The LIM-Homeodomain transcription factor Islet-1 is required for the development of sympathetic neurons and adrenal chromaffin cells

Abstract

Islet-1 is a LIM-Homeodomain transcription factor with important functions for the development of distinct neuronal and non-neuronal cell populations. We show here that Islet-1 acts genetically downstream of Phox2B in cells of the sympathoadrenal cell lineage and that the development of sympathetic neurons and chromaffin cells is impaired in mouse embryos with a conditional deletion of Islet-1 controlled by the wnt1 promotor. Islet-1 is not essential for the initial differentiation of sympathoadrenal cells, as indicated by the correct expression of pan-neuronal and catecholaminergic subtype specific genes in primary sympathetic ganglia of Islet-1 deficient mouse embryos. However, our data indicate that the subsequent survival of sympathetic neuron precursors and their differentiation towards TrkA expressing neurons depends on Islet-1 function. In contrast to spinal sensory neurons, sympathetic neurons of Islet-1 deficient mice did not display ectopic expression of genes normally present in the CNS. In Islet-1 deficient mouse embryos the numbers of chromaffin cells were only mildly reduced, in contrast to that of sympathetic neurons, but the initiation of the adrenaline synthesizing enzyme PNMT was abrogated and the expression level of chromogranin A was diminished. Microarray analysis revealed that developing chromaffin cells of Islet-1 deficient mice displayed normal expression levels of TH, DBH and the transcription factors Phox2B, Mash-1, Hand2, Gata3 and Insm1, but the expression levels of the transcription factors Gata2 and Hand1, and AP-2ß were significantly reduced. Together our data indicate that Islet-1 is not essentially required for the initial differentiation of sympathoadrenal cells, but has an important function for the correct subsequent development of sympathetic neurons and chromaffin cells.

Keywords: Chromaffin cell; Islet-1; Mouse; Sympathetic neuron; Sympathoadrenal cell lineage.

Fig. 1

Islet-1 is expressed in primary sympathetic ganglia (arrowheads) of E10.5 mouse embryos (A). In situ hybridizations for NF68 (C) and TH (E) were performed on near adjacent sections. In the developing adrenal gland Islet-1 can be detected at E12.5 (B). To identify the area of the developing adrenal gland the adreno-cortical marker Sf1 was used as a marker on an adjacent section (not shown). Islet-1 expression can be detected at least until E16.5 in sympathetic ganglia (D, thoracic sympathetic ganglion) and in adrenal chromaffin cells (F) albeit at lower levels. Insert: higher magnification. The dashed line indicates the border of the adrenal gland anlage in (B) and the adrenal medulla in (F). (ac) Adrenal cortex, (ag) adrenal gland anlage, (am) adrenal medulla (da), dorsal aorta, (sg) sympathetic ganglion, (srg) suprarenal ganglion; bars: (A, B, C, E, and F) 100 μm, (D) 50 μm.

Fig. 2

Islet-1 expression is lacking in the area of sympathetic ganglia of Phox2B^LacZ/LacZ mice at E11.5. Photomicrographs show cross sections through thoracic sympathetic ganglia (arrowheads) of E11.5 wildtype (A and B) and Phox2B^LacZ/LacZ mouse embryos (C and D). In-situ-hybridizations for Phox2B, LacZ and Islet-1 were performed in near adjacent sections. (da) Dorsal aorta. Bar: 100 μm.

Fig. 3

The initial differentiation of SA cells is grossly normal in Islet-1^wnt1cre mouse embryos. Photomicrographs show sections through thoracic primary sympathetic ganglia of control (A, C, E, G, I, K, M, O, Q, and S) and Islet-1^wnt1cre (B, D, F, H, J, L, N, P, R, and T) mouse embryos at E10.5. In situ hybridizations were performed for Phox2B (A and B), neurofilament 68 (E and F), c-Ret (I and J), TH (M and N), DBH (Q and R), Mash1 (C and D), Hand2 (G and H), Phox2A (K and L), Insm1 (O and P) and Gata3 (S and T). Bar: 100 μm.

Fig. 4

Sympathetic ganglia of Islet-1^wnt1cre mouse embryos become progressively atrophic. The size of sympathetic ganglia, as visualized by Phox2B ISH, is reduced in Islet-1^wnt1cre mouse embryos (B, D, F, H, and J) as compared to control embryos (A, C, E, G, and I) at E11.5 (A and B) and E13.5 (C–J). (K–N) Double immunofluorescence-staining using antibodies against TH (red cytoplasmic stain) and Phox2B (green nuclear stain) of sympathetic ganglia of control and Islet-1^wnt1cre mouse embryos at E11.5 (K and L) and E13.5 (M and N). Note that in both control and Islet-1^wnt1cre mouse embryos virtually all Phox2B-immunoreactive cells are also positive for TH. (O) Counts of TH-immunoreactive cells in the thoracic sympathetic chain of control and Islet-1^wnt1cre mouse embryos. Data represent the number of TH positive cells per section and are presented as means±s.e.m. Every 10th section throughout the thoracic sympathetic chain of at least 3 embryos per group was analyzed. Bars: (A–J) 100 μm, (K–N) 50 μm. ^**p<0.01; ^***p<0.01.

Fig. 5

Apoptosis is enhanced and proliferation is reduced in sympathetic ganglia of Islet-1^wnt1cre mouse embryos at E11.5. (A and B) Photomicrographs showing TUNEL (green) and TH-immunofluorescence-staining (red) of sympathetic ganglia of a control (A) and an Islet-1^wnt1cre mouse embryo (B) at E11.5. (C) Relative number of TUNEL-positive cells per TH-positive area in thoracic sympathetic ganglia of control and Islet-1^wnt1cre mouse embryos. (D and E) Double immunofluorescence-staining using antibodies against TH (red) and phosphohistone 3 (pH3, green) of sympathetic ganglia of control (D) and Islet-1^wnt1cre mouse embryos (E). (F) Number of pH3- positive cells (% of TH positive cells) in thoracic sympathetic ganglia of control and Islet-1^wnt1cre mouse embryos. Data are presented as means±s.e.m. Every 10th section throughout the thoracic sympathetic chain of at least 3 embryos per group was analyzed. Bar: 50 μm. ^**p<0.01.

Fig. 6

In situ hybridization for Phox2B (A and B) and TrkA (C and D), c-Ret (E and F), DBH (G and H) and neurofilament 68 (I and J) on sections through the thoracic sympathetic chain of E16.5 control and Islet-1^wnt1cre mouse embryos. Note that the majority of cells in control sympathetic ganglia express TrkA and a small subpopulation of cells expresses high levels of c-Ret. In contrast the few remaining sympathetic neurons in Islet-1^wnt1cre mouse embryos lack TrkA and uniformly express high levels of c-Ret, while the expression levels of DBH and neurofilament 68 appear unaltered. The area of the sympathetic ganglion is demarcated by a dashed line in (D). Bar: 100 μm.

Fig. 7

Photomicrographs showing cross sections through the adrenal glands of control (A and C) and Islet-1^wnt1cre mouse embryos (B and D) at E13.5 (A and B), and E16.5 (C and D) stained with an antibody against TH (red cytoplasmatic stain) and DAPI (blue nuclear stain). (E) Counts of TH-immunoreactive cells in sections of the adrenal glands of Islet-1^wnt1cre mouse embryos and control littermates. Data are presented as means±s.e.m. Every 5th section of at least 6 adrenal glands of 3 different animals for each group has been analyzed. Bars: 100 μm. ^**p<0.01.

Fig. 8

In situ hybridization for TH (A and E), PNMT (B and F), DBH (C and G) and chromogranin A (D and H) on sections of the adrenal glands of E16.5 control (A–D) and Islet-1^wnt1cre mouse embryos (E–H). Note that in the adrenal gland of Islet-1^wnt1cre mouse embryos the expression of PNMT is virtually lacking and expression levels of chromogranin A appear reduced. The approximate border of the adrenal medulla is demarcated by a dashed line. (I and J) Electronmicrographs of adrenal medullae of E16.5 control (I) and Islet-1^wnt1cre mouse embryos (J). SA cells in the adrenal gland of Islet-1^wnt1cre mouse embryos have undergone grossly normal chromaffin cell differentiation as indicated by the presence of chromaffin granules (arrowheads); insert: higher magnification. Bars: (A–H) 100 μm and (I and J) 2 μm.

Fig. 9

(A–D) In situ hybridization for Gata2 on sections through thoracic primary sympathetic ganglia (arrowheads) of E10.5 control (C) and Islet-1^wnt1cre mouse embryos (D). Phox2B in situ hybridizations (A and B) have been carried out on adjacent sections. (E and F) In situ hybridization for Gata2 on sections of the adrenal gland of E13.5 control (E) and Islet-1^wnt1cre embryos (F). The border of the adrenal gland is demarcated by a dashed line. Bars: 100 μm.