Investigation of Possible Anatomical Alterations in the Brainstem: Lungs and Intestines of Hap-1 Deficient Mice
Lesly Hendershot, Edmon Chan, Raphael James, Michael Hayden, and Claire-Anne Gutekunst
Department of Neurology, Emory University, Atlanta, GA

Abstract

Huntingtin associated protein 1 (Hap1) is expressed mainly in the central and peripheral nervous system. The cellular function(s) of Hap1 is not known although several studies have suggested that it may participate in neuronal growth and differentiation. The importance of Hap1 is clearly evident in animals with homozygous disruption at the Hap1 locus. After normal intrauterine development and birth Hap1 knockout pups fail to thrive and do not survive past postnatal day 8. On postnatal day 1 Hap1 protein levels are highest in the hypothalamus and brain stem regions. The hypothalamus is critical for homeostatic control and several knockout of hypothalamic relevant genes result in postnatal death. Likewise brain stem nuclei are critical in the control of breathing and other autonomic modulation and some of these nuclei are shown to be affected in early postnatal lethal mouse models. These experiments aimed to identify potential anatomical abnormalities in brainstem nuclei enteric neurons and lungs of Hap-1 knockout mice. Tissue samples from mice homozygous knockout (Hap-1 -/-) heterozygous (Hap-1 +/-) and homozygous control (Hap-1 +/+) were collected and processed for pathological examination using histochemical and immunocytochemical techniques. Results showed no significant alterations of the brainstem lung or intestine leaving the cause of the postnatal lethality effect of Hap1 depletion yet to be determined.

Introduction

Huntingtin associated protein 1 (Hap1) is expressed mainly in the central and peripheral nervous system. The cellular function(s) of Hap1 are not known although several studies have suggested that it may participate in neuronal growth and differentiation. The importance of Hap1 is clearly evident in animals with homozygous disruption at the Hap1 locus. After normal intrauterine development and birth Hap1 knockout pups fail to thrive and do not survive past postnatal day 8. On postnatal day 1 Hap1 protein levels are highest in the hypothalamus and brain stem regions. The hypothalamus is critical for homeostatic control and several knockout of hypothalamic relevant genes result in postnatal death. Likewise brain stem nuclei are critical in the control of breathing and other autonomic modulation and some of these nuclei are shown to be affected in early postnatal lethal mouse models. These experiments aimed to identify potential anatomical abnormalities in brainstem nuclei enteric neurons and lungs of Hap-1 knockout mice. Tissue samples from mice homozygous knockout (Hap-1 -/-) heterozygous (Hap-1 +/-) and homozygous control (Hap-1 +/+) were collected and processed for pathological examination using histochemical and immunocytochemical techniques. It was anticipated that results would help to better understand the possible mechanisms involved in the postnatal lethal effect of Hap1 depletion and shed light on the potential role of Hap1 in early development.

Methods and Materials

Animals/Subjects:

The Hap1 heterozygous mice were generated by Dr Michael Hayden’s group at the Center for Medicine and Molecular Therapeutics at the University of British Columbia Vancouver Canada where the mouse colony is presently housed. Knockout mice for the Hap-1 gene were generated by heterozygous x heterozygous mating and genotyping of the progeny described elsewhere was performed by personnel in Dr. Hayden’s laboratory. Once the animals were perfused bodies were shipped to our laboratory for analysis.

Perfusion and tissue collection:

P1 pups were euthanized using isofluorane. Pups were fixed using either immersion fixation in Boin’s solution for several weeks or perfusion of 4% paraformaldehyde through the heart’s left ventricle. Pups that were paraformaldehyde fixed were left overnight in fixative then switched to sterile phosphate buffered saline until use. All procedures were in accordance with the Institutional Animal Care and Use Committee of Emory University. Dissection of the brain the lung and the intestinal tissue was performed under a dissecting microscope. Dissected tissue was placed in phosphate buffered saline (PBS) and stored at -80oC.

Tissue preparation:

Tissue was dehydrated and rehydrated with paraffin over a 12-hour cycle in a tissue processor. Samples were then embedded in paraffin blocks cut into 8 microme sections using a rotating microtome and air dried overnight or dried in an oven for 1 hour at 60oC and either stained by H&E or cresyl violet (brain and lung tissue) or processed for ICC (intestinal tissue) as described below.

Immunocytochemistry:

The H&E staining does not allow for the identification of the enteric neurons. To visualize enteric neurons immunocytochemical staining was used as described elsewhere. Analysis: Slides were visualized using an AO Spencer compound microscope at 2.5 10 20 and 40x magnification. Identification of nuclei was done using the Atlas of the mouse brain and spinal cord11 as a reference. Brainstem regions from Hap1 KO pups were compared with that of wildtype littermate controls. Areas where potential differences were seen were further analyzed using neuronal counts and nuclei area measurements. Images of the nuclei were collected via a CCD camera attached to a computer and pictures analyzed using AIS Image software. Means from neuronal counts and brainstem nuclei size were obtained and compared between Hap1 KO and wildtype littermate controls using Student’s t t-tests. A p value less than 0.05 was considered significant.

Results

1. Investigation of Brainstem: Horizontal Sections suggests a decrease in the size of the Dorsal Tegmental Nucleus A first analysis of horizontal sections through the brainstem suggested no gross anatomic differences except for a potential decrease in the size of the dorsal tegmental nucleus (von Gudden). A more detailed analysis of the dorsal nucleus comparing the mean area and estimated total cell count was obtained by measurements from all slides in which the nucleus was present.

2. Analysis of the Dorsal Tegmental Nucleus in Coronal Sections through the Brainstem: Additional tissue sections were cut coronally and collected serially to further analyze the dorsal tegmental nuclei in the brain of one wildtype and one Hap1-/- pup. Mean area and estimated cell counts were obtained through all slides in which the nucleus was present.

3. Inspection of Lung Tissue: reveals normal lung anatomy in Hap1-/-. To determine whether the Hap1 deficient pups were dying of respiratory distress we examined lung tissue for potential hyperplasia by evaluating the thickness of the epithelium.

4. Analysis of Intestinal Tissue: To determine whether the intestines of the Hap1-/- pups might be affected we measured the mean area and length of microvilli from intestinal sections collected serially. In doing this we were trying to determine if the intestine contributes to the malnutrition and subsequent death of the pups.

5. Immunocytochemistry of Intestinal Tissue: Hap1 proteins have been described in the enteric neurons localized around the intestines. As a means to further investigate whether the absence of Hap1 affects the localization or number of enteric neurons we set out to stain enteric neurons with antibodies to the neuronal specific antigen PG9.5 which has been used to specifically label enteric neurons. PG9.5 (kindly provided by Dr Keith Wilkinson) is a mouse monoclonal antibody specific to ubiquitin hydrolase 1.

Conclusions and Future Studies

Our results suggest a trend towards a decrease of the Dorsal Tegmental Nucleus in the Hap1-/- pups (p=0.067) as compared to controls. Further investigations should be conducted however to confirm this trend.   The Hap1-/- pups have not been observed to die of respiratory distress and we further demonstrate no difference in lung anatomy between Hap1-/- pups and littermate controls.   Additional research should also include further investigation with immunocytochemistry on fresh tissue samples to determine if the antibody will successfully stain the enteric neurons and allow examination of these Hap1 containing neurons between the intestinal tissue of both the control and Hap1-/- mice.

Acknowledgements and Funding Attributions

This material is based upon work supported by the Howard Hughes Medical Institute# 52003727.

In Plain English

When the gene for the Huntingtin Associated Protein (Hap1) is not present in a mouse the babies do not live beyond the eight day following birth. The cause of death is unknown however it is believed to be due to decreased feeding behaviors. The experiments I did looked at the brainstem lungs and intestines as possible areas that may be affected in the development of the knockout mice. After investigation there was no significant difference in the anatomical appears of the brainstem lungs or intestines however there was a trend towards signficance in a particular nuclei the dorsal tegmental nuclei located in the brainstem. Further studies will need to be performed in order to determine whether this area is altered in Hap1 deficient mice as compared to wildtype littermate controls.