 4 C. In the morning, the fixative was replaced with 0.1 M PB (pH 7.4) and stored at 4 C until use. After perfusion of the older animals, the brains were removed and postfixed overnight similar to the younger tissue. The University of Massachusetts Medical School Institutional Animal Care and Use Committee approved all animal procedures.

Immunocytochemistry
All brains were embedded in 5% agarose and cut into 50-µm-thick sections using a vibrating microtome (Leica VT-1000S, Leica Instruments, Nussloch, Germany). Tissue sections from younger animals (embryos and neonates) were alternately distributed into two containers for immunocytochemistry, whereas tissue sections for older animals (weanlings and adults) were distributed into three containers, so that each had a complete set of sections from the anterior hypothalamus through the region of the VMH. Sections were then run through an immunocytochemistry protocol that has been detailed previously (24, 32). Briefly, sections were incubated in 1% glycine in PBS for 30 min and, after rinsing, placed into a 0.5% solution of sodium borohydride in PBS. After more PBS washes, sections were placed into a block containing 1% hydrogen peroxide, 5% normal goat serum, and 0.3%Triton X-100 in PBS for no less than 30 min. Sections were then incubated in primary antibody for 2–3 d at 4 C under constant agitation. After incubation in primary antibody, sections were washed in room temperature PBS containing 0.02% Triton X-100 and then incubated in the appropriate secondary antibody for 2 h at room temperature, followed by either a fluorescent conjugated third antibody or an avidin horseradish peroxidase conjugate (Vectastain ABC, Vector Laboratories, Burlingame, CA). For the E17 and P0 tissue, the concentration of Triton X-100 was 0.3%, but it was 0.5% for all older tissue to ensure antibody penetration. Polyclonal rabbit anti-ER{alpha} (C1355; Upstate BioTechnology Inc., Lake Placid, NY) was used at a 1:1000 dilution for fluorescence and 1:5000 dilution for horseradish peroxidase. For Isl-1, the monoclonal antibody 39.4D5, developed by T. Jessell and colleagues, was obtained from the Developmental Studies Hybridoma Tissue Bank, which was developed under the auspices of the National Institute of Child Health and Human Development and is maintained by the University of Iowa (Department of Biological Sciences, Iowa City, IA), and was used at 1:30 dilution for fluorescent and 1:300 dilution for nickel-enhanced diaminobenzidine (DAB) visualization. The double-label fluorescent immunocytochemistry was performed sequentially. Sections were first incubated in the Isl-1 antibody for 2–3 d and then visualized using a biotinylated secondary antibody (1:2500) and an avidin conjugated fluorescent tertiary antibody (1:500). After washing, sections were incubated in the ER{alpha} antibody for 2–3 d, and then immunoreactive ER{alpha} was visualized using an antirabbit fluorescent conjugated secondary antibody (1:250). Fluorescent sections were again rinsed, mounted onto gelatin-subbed slides, and coverslipped using Vectashield media (Vector Laboratories). Sections developed using DAB were rinsed, mounted, dehydrated, and coverslipped using Permount (Fisher Scientific, Hampton, NH). For these studies, female mice of four different ages were used, including five E17, four P0, three weanlings, and three adults.

Analysis
Nickel-enhanced DAB sections.
For the age comparisons of the amount of immunoreactive Isl-1 in the VMH, bright-field images were taken from sections reacted using nickel-enhanced DAB for visualization. Digital images were obtained using a Zeiss Axioplan microscope (Carl Zeiss, Thornwood, NY) with a Prior automated X, Y, Z stage (Prior Scientific Inc., Rockland, MA) and a SPOT-RT Slider digital camera (1520 x 1080 pixel resolution; Diagnostic Instruments, Inc., Sterling Heights, MI). Analysis of the images used IPLab (Scanalytics, Inc., Fairfax, VA) for segmentation of the areas to be measured. The images were normalized to ensure that the pixel intensities were spread across the dynamic range, and then the darkest one third of the pixels were segmented for quantification of the area occupied by positive cells. To enable positional analysis, each digital image had a grid overlay made up of 80-µm x 80-µm boxes (Fig. 1Go). The grid allowed medial to lateral comparisons of cell position from the third ventricle. Columnar data were determined by tallying all of the values from each box contained in a column using the data contained in rows 2–5 only (row 1 is the most ventral in the grid).



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  		FIG. 1. Digital image of a coronal section from a P0 female mouse brain showing a grid overlay on the posterior hypothalamus. Using a grid-based positional analysis, the amount of islet immunoreactivity lateral to the third ventricle was determined in increments of 80 µm (each bar represents 80 µm). This positional system allowed examination of the same regions in different ages. The lateral ventricle is located to the left of the image.

 
VMH width measurement.
Digital images of immunoreacted sections of mouse brains from E17, P0, weanling, and adult animals were measured to determine changes in hemisphere diameter in the posterior hypothalamus to more accurately interpret changes in cell position. Using IPLab, a line was drawn on each image. The section was measured from the medial edge of the tissue (just lateral to the third ventricle) to the lateral edge of the medial basal hypothalamus, 160 µm above the base of the brain. This location allows for a line that crosses through the entire VMH from medial to lateral and is above the area of the arcuate nucleus (ARC) for all tissue ages, but is still within the area of the VMH. The length was measured in micrometers.

Fluorescent sections.
Digital images were obtained using a Zeiss Axioplan microscope with a Prior automated X, Y, Z stage and a SPOT-RT Slider digital camera (1520 x 1080 pixel resolution; Diagnostic Instruments, Inc.). Monochromatic fluorescent images were digitized independently for Texas Red isothiocyanate and fluorescein isothiocyanate channels and then merged in Adobe Photoshop (Version 5.0, San Jose, CA) to obtain combined green and red dual-color images. Double-label cells were identified by their yellow-orange appearance. The double-label cells in each image were counted by hand in at least one bilateral section per animal per region. Regardless of how many sections were counted, the hemisphere with the greatest number of double-labeled cells was used, so that one number per region per animal was included in the analysis. This methodology allowed for analysis of the highest degree of double label but did not take into comparison any differences within different rostral-caudal regions of the ventrolateral VMH.

Statistics
Nickel-enhanced DAB sections were compared using a three-way ANOVA, using age, row, and column as the factors (JMP 4.0 Statistical Package; SAS Institute Inc., Cary, NC). For the fluorescent sections, the ER{alpha}-positive cells, the total number of cells, and the percent of VMH cells double labeled were all compared using a one-way ANOVA. For comparison of immunoreactive Isl-1 areas within specific locations across ages, a three-way ANOVA using age by row by column was used. When applicable, a post hoc test (Tukey HSD) was used to examine differences between groups. All values are reported as mean ± SEM. In all cases, {alpha} level was set at P < 0.05 for statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isl-1 and ER{alpha} double-labeled cells
Immunoreactive Isl-1 was present within cell nuclei inside the POA and anterior hypothalamus (Fig. 2Go), including the AVPv (Fig. 2AGo), the medial POA (Fig. 2BGo), and the bed nucleus of the stria terminalis (BST) (Fig. 2CGo). Cells containing immunoreactive Isl-1 were also found in more caudal regions, specifically in the ventromedial and arcuate hypothalamic nuclei (Fig. 3Go).



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  		FIG. 2. Digital color images show cells containing immunoreactive Isl-1 and ER{alpha} in the anterior regions of the mouse hypothalamus. Images are taken from female mice at E17 (A, B, and C) or E20 (inset in B). ER{alpha}-containing cells (red) and Isl-1-containing cells (green) were located in many regions of the hypothalamus. However, the number of double-label cells (as indicated by yellow-colored cells and the arrows in the inset in panel B) varied greatly by region. Few double-label cells were located in the anterior hypothalamus, specifically the AVPv (A), POA (B), or BST (C). Rather, in these regions, cells containing immunoreactive ER{alpha} or Isl-1 were located adjacent to one another. The panel scale bars indicate 200 µm, and the inset scale bar indicates 100 µm. V, Ventricle.

 


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  		FIG. 3. Digital color images show cells containing immunoreactive Isl-1 and ER{alpha} in the caudal hypothalamus. Both ER{alpha} (red, A) and Isl-1 (green, B) were located in cells of the VMH and ARC, but the degree of double-labeled cells was dramatically different than it was in the anterior hypothalamus. At this stage of development (E17), the number of ER{alpha}-containing cells that also express Isl-1 was near 100% in the VMH (C). D, Another double-labeled VMH is shown at higher magnification in which all ER{alpha}-containing cells also expressed Isl-1. Scale bars represent 200 µm.

 
Although cells containing immunoreactive ER{alpha} and Isl-1 were found in many hypothalamic regions, the degree of double label differed across regions. For example, in the AVPv and POA, few cells contained both Isl-1 and ER{alpha}. These two cell populations were strikingly adjacent rather than overlapping (Fig. 2Go, A and B). In the region of AVPv, the distribution of cells containing immunoreactive Isl-1 was notably broader than that of ER{alpha}. In the BST, a greater number of cells were double labeled, but the majority of the cells containing immunoreactive ER{alpha} did not contain immunoreactive Isl-1 (Fig. 2CGo). More caudally in the hypothalamus, there were some dual-labeled cells in the ARC (Fig. 3Go); however, the degree of overlap was not as great as that in the neighboring VMH (Fig. 3CGo). In the perinatal VMH, virtually all cells containing immunoreactive ER{alpha} in the ventrolateral region also contained immunoreactive Isl-1.

We quantified the degree of single label and colocalization of immunoreactive ER{alpha} and Isl-1 in cells of the ventrolateral VMH across development. The total number of cells expressing ER{alpha} (total of single and double labeled) per brain hemisphere section did not change with age (E17, 132.5 ± 16.3 cells; P0, 118 ± 19.9 cells; weanling, 97.0 ± 18.0 cells; adult, 130.3 ± 15.9 cells; F(3,10) = 0.74; P > 0.50; Fig. 4AGo). However, there was an effect of age on the number of ER{alpha}-only labeled cells (F(3,10) = 11.85, P < 0.01). At E17, there was an average of 6.0 ± 2.1 ER{alpha}-only cells. At P0, there were 2.8 ± 1.6 ER{alpha}-only cells. In the weanlings, the number of ER{alpha}-only cells had increased to 28.0 ± 3.8, and in the adult females, there were 41.0 ± 11.3 ER{alpha}-only cells. Using this data, the percent of ER{alpha}-containing cells that also contain Isl-1 was calculated by dividing the number of double-labeled cells by the total number of ER{alpha}-containing cells and multiplying by 100. There was a significant effect of age on the percent of double-label cells ranging from almost 100% (E17, 96%; P0, 98%) to as low as 68% (adult, 68%; weanling, 70%; F(3,10) = 12.86; P < 0. 001; Fig. 4BGo).



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  		FIG. 4. Age-related change in the amount of ER{alpha} and Isl-1 double-label cells. Although the total number of cells containing immunoreactive ER{alpha} (in one hemisphere of one section) was not different at the ages examined (P = 0.55; A), there was a significant decrease in the percentage of cells containing immunoreactive ER{alpha} and immunoreactive Isl-1 (as determined by double label) in the weanling and adult animals when compared with either the E17 or P0 mice (P < 0.05; B).

 
Comparison of immunoreactive Isl-1 by age
To determine whether there was a change in the amount of immunoreactive Isl-1 in the lateral VMH with age, we used a grid system to examine differences in amount and/or location of Isl-1-positive cells (Fig. 1Go). To ensure that differences in medial and lateral locations were not due to changes in the size of the medial basal hypothalamus as a whole, we measured the width of the medial basal hypothalamus on one side from the sections used in the VMH analysis. At E17, the average width was 446 ± 17 µm. All of the older animals had a similar width (P0, 543 ± 18 µm; weanling, 517 ± 16 µm; and adult, 541 ± 13 µm). There was a significant difference in the width of the medial basal hypothalamus among all ages (F(3,14) = 8.9, P < 0.01) that was due entirely to the smaller width at E17. The average width at P0 was not significantly different than the older ages examined. For this reason, the positional differences in immunoreactive Isl-1 are limited to examining only P0, weanling, and adult animals.

The area of immunoreactive Isl-1 protein and its distribution was compared at all three ages (P0, weanling, and adult animals) in the region located between 320 and 540 µm from the third ventricle (Fig. 5Go, area lateral from arrow for B–D). This area included the Isl-1 cluster in the ventrolateral region. There was significantly more immunoreactive Isl-1 at P0 than at either weanling or adult ages (F(2,120) = 20.9, P < 0.01). There were no significant interactions of age with row (F(3,120) = 0.23, P > 0.90; data not shown) or column (F(2,120) = 0.24, P > 0.90; Fig. 6Go). There was no shift in the position of the greatest amounts of immunoreactive Isl-1, implying that the amount of immunoreactive Isl-1 in the VMH decreased with increasing age rather than changing its location.



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  		FIG. 5. Digital images showing changes in immunoreactive Isl-1 with age. We examined the area of immunoreactive Isl-1 in the region of the VMH in E17 (A), P0 (B), weanling (C), and adult (D) animals. The area lateral to the arrow (located 400 µm from the third ventricle) to the edge of the brain was used to determine whether there was any change in the area of immunoreactive Isl-1 with age. Scale bar, 80 µm. V, Ventricle.

 


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  		FIG. 6. Isl-1 immunoreactivity in the lateral VMH decreased with increasing age. Using a columnar analysis, there was a significant decrease in the amount of Islet-1 immunoreactive area with age in the lateral VMH. The lateral VMH was divided into three 80-µm-wide sections, and the amount of immunoreactive area was determined using an automated system. P0 animals contained significantly greater immunoreactive Isl-1 in each column when compared with weanling and adult animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The mechanisms that control brain development are continually being examined and involve cascades of diffusible factors, signaling molecules, and transcription factors (27). Isl-1 is a LIM homeodomain-containing transcription factor that we have found in select nuclear patterns in the developing hypothalamus. It has also been shown to interact with and modify the function of ER{alpha} (31). In this study, we have shown that the colocalization of ER{alpha} and Isl-1 in the cells of the developing hypothalamus varies by brain region. In more anterior hypothalamic cell groups (POA, AVPv, and BST), fewer cells were double labeled, and the two cell populations were strikingly adjacent to each other. However, in more caudal hypothalamic cell groups (ARC and VMH), the degree of double label is more significant. Furthermore, this degree of colocalization decreased with age, suggesting that interactions between Isl-1 protein and ER{alpha} protein may be a mechanism that underlies changes in ER{alpha} function that may occur with increasing age in neurons of the ventrolateral VMH.

Using Nissl stains, the VMH becomes discernible as a nucleus between E15 and E17 in mice. Several of its well-characterized cell populations, including the ER{alpha}-expressing cells in the ventrolateral region, are in the correct location within the nucleus before this time in development (24). Cells in the VMH also express Isl-1 at this time, suggesting that this protein could play a role in the formation or organization of the nucleus. Because Isl-1 has been shown to influence motor neuron differentiation (5, 33) and cell-type specification (2, 8, 34), it is tempting to speculate that Isl-1 expression regulates specific cellular characteristics or protein expression in the VMH.

The anterior portions of the hypothalamus are known to contain regions that have sexually dimorphic characteristics. For example, in rats the POA contains the SDN-POA, a region of densely packed cells whose total volume is usually 5–7 times greater in males than females (15). It is known that the establishment of a volumetric sex difference in this region is dependent upon ER{alpha} activation (17, 35). We examined the murine hypothalamus for sex differences in the number or distribution of cells containing immunoreactive Isl-1 in the POA and hypothalamus but did not find any reliable differences at E15 and E17 (Walker, H. J., and S. A. Tobet, unpublished observations). It is unknown whether the rat SDN-POA contains cells that express Isl-1 and whether this cell population is sexually dimorphic. Nonetheless, it is interesting to note the dramatic closeness of Isl-1 and ER{alpha} cell populations in the anterior hypothalamic regions. Although Isl-1 and ER{alpha} may not interact in the same cells, the proximity of the cells expressing the two transcription factors allows for the possibility that they may be involved in regulating the interactions of these two cell populations.

Previous in vitro studies indicate that Isl-1 and ER{alpha} can interact at the protein level, and Isl-1 can interfere with ER{alpha}-induced transcription (31). With the exception of the ARC, where overlap was reported in rats (31), the degree of colocalization between these two proteins was unknown. Previous examination of double-label cells in the ARC of adult female Wistar rats concluded that there were very few exceptions to cells colocalizing both ER{alpha} and Isl-1 (31). Our results in mice show double-labeled cells in the ARC, but there are many cells (at every age examined) containing immunoreactive ER{alpha} that did not contain immunoreactive Isl-1. These differences in results may stem from age and/or species differences in the two studies.

In the VMH of mice, there was a large degree of double labeling for ER{alpha} and Isl-1, especially perinatally. The amount of ER{alpha} and Isl-1 double label varied by age, with the younger animals having almost 100% of ER{alpha} cells also expressing Isl-1 and the older animals containing less than 70% double-label cells. Because there was no change in the total number of ER{alpha}-containing cells in the VMH with age, it is likely that this age difference is caused by a loss of Isl-1 expression in a subpopulation of ER{alpha}-containing cells rather than the appearance of a new ER{alpha}-containing cell population. A similar down-regulation of Isl-1 protein with age occurred in the striatum (8), and the number of motor neurons that are Isl-1 positive decrease as they develop (36).

The hypothalamus regulates many basic functions including (but not limited to) reproduction, food intake, and cardiovascular regulation. If Isl-1 functions to decrease ER{alpha} activation, then it would be predicted that the age-related decline in colocalization may be particularly important for enabling some ER{alpha}-dependent function. It is interesting to note that progesterone receptor induction by estrogen in the VMH of rats is also age dependent, becoming more prevalent in adulthood than during development when colocalization of immunoreactive ER{alpha} and Isl-1 was high in the current study. At the same time, progesterone receptor induction in the POA, where colocalization in the current study was low, is similarly inducible at both time points (Quadros, P. S., and C. K. Wagner, personal communication). It is possible that a loss of Isl-1 expression with development is important for particular ER{alpha}-dependent molecular actions in the ventrolateral VMH to fully emerge. For example, a subpopulation of these ER{alpha}- and Isl-1-containing cells may stop expressing Isl-1 as the animals become reproductively competent and may play a role in facilitating progesterone receptor induction and/or the ability to exhibit reproductive behavior (lordosis).

In conclusion, we have found that the LIM homeodomain transcription factor Isl-1 is present throughout the developing hypothalamus in selective cell groups, and there is significant overlap of immunoreactive ER{alpha} and Isl-1 in the developing and adult VMH. However, the number of cells colocalizing these proteins decreases with increasing age. Given that these two proteins have been shown to interact (31), these data are consistent with a hypothesis that changing expression of Isl-1 may play a role in activation of ER{alpha}-dependent gene regulation. Isl-1 adds another potentially important piece to the molecular profile of hypothalamic neuronal identity, function, and nuclear organization in the neuroendocrine brain.


   Footnotes
 
This work was supported by Grants MH57748 and MH61376 (to S.A.T.).

Abbreviations: ARC, Arcuate nucleus; AVPv, anteroventral periventricular preoptic area; BST, bed nucleus of the stria terminalis; DAB, diaminobenzidine; E17, embryonic d 17; ER, estrogen receptor; Isl-1, Islet-1; P0, postnatal d 0; POA, preoptic area; SDN, sexually dimorphic nucleus; VMH, ventromedial nucleus of the hypothalamus.

Received August 4, 2003.

Accepted for publication October 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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