Honours in Anatomy and Cell Biology

Contact for 2004

Dr Jane Black (Coordinator) Tel: 9905 9225 email: jane.black@med.monash.edu.au
or Chris Kelly (Administration) Tel: 9905 2751 email: chris.kelly@med.monash.edu.au
Not all projects are listed in this circular. Other projects of possible interest to the student may be discussed with
the appropriate supervisor.
The Department of Anatomy and Cell Biology runs an active Bachelor of Science (Hons), Bachelor of
Biomedical Science and Bachelor of Medical Science Honours program. Our students carry out their projects at
the Clayton campus. If enrolling with one of our collaborative Centres, students conduct their research
component at the venue and complete their coursework through the department. Our collaborative groups
include, Monash Accident Research Centre, Monash Institute of Reproduction and Development (MMC),
Prince Henrys Institute (MMC), Box Hill Hospital, Alfred Hospital, the Baker Heart Institute and the Victorian
Institute of Forensic Medicine.
Students who have completed their Honours projects in this Department have had very successful career
outcomes. Most have gained employment, continued on to complete their PhD or enrolled into postgraduate
Medicine or allied Medical courses.
To be eligible to undertake Honours, students must have a distinction level average in at least 24 points (or
equivalent) of relevant third year units.
To apply to undertake an Honours year, students should apply to the Department by the 7th November 2003 by
submitting the Faculty of Science Application form. Application forms can be downloaded from the Faculty of
Science web page, or be collected from the Faculty office or from the Administrative Officer, Department of
Anatomy & Cell Biology office, Room 138.

DEPARTMENT OF ANATOMY & CELL BIOLOGY,

CLAYTON CAMPUS
THE RENAL DEVELOPMENT AND REGENERATION RESEARCH GROUP
The Renal Development and Regeneration Research Group is a large group of energetic scientists researching
the molecular regulation of kidney development, the potential use of stem cells to repair damaged kidneys and
grow new kidneys, and the consequences of suboptimal fetal kidney development on adult health. During 2003
the group consisted of Professor John Bertram, four Research Fellows (Dr. Sharon Ricardo, Dr. Georgina
Caruana, Dr. Karen Moritz and Dr. Luise Cullen-McEwen), 7 PhD students, 1 Honours student, 5 technical staff
and 1 visiting international student. The group receives funding from the NHMRC, NIH (USA), National Heart
Foundation, Australian Kidney Foundation, Janssen-Cilag Pty. Ltd. and Monash University. Our main research
collaborators are based in the Department of Physiology (Monash), the University of Queensland, University of
Mississippi and Ecole des Mines (Paris). The group has a strong national and international reputation and is
very productive, publishing in excess of 75 papers in the past 5 years.
We utilise a full range of molecular and morphological techniques in our research. These include cell and tissue
culture (including culture of whole embryonic mouse and rat kidneys, culture of specific subcompartments of
developing kidneys, culture of specific cell lines), fluorescence-activated cell sorting FACS), laser capture
microdissection, manual microdissection, imaging (including phase, DIC, fluorescence and confocal
microscopy), whole mount and section RNA in situ hybridisation and immunohistochemistry, RT-PCR, real
time PCR, Northern and Southern blotting, gene microarrays, bioinformatics, and quantitative microscopy
(image analysis, stereology). We conduct research in human kidneys and renal biopsies, wildtype, transgenic
and knockout mice, rats and sheep.
In 2004, the following projects are available in our research group for Honours students. For further
information, contact the supervisors and/or visit our website at
http://www.med.monash.edu.au/anatomy/research/kidneydevelopment.html
Project 1. Characterisation of genes differentially expressed in various sub-compartments of the
developing kidney identified using microarrays.
Supervisors: Dr. Georgina Caruana (georgina.caruana@med.monash.edu.au) & Professor John Bertram
(john.bertram@med.monash.edu.au)
One of the major focuses of our research is to perform expression profiling of various sub-compartments of the
developing kidney to identify soluble secreted proteins and surface markers that can be used to differentiate
embryonic stem cells into renal tissue and isolate these differentiated cells. To date, we have successfully
isolated sub-compartments of the developing kidney that are involved in branching morphogenesis and
nephrogenesis using a variety of techniques such as fluorescence-activated cell sorting, laser capture
microdissection and manual microdissection. Using microarrays we have identified known and novel genes that
are differentially expressed in various sub-compartments of the kidney. This project will involve further
characterisation of a subset of these genes. This will involve validation of the microarray data using kidney
wholemount and section RNA in situ hybridisation and RT-PCR. Analysis of the function of genes encoding
secreted proteins will involve the production of cell lines that are able to secrete these proteins and adding
conditioned media to embryonic kidney (metanephric) organ cultures. The effect this exogenously added
soluble protein has on branching morphogenesis and nephrogenesis will be analysed using wholemount

immunohistochemistry. Other assays used to analyse the role genes have on branching morphogenesis will
include transfecting mIMCD-3 cells with full length cDNAs of the genes of interest and assaying the ability of
these cells to undergo tubulogenesis in a collagen gel matrix.
PROJECT 2: Exploring mechanisms through which glucocorticoids affect the growth and development
of the kidney.
Supervisors Dr. Karen Moritz (Karen.moritz@med.monash.edu.au),
Dr Luise Cullen-McEwen (Luise.cullen@med.monash.edu.au) and Prof John Bertram
(john.bertram@med.monash.edu.au)
Many adult onset diseases, including much cardiovascular and renal disease, can be linked to exposure to a suboptimal intrauterine environment. When pregnant rats or sheep are exposed to high levels of glucocorticoids
early in development of the kidney, the offspring go on to develop hypertension after birth. This has been found
to be associated in many instances with decreased nephron number in the offspring as well as changes in gene
expression in the developing kidney. However, the exact mechanism through which glucocorticoids cause a
decrease in nephron endowment is unknown. The overall aim of this project is to define the association
between glucocorticoids, ureteric branching morphogenesis and nephron number. The development of the
kidney involves complex interactions between the ureteric bud epithelium and the metanephric mesenchyme.
The branching of the ureteric tree is the critical process that determines the final number of nephrons as new
nephrons are only formed adjacent to ureteric tips. Research within our laboratory has identified the role/s of
factors essential for normal branching morphogenesis. Using metanephric organ culture, this project will
examine the effects of increased levels of glucocorticoids on branching morphology of the mouse ureteric tree
in vitro. We will also determine if glucocorticoids cause alterations in expression of certain genes known to be
critical to ureteric branching morphogenesis both in vitro and in vivo using molecular techniques including
qualitative and quantitative PCR.
PROJECT 3: Effects of inhibition of the renin-angiotensin system on development of the kidney.
Supervisors - Dr Karen Moritz (Karen.moritz@med.monash.edu.au) and Prof John Bertram
(john.bertram@med.monash.edu.au)
The renin-angiotensin system (RAS) has been shown to be critical for normal fetal kidney growth and
development in many species including human, rat and sheep. In the rat, an intact RAS is critical for normal
growth of the tubules and renal interstitium. Both the AT1 and AT2 receptors are expressed in the interstitial
cells of the developing sheep kidney as well as the embryonic rat kidney. Recently, in the ovine fetus it has been
shown inhibition of this system at specific times during pregnancy caused effects on the growth and functioning
of the kidney. A reduction in weight of the kidney was observed after inhibition between 30-40 days of
gestation, however no information was gained as to whether this was due to a decrease in nephrogenesis, a
decrease in tubular formation or resulted from an increase in apoptosis. A detailed study to ascertain the
mechanisms by which angiotensin inhibition affects renal growth is therefore warranted. In the sheep,
differences in renal function, as evidenced by changes in urinary/allantoic fluid composition, were found after
RAS inhibition between 40-50 days suggesting alterations in cellular function and/or gene expression. This
project will examine how inhibition of the RAS may affect growth of the kidney using a range of techniques
including whole animal physiology, real-time PCR, unbiased stereology and immunohistochemistry.
PROJECT 4. Regeneration of the renal papilla following chemical ablation.
Supervisors Dr. Sharon Ricardo (sharon.ricardo@med.monash.edu.au),
Dr. Michelle Kett (michelle.kett@med.monash.edu.au),
Professor Warwick Anderson (warwick.anderson@med.monash.edu.au) and
Professor John Bertram (john.bertram@med.monash.edu.au)

Very little is known about the presence or identity of stem or progenitor cells in the adult mammalian kidney.
We are keen to identify such cells and ultimately evaluate their clinical potential for patients with chronic renal
disease. It is known that the inner medulla of the kidney (renal papilla) has a remarkable capacity for
regeneration. In this project, the renal papilla of mice will be ablated or partially ablated following
administration of bromoethylamine (BEA). The papillae will then be allowed to recover. Careful histological
studies and cell proliferation assays (PCNA) will be performed to document the process of recovery and the cell
kinetics of the region. The contribution of infiltrating monocyte-derived macrophages in this regeneration will
also be assessed using cfms-GFP transgenic mice. The aim is to understand the process of papillary regeneration
and try to identify the cells primarily responsible for the regeneration. Principal techniques will include
histology, immunofluorescence and PCNA proliferation analyses.
PROJECT 5. The role of Sema 4D(CD100)/plexin B1 interactions in renal development.
Supervisors Prof John Bertram (john.bertram@med.monash.edu.au) and Dr Richard Kitching (Dept
Medicine) (Richard.kitching@med.monash.edu.au)
Semaphorins, originally found to be involved in axon guidance, have emerging roles in inflammation and
branching morphogenesis. Both Sema 4D and its ligand plexinB1, are strongly expressed within the kidney,
although nothing is known about their roles in development and disease. Using molecular biological techniques,
immunofluoresence, whole animal models, and cell culture the role of Sema 4D in renal development will be
determined by examining the expression/localization of Sema 4D/plexin B1 in developing and adult murine
kidneys. Renal development and nephron number will be assessed in Sema 4D-/- mice. Alterations in Sema
4D/plexin B1 expression in renal inflammation will be assessed.
PROJECT 6. The role of BMP3 in the developing mouse kidney in vitro
Supervisors Dr Luise Cullen-McEwen (luise.cullen@med.monash.edu.au) and Prof John Bertram
(john.bertram@med.monash.edu.au)
One of the major areas of our research involves studies into the roles of specific genes in metanephric
development. Bone morphogenetic proteins (BMPs) / osteogenic proteins (OPs) are growth and differentiation
factors and comprise the largest subfamily of the transforming growth factor-beta (TGF-) superfamily of
secreted proteins. These proteins were identified based on their ability to induce bone and cartilage formation
Several BMPs/OPs are also present at sites of epithelial-mesenchymal interaction during mammalian
development. Members of the BMP family (BMP2-7) display dynamic expression patterns and in recent years,
BMP2, 4 and 7 have been shown to play central roles in mammalian kidney development. There is preliminary
evidence that BMP3 (osteogenin) is expressed in the developing kidney in the trunk of the ureter and collecting
ducts, but not in the tips of the ureteric tree. This expression pattern raises questions on the possible role of
BMP3 during metanephric development. This project will confirm expression of BMP3 during kidney
development in vivo and in vitro using RT-PCR and in-situ hybridization. Secondly, this project will utilize in
vitro approaches to analyse the role of BMP3 in metanephric development. Kidneys will be dissected from
embryonic day 12 transgenic mice that specifically express GFP (Green Fluorescent Protein) in the ureteric
epithelial 'tree'. BMP3 recombinant protein will be added to whole mouse metanephric organ culture and the
effects of exogenous BMP3 on ureteric branching morphogenesis and nephron induction will be examined using
fluorescence and confocal microscopy. We will also determine if excess BMP3 cause alterations in expression
of certain genes known to be critical to ureteric branching morphogenesis using real-time (quantitative) PCR.

PROJECT 7: Macrophages as a Potential Stem Cell in Kidney Injury and Repair

Supervisor: Dr Sharon Ricardo (sharon.ricardo@med.monash.edu.au)
Macrophages play an important role in the development of renal injury leading to disease and scarring.
Infiltration of macrophages from the blood stream into the kidney is controlled by an array of cytokines and
adhesion molecules. Macrophages are capable of producing a variety of inflammatory mediators important in
the development of fibrosis and cell injury. In addition to the detrimental inflammatory effects of macrophages,
they are also important in connective tissue remodeling, removal of cell debris and vascularization. It is
therefore likely that the balance of productive and destructive potential of macrophages is the key to the end
result of renal damage.
There is also evidence that cells from the monocytic lineage of the bone marrow may infiltrate into the kidney
in response to injury and have the ability to differentiate into kidney cell types such as tubular and glomerular
cells. This project will use a transgenic mouse line carrying green fluorescent protein (GFP) driven by the c-fms
(CSF-1) promoter, an early macrophage-specific marker, to elucidate the capacity of macrophages to contribute
to renal regeneration. Immunofluorescence microscopy will be used to examine the presence of GFP-positive
macrophage cells in mice with chronic renal disease. In situ hybridization and immunofluorescence microscopy
will provide insight into the role of macrophages and growth factors important in renal damage and repair.
CARDIOVASCULAR AND RENAL CELL BIOLOGY LAB
Project Title:
Vitamin A and nephrogenesis
Supervisor:
Dr Jane Black
Location:
Department of Anatomy & Cell Biology
Phone:
99059225
Email:
Jane.Black@med.monash.edu.au
Project:
Reduced numbers of nephrons in the kidneys of low birth weight infants have been linked to increased
incidence of hypertension and renal disease later in life. Hence, it is important to maximise nephron
endowment at birth. In our laboratory, we have shown that a low protein diet administered to rats during
pregnancy leads to low birth weight, reduced kidney weight and reduced nephron endowment in the offspring.
We have recently shown that administration of vitamin A during pregnancy prevents the reduction in nephron
endowment associated with this maternal protein restriction. The aim of this project is to investigate the
potential mechanisms whereby vitamin A stimulates nephrogenesis in the kidneys. The project will involve
animal treatment studies and subsequent molecular analyses in the kidneys.
Project Title:
Supervisor:
Location:
Phone:
Email:

Does intrauterine growth restriction lead to delayed renal maturity in sheep kidneys?
Dr Jane Black
Department of Anatomy & Cell Biology
99059225
Jane.Black@med.monash.edu.au

Project:
We have recently shown in lamb fetuses, that intrauterine growth restriction (IUGR) as a result of
experimentally restricting blood supply to the placenta appears to lead to delayed development of the fetal
kidney. The glomeruli and associated tubules appear not as well developed and there are differences in the
macula densae. This could have important implications later in life. In this project we aim to quantify these
observations in fetal kidneys and also look at kidney morphology later in life, when the IUGR lambs grow out
to adulthood. This project will involve histological and stereological analyses of IUGR and control fetal and
adult sheep kidneys.

Project Title:
Intrauterine growth restriction and the heart: effects later in life
Supervisor:
Dr Jane Black
Location:
Department of Anatomy & Cell Biology
Phone:
99059225
Email:
Jane.Black@med.monash.edu.au
Project:
Population studies have shown a strong association between low birth weight and development of heart disease
later in life. Recently in our laboratory we have shown that intrauterine growth restriction (IUGR) in rats as a
result of maternal protein restriction leads to reduced birth weight, heart weight and reduced number of
cardiomyocytes (heart muscle cells) in the offspring. Most of the proliferation (cell division) of cardiomyocytes
occurs before birth and most of the growth of the heart after birth is due to enlargement (hypertrophy) of the
cardiomyocytes. As there are fewer cardiomyocytes in the IUGR hearts we hypothesise that this could lead to
problems later in life, especially if the heart undergoes abnormal enlargement in adulthood as is observed
following the induction of hypertension. In this project, we will induce IUGR and reduced cardiomyocyte
number in rat offpring by maternal protein restriction. The offspring will be allowed to grow to adulthood when
hypertension will be induced. We expect that there will be greater pathological changes in the IUGR hearts that
have fewer cardiomyocytes to begin with. Using histological techniques we will investigate the pathological
changes in the heart.