The photo above are of an in situ hybridization I did of early Xenopus embryos using HoxD1 as a probe. In situ hybridization is a staining procedure that identifies the specific site of expression of a gene, in this case  HoxD1 at a given stage of development. This is part of a study to examine the effect of retinoic acid on the development of the lens. Below is an outline of a talk I gave on this research. 

USE OF DOMINANT NEGATIVE CONSTRUCT TO STUDY ACTION OF RETINOIC ACID     ON LENS FORMATION IN VIVO

I. Introduction

This is some of the work I did on my sabbatical that started my work with retinoic acid and vertebrate development. I had applied for and was awarded a NSF ROA (research opportunity award) for a project that I developed with Ron Grainger's lab at the University of Virginia. This work turned out to be interesting enough for me to bag my calcium projects with the fathead minnow and start to follow the lead of retinoic acid in the African clawed frog, Xenopus.

I spent the summer in Charlottesville and continued with visits to the lab during the Fall. This work was done primarily in two medium sized rooms that made up Dr. Grainger's office/lab complex. In those rooms were located most of the supporting instrumentation, chemicals, microsurgery stations and working benchspace for 13 people. These included visiting scientists like myself, post-doctoral students and graduate students, it was a tight fit.

Dr. Grainger's lab had been investigating some basic questions about development. What mechanisms are utilized that cause a single cell, the fertilized egg, to develop into an adult multicellular organism. Or in my case what is the nature of the tissue interactions that cause the development of the lens of the eye in the vertebrates.

There appears to be a complex cascade of signals between the early retina forming tissue and the lens forming tissue that are essential for normal development. Crystallines are a common group of soluble lens proteins indicative of lens differentiation. An understanding of the nature of these signals will give us insight into how the expression of specific groups of genes and hence the fate of a cell or tissue group.

Retinoic acid is a derivative of vitamin A that has profound effects on the development of vertebrate embryos. These effects include the differentiation of anterior structures of the nervous system including the eye. Much of retinoic acid's action is mediated through a group of cytoplasmic receptor proteins.

The product of the c-erbA gene is a protein that can bind to and inactivate cytoplasmic retinoic acid receptors. The objective of the present study is to examine the effect of inactivating the retinoic acid receptors in presumptive lens ectoderm and to characterize the subsequent lens development in the African clawed frog Xenopus Laevis.

HoxD1 is a gene that provides positional information along the anterior/posterior axis of the embryo. It was recently shown that RA strongly induces the expression of HoxD1. We can use HoxD1 to test our model by seeing if we can alter the normal expression of HoxD1 with c-erbA.

II. Experimental Design

A. OBJECTIVES:

The objective of the present study is to see if retinoic acid is responsible for the expression of lens crystalline proteins in Xenopus in vivo.

1. We know that the crystallines are a common group of soluble lens proteins indicative of lens differentiation.

2. Retinoic acid response elements (RARE's) have been identified in the promoter of the alpha crystalline gene. Hence RA may be involved in the       expression of this gene during lens formation.

3. HoxD1 is a protein whose expression has been blocked with the application of c-erbA.

4. c-erbA is a thyroid hormone receptor that can bind RXR which prevents heterodimer formation with RA, hence prevent binding of the RA complex to the RARE and blocking gene expression of target genes.

I have access to the alpha-crystalline, HoxD1 and C-erbA genes contained in plasmids. A plasmid is a loop of DNA that contains our gene of choice. These plasmids were provided by other laboratories and are available in limited numbers. The first part of the experiment involves growing up the plasmids and demonstrating that they work either as probes for in situ hybridization or as mRNA for injection.

Once it has been demonstrated that they are working properly I can transplant presumptive lens ectoderm (PLE) containing c-erbA to a labeled host embryo which will be maintained till stage 35 and stained for crystalline. My hypothesis is that c-erbA will block the induction of lens crystallines. Now, where did these plasmids with our genes of interest come from?

To get at the question of how RA effects lens formation we first need to see if the probes I have made up work and if they work I can go on to the main experiment.

I. What is the effect of RA on lens induction?

1. Does the alpha-crystalline probe work?

2. Does the HoxD1 probe work?

3. Does c-erbA mRNA exhibit biological activity?

4.. What is the half life of sense c-erbA mRNA?

c. Can c-erbA alter HoxD1 expression in isolated animal caps?

What is the effect of c-erbA on lens induction?

One group of embryos will be injected with a labeling dye, another with c-erbA. At stage 11 presumptive lens ectoderm (PLE) from the c-erbA injected embryos will be transplanted to the dye injected embryos.

Embryos will be grown up to stage 30-35 and whole mount in situ's for crystalline will be performed. If lens induction is the result RA signaling, c-erbA in the transplanted PLE should block RA's induction and no lens should form. It is essential to have controls that will allow me to interpret a negative result.

B. METHODS

1. Does the alpha-crystalline probe work?

The crystalline gene is carried in a loop of DNA along with a few other genes and some cutting or restriction sites in a structure called a plasmid. The crystalline plasmid was replicated, harvested, cut and transcribed for anti-sense mRNA.

Control and RA acid treated embryos were then preped for whole mount in situs. The results demonstrated that the probe worked and that there is a period of sensitivity to RA's teratogenic effects. See table 1 and 2.

Crystalline Gene in Plasmid

Stick Plasmid in Bacterial Cell

Cells replicate and with them plasmids

Harvest/clean up plasmid prep

Cut plasmid with restriction enzyme

Transcribe plasmid DNA into anti-sense mRNA

Clean up anti-sense mRNA prep, this is the probe molecule

                                                                           Expose Xenopus embryos to 10^-5 RA

Fix embryos and prepare for In Situ Hybridization with probe

2. Kill a male frog, remove its testis, mince them up and place the sperm suspension on the eggs to be fertilized.

3. Allow to develop to the 2-cell stage and sort for viable embryos.

4. Inject c-erbA and or dose with 10-5 RA at appropriate stage.

5. Fix embryos and prepare for in situ hybridization.

B. Very, very light stripes are present in some cases, but one could almost say they were not present.

C. Stripes are present, but are lighter staining than the controls.

D. Many of the embryos with two lenses have one or both of the lenses smaller compared with the controls, but these are not considered "micro" lenses.

* All treated embryos received 1 dose of 10^-5 RA at stage 18, 20, or 22 (stage of exposure is shown on the left side of the table 1) and were allowed to develop until stage 24 or 35 and In Situ stained for Krox-20 or Crystalline respectively.

Table 2. RA and Alpha-Crystalline Expression # 2*

C. Stripes are present, but are extremely faint.

D. Two of the embryos had two micro lenses in the left eye.

E. Many or most of the lenses on one or both sides are very light staining and one had two micro lenses on one side.

F. Stripes are present, but are very faint.

G. Three embryos had two lenses, but on one side staining was half the intensity. The lenses were about full size, but were lighter staining.

* All treated embryos received 1 dose of 10^-5 RA at stage 16, 18, 20, or 22 (stage of exposure is shown on the left side of table 2) and were allowed to develop until stage 24 or 35 and In Situ stained for Krox-20 or Crystalline respectively. Therefore, based on the data provided, the crystalline probe works and elevated RA concentrations can result in abnormal lens development.

2. Does the HoxD1 probe work?

This experiment was designed to determine the normal expression patterns of HoxD1. HoxD1 plasmid was received, grown up, cleaned up and anti-sense mRNA probes were transcribed for use in subsequent experiments.

A recent paper had demonstrated that c-erbA was able to alter the normal pattern of HoxD1 expression in Xenopus. Therefore it was necessary to demonstrate that the normal staining pattern of HoxD1. This was done three times because of poor yields and or purity on initial attempts. See Table 3.

HoxD1 Gene in Plasmid

Stick Plasmid in Bacterial Cell

Cells replicate and with them plasmids

Harvest/clean up plasmid prep

Cut plasmid with restriction enzyme

Transcribe plasmid DNA into anti-sense mRNA

Clean up anti-sense mRNA prep

Allow embryos to grow to appropriate stage

Fix embryos and prepare for In Situ Hybridization with HoxD1 probe

3. Does c-erbA exhibit biological activity?

c-erbA is a new gene that has not been tested in many experimental setups. Therefore it is essential that we get a handle on its biological activity if my experiments based on its function are at stake. A number of indicators of c-erbA's biological activity are being examined; its ability to protect embryos from RA exposure; its ability to alter the normal patterns of HoxDl expression; its half life in vivo and its ability to alter HoxD1 expression in isolated animal caps.

A. A series of microinjection experiments were done to see if c-erbA was able to alter the normal pattern of HoxD1 expression or protect the embryo from the action of retinoic acid. A positive of either of these experiments would indicate that the c-erbA message was biologically active.

The experiments used 1 ng and 2 ng c-erbA/embryo and both 10^-6 and 10^-5 concentrations of RA (when used). Mortality and abnormals appeared equally high in the injected and the injected + RA group. Thus, either the c-erbA is inactive or at too low a dose or the level of RA was still too high.

c-erbA Gene in Plasmid

Stick Plasmid in Bacterial Cell

Cells replicate and with them plasmids

Harvest/clean up plasmid prep

Cut plasmid with restriction enzyme

Transcribe plasmid DNA into sense mRNA

Inject 2ng sense c-erbA mRNA into 2-cell embryo

Dose embryos with RA/allow to develop normally

Fix embryos and prepare for In Situ Hybridization with HoxD1 probe

Pull needles from RNase free capillary tubing

Load needle in arm and break off tip

Put 1.0 ul H₂O on sterile parafilm on dissecting scope stage

Insert needle in water and suck up all H₂O

Set inject time to about 30 msec

Insert needle under buffer and inject 30 msec volumes till needle is empty

Determine injection time necessary to give 10 nl and set appropriate injection time

Load 1.5 ul c-erbA into needle

Place embryos in 5% Ficol in 3/4 NAM for injections

Inject half the total volume in each blastomere of 2-cell embryo (<1 hr time window)

B. What is the half life of sense c-erbA mRNA?

These experiments were designed to see how long c-erbA mRNA lasted in vivo or in the developing embryo. The idea was to inject the mRNA into an early embryo and then look for it at different time intervals using Northern Blot analysis techniques. c-erbA mRNA (1, 2, 4, and 5.35 ng/embryo) was injected into each blastomere of a 2-celled embryo. Embryos were harvested at Time = 0, stage 10, stage 20, stage 30.

Embryos were frozen in liquid nitrogen, then stored at -80C. RNA's were extracted and Northerns run using the protocol of Condie and Harland (1987, Dev. 101:93-105) to determine amount of c-erbA mRNA present.

NORTHERN BLOT PROTOCOL

Grow up c-erbA plasmids, cut, transcribe for anti-sense mRNA

Inject 2 ng sense c-erbA mRNA into 2-cell embryo

Allow embryos to develop to appropriate stage

Harvest total embryo mRNA (sense)

Grow up new c-erbA plasmids, cut and transcribe to make radioactive anti-sense c-erbA probes

Run total embryo mRNA's on a RNA gel and electrophoresis to separate

Transfer mRNA's to nitrocellulose via blotting techniques

Allow "hot" anti-sense mRNA probe to hybridize to total mRNA's on nitrocellulose blot

Place photographic film on nitrocellulose and "hot" probes will expose film where hybridization occurs

Exp # Injected C-erbA Northern Analysis Morphology In Situ RA Levels Outcome

C. Can c-erbA alter HoxD1 expression in isolated animal caps?

The final transplantation experiments will require the c-erbA construct to be active in isolated transplanted animal caps. Therefore the activity of c-erbA in isolated animal caps needs to be demonstrated. Embryos will be injected with 2-4 ng c-erbA mRNA, raised to stage 10 and their animal caps will be removed. Some of the caps will be treated with RA. This experiment is designed to determine if c-erbA is able to protect from the effects or RA or alter the normal expression of HoxD1 in isolated animal caps. This experiment has not yet been performed, pending the completion of previous experiments.