Sex influence on brain defects in the fetus – a developmental biology analysis

Background:
Sex differences in disease frequency and severity are common. Although they can result from sex hormone influence, a gender bias may occur in clinically-important birth defects before the stage when sex steroids first appear. In humans, the brain defects holoprosencephaly (HPE, small, narrow, closed brain) and anencephaly (ANC, open brain) and are common, severe defects of embryonic and fetal development. HPE is seen most often in males, and can cause cognitive disability, whereas ANC is commonest in females, and usually leads to stillbirth. This difference in sex bias has never been explained and the overall goal of this PhD project is to determine how it arises in the embryo.

The project will study a mouse strain, Zic2-kumba, in which the Zic2 gene is non-functional. Homozygotes lacking Zic2 protein develop brain defects but the precise nature of the defect varies: it can be either HPE (in 25-30%) or ANC (in 70-75%). In humans, ZIC2 mutations are a cause of HPE [1]. In other systems, forebrain defects (like HPE) are male preponderant, and hindbrain defects (like ANC) occur mainly in females. However, we do not yet know the sex ratio in HPE and ANC in Zic2-kumba fetuses. We also do not understand the relationship between development of HPE, where there is early failure of ventral midline specifiction in the developing brain [2], and ANC which results from later failure of the neural tube closure [3].

Aims/Objectives:
The following questions will be addressed in this project:
1. What is the sex distribution of HPE and ANC in the Zic2-kumba strain, and does it mirror the female-male differences seen in humans?
2. Are differences identifiable in early brain development of males vs females: (i) at the stage of ventral midline specification, and (ii) at the stage of dorsal neural fold closure?
3. If sex differences are not identifiable, nevertheless can we determine what causes some mutant embryos to embark on the pathway to HPE and others to ANC?
4. Can these morphological defects be related to molecular events occurring in the early embryonic brain as it develops?

Methods:

The project will be based within the Neural Tube Group at the Institute, which has many years’ experience of studying brain and spine development in mouse mutant strains [4]. The student will learn methods of mouse genetics, with breeding and genotyping for mutant genes and embryo sex. Developmental biology methods will include dissection and imaging, including confocal microscopy, with a possibility of also using whole embryo culture to allow experimental procedures outside the confines of the pregnant female [5]. Molecular methods will involve biochemical and molecular biology analysis, with immunohistochemistry and the possibility of using RNA sequencing or other ‘omics techniques. All techniques, and the Zic2-kumba mouse, are available in our lab, with several postdoc fellows to provide training in particular techniques.

References:

1.  Barratt, K.S. et al. ZIC2 in holoprosencephaly. Adv. Exp. Med. Biol 1046, 269-299 (2018).

2.  Houtmeyers, R. et al. Zic2 mutation causes holoprosencephaly via disruption of NODAL signalling. Hum Mol Genet 25, 3946-3959 (2016).

3.  Copp, A.J. Neurulation in the cranial region - normal and abnormal. J. Anat 207, 623-635 (2005).

4.  Nikolopoulou, E. et al. Neural tube closure: cellular, molecular and biomechanical mechanisms. Development 144, 552-566 (2017).

5.         Culshaw, L.H. et al. Mouse whole embryo culture: Evaluating the requirement for rat serum as culture medium. Birth Defects Res 111, 1165-1177 (2019).